Barcelona: 10th AD/PD Conference Hosts a Shaken, Restive Field

24 Mar 2011

When 2,711 scientists from 68 countries around the globe gathered in the beautiful Catalan capital of Barcelona, Spain, on 9-13 March 2011 for the International Conference on Alzheimer’s and Parkinson’s Diseases (AD/PD), the buzz they generated during five packed days of talks, discussion, and socializing was remarkable for how it sounded opposing notes at equal volume. Distressed about a decade of trial failures in Alzheimer’s disease, a growing number of scientists across the field voiced doubt about the power of the amyloid hypothesis. Ezio Giacobini of Southern Illinois University School of Medicine, Springfield, spoke for many when he asked, “Is Aβ42 the best therapeutic target? There is a great question about this in our minds now, even if we do not want to state it openly.” Other scientists counseled patience at a time when second-generation therapeutics are in the clinic and biomarkers are making more focused trials feasible. “Cholesterol was discovered in 1890, insulin in 1920. Aβ in brain was discovered in 1984 and circulating in body fluids in 1992, Aβ protofibrils in human AD brain in 2008 and in human CSF in 2010. I want to give some optimism to the field. It is very difficult to develop CNS drugs, but it will happen,” said Lars Lannfelt of Uppsala University in Sweden. Anti-amyloid approaches continue to multiply, but industry is clearly looking for alternatives as well. The call for combination therapies issued loudly from the lectern many times, as did calls for intervening earlier in the disease when the brain is less damaged.

Behind the front lines of clinical research, meanwhile, basic and translational researchers displayed a burgeoning of ideas, and in the process, they openly presented plenty of new, unpublished data. Innate immunity, a strengthening connection between α-synuclein and tau, in-vivo metabolism of pathogenic proteins, and mechanistic studies of risk genes ranging from clusterin to LRRK2 and GBA were prominent themes. So were the important but poorly defined cognitive deficits in Parkinson’s, biomarkers for dementia with Lewy bodies, and experimental therapies across the AD-PD spectrum.

After its initial, modest beginning in 1985 in Israel, this 10th AD/PD Conference has grown to feature 1,520 abstracts. Abraham Fisher of the Israel Institute for Biological Research in Ness-Ziona organizes it, along with Israel Hanin of the University of Arizona, Roger Nitsch of the University of Zurich, Switzerland, and Manfred Windisch of the contract research organization JSW-Research Ltd. in the Austrian city of Graz. AD/PD visits a different European country every other year, using local scientists to help host the program. This year, Jesus Avila of Universidad Complutense de Madrid represented Alzheimer’s and Jaime Kulisevsky of the Hospital de la Santa Creu i Sant Pau, Barcelona, represented Parkinson’s disease.

In the opening ceremony, Ibrahim Ferrer of Universitat de Barcelona placed Spanish dementia research into the context of that country’s turbulent history. The great master Francisco Goya frequently portrayed age and madness. Some of his works are likely to have featured people with dementia, Ferrer said, for example, the painting Viejos Comiendo Sopa.

Viejos Comiendo Sopa by Francisco de Goya. Image credit: Wikimedia

The artist Salvador Dali had parkinsonism, Ferrer said, though in his case it could have partly been a secondary consequence of Dali’s habit of self-medicating his tremor. As for research, Ramon Y Cajal developed his seminal neuron doctrine while living in Barcelona for five years, but then moved to Madrid. “He made major contributions to neuroanatomy at the University of Barcelona, but was not recognized as an important scientist. His stay here was not that happy,” Kulisevsky told the audience. “Cajal knew about Alzheimer’s work, but was not interested in diseases,” Ferrer noted. In the past century, the Spanish Civil War and its aftermath set back Spanish science, though the dictator Francisco Franco died of Parkinson’s and the first president of the democratic period, Adolfo Suárez, developed Alzheimer’s.

The AD/PD Conference is popular with young scientists, who respond to added incentives ranging from the financial to the social and the culinary. Each invited speaker can bring a student without paying registration. The conference this year awarded 22 cash prizes to junior researchers, who are listed with photos on the AD/PD 2011 website. AD/PD serves snacks and lunch daily. Researchers, therefore, don't venture away from the conference center in search of food, but tend to stay together and talk science. Generous helpings of paella, croquettes, and cured meats helped students live on a shoestring; 540 of them attended the conference, according to Fisher. AD/PD 2011 offered daily networking sessions where students and young scientists could meet principal investigators. The first day kicked off with a Flamenco dance performance at the conference center. Diehards who had the stamina for an evening at the center after a relentless 8:30 a.m. to 7:10 p.m. scientific program could avail themselves of a free concert with local singers one night, and a ticketed dinner party featuring a rock band that kept the dance floor crowded until midnight on the last night. “As a new investigator in PD, I had an exceptional experience attending the AD/PD Conference in Barcelona,” Jinhua Zhang of the University of Alabama in Birmingham, wrote to Alzforum (see full comment below).

Fun aside, what were the major scientific themes? Taking stock on the last afternoon of the meeting, Bengt Winblad of Karolinska Institutet, Stockholm, said that Phase 3 trial failures reinforced that late-onset Alzheimer’s is a heterogeneous disease. This was a commonly heard sentiment. Viewed that way, the standard randomized controlled Phase 3 trial is arguably set up to fail. For one, it treats a clinically diagnosed group of patients who bring a mix of uncharacterized pathologies into the trial as if they were all the same. The conference featured numerous sessions on trying to disentangle mixed disease and defining markers for the various dementing and movement disorders that fall under the AD/PD umbrella.

For another, the single-target approach, e.g., only anti-amyloid, only NSAID, only Dimebon, may be too weak to make much difference. “The field is shifting from the one-protein-one-target-one-drug-approach to a multi-target approach. We should look for combination drugs. We could have formulations that contain two or more ingredients, or single compounds that have a selective polypharmacology,” Winblad says. Combination therapy to date means testing approved drugs together, and in the case of AD, two modest drugs, such as a cholinesterase inhibitor and memantine, still add up to a weak therapy. To be fair, regulatory agencies have discouraged simultaneous testing of unapproved investigational therapies in the past. The U.S. Food and Drug Administration has recognized the stalemate, however, and last December issued guidance encouraging scientists to begin doing exactly that (see on Google Docs).

Finally, Phase 3 trials can sink under the weight of center-to-center variability that drowns out whatever small signal a trial’s outcome measures might otherwise have yielded. “Europe alone has 27 countries with different cultures and different languages, where scales need to be validated,” Winblad said. He urged the field to publish negative studies and side effects quickly and to improve collaboration among regulatory, pharma, and academic researchers for the greater good.

The idea of both Alzheimer’s and Parkinson’s being diseases of networks also echoed throughout the conference. Scientists are grappling with the idea that disease results from a breakdown of dysfunctional networks more than from a linear disease process. In the case of AD, scientists said, APP processing, tau hyperphosphorylation, microglial activation, and oxidative stress intertwine with aging processes. How a given person ages is marked by infectious and other environmental influences as well as by a lifetime of epigenetic responses. This adds complexity, but also potential drug targets. Late-onset AD derives to a significant degree from environmental risk factors, said Christine van Broeckhoven, University of Antwerp, Belgium. Even the risk genes that have been identified in LOAD suggest as their modus operandi a role in innate immunity and a person’s ability to respond to infection, said Rudolph Tanzi. As such, they might act early in life and set the stage for neurodegenerative disease later.

Systems complexity is difficult to study, but scientists now are tackling interacting and dysregulated networks at different levels of analysis. For example, Lawrence Rajendran of the University of Zurich presented a cell-based screening approach that allows his group to knock down by RNAi each one of a given group of genes—every kinase, say, or every phosphatase in the human genome—and systematically catalogue its effect on APP processing as a core factor in AD pathogenesis. For example, the scientists silenced each of the body’s 1,270 kinases. In the process, they fingered Cdk5 as a master regulator of Aβ production through β-secretase, confirming a paper by Karen Duff (Wen et al., 2008). Akt1 and 2 affected APP processing at the γ-secretase level, and combined screens affected production of both Aβ and tau. “Alzheimer’s is not a cascade. It is more like what is behind your watch, with Aβ being one wheel in a complex machine. An FAD mutation can have a great impact on the machine, but in late-onset AD it is more multifactorial,” Rajendran said. At the level of mouse electrophysiology, Lennart Mucke’s and other labs presented their studies, closely covered on Alzforum, of how Aβ and tau dysregulation affect synaptic transmission, leading to dampened firing from inhibitory interneurons and an increase in hypersynchronous network activity and seizures (e.g., ARF related news story; Palop et al., 2011). And at the human level, Reisa Sperling of Brigham and Women’s Hospital, Boston, and others talked about how multimodal imaging is beginning to visualize the way in which functional connectivity networks are gradually being degraded as aging people accumulate pathology in their brain (for detailed ARF coverage, see ARF Miami series). Here is how Barry Greenberg of University Health Network, Toronto, Ontario, summed up the challenge. “These are diseases of networks, with slightly overlapping nodes in the different diseases. The neuronal networks are the substrates of the diseases, and they are what we should work on.”

Other ideas that have been around for some time bubbled more prominently to the surface at AD/PD 2011. For example, there is now considerable consensus that APP and presenilin mouse models simulate the pre-dementia stage of AD; hence, they might predict treatment success better at that stage than at the mild to moderate stage. Biomarkers carry great hope of improving trial outcomes, but it is important to remember that despite accelerating research in this field, at this point, no phase trial has as yet robustly coupled a biomarker effect with a desirable cognitive outcome. Just as biomarkers are being standardized and qualified for use—at considerable cost—in treatment trials at earlier stages of AD, a disquieting debate has arisen in the wake of an inconvenient paper by Lon Schneider at the University of Southern California, Los Angeles. It challenges the common assumption that Aβ42 biomarkers will improve prodromal trials (Schneider et al., 2010), and opinions about it could be heard in the hallways and in scientific talks.

In the field of PD, the conference showed that AD and PD have much in common, Winblad said. PD researchers are focusing on better understanding the cognitive component of what is now known to be a disease affecting many parts of the nervous system, including the digestive system, the heart, and perhaps even the skin. In the process, they are grappling with the entire spectrum of overlapping Lewy body diseases, and many such presentations were on display at AD/PD 2011. Like in AD, the PD field is racing to define early biomarkers; in PD that means pre-motor markers that precede the current clinical diagnosis. For a regulatory perspective on PD compared to AD, see the next AD/PD story.—Gabrielle Strobel.

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Barcelona: Straight Talk From a Regulator on Trials in Parkinson’s

25 Mar 2011

It’s the rare occasion that scientists get unvarnished advice from their colleagues at the drug approval authorities at public conferences. The thinking that goes on at these underfunded, much-maligned, yet all-important government agencies can seem opaque to most working scientists, especially those toiling at the benches. So when the 10th International Conference on Alzheimer’s and Parkinson’s Diseases (AD/PD), held 9-13 March 2011 in Barcelona, put up Cristina Sampaio of the European Medicines Agency (EMA) to share her perspective on where clinical trials in Parkinson’s disease stand at the moment, your roving reporter whipped out her pen and paper. Sampaio is a Parkinson’s expert at the University of Lisbon who brings a penchant for frank language to her service on the EMA’s Committee for Medicinal Products for Human Use (CHMP). Previously, Alzforum covered Sampaio’s take on Alzheimer’s disease trial failures (see ARF Geneva story), and on planned preclinical trials in genetic populations (see ARF London DIAN story; ARF DC API story). Curious about the similarities and differences between AD and PD? Below are extended excerpts of Sampaio’s talk, followed by excerpts of a subsequent panel discussion.—Gabrielle Strobel.

“I take an optimistic view on the prospects for clinical trials in PD,” Sampaio began. “In this disease we have plenty of effective treatments. We have drugs and surgery, which are highly effective and can be used with wisdom to improve our patients’ quality of life. [Editors note: DBS; see ARF series.] The challenge in PD is to find a further drug that makes a difference. That is much different from AD, where the drugs available have made little impact on the disease so far.

“In PD, people want to find a disease-modifying (DM) drug that will change the course of the disease. In that sense, the prospects are not different from AD. But the background of already having effective drugs changes the way we look to disease modification in PD.

“Unlike in AD, the pipeline for PD is commonly said to be empty. Few drugs are coming out, and the ones that do are not particularly novel. That is true in the sense that most drugs in the last phases of development are targeting symptomatic treatment, and most drugs that you can expect to see approved in 2012, 2013 are new formulations of well-known drugs.

“If you read about early-stage compounds, you will count 181 in the pipeline across all stages. Most of these are in very early stages of development. Most also appear in the pipeline of many neurodegenerative disorders, so are not specific for PD. For example, glutamatergic compounds are in the pipeline for AD, PD, and Huntington’s disease; sirtuin antagonists are being tried for several indications. Companies are trying a broad approach to certain compounds, and so this 181 number of compounds that can be potentially developed for PD is somewhat artificial. The fact is, in the next few years we will see mostly symptomatic treatments.

“The hottest issue in the regulatory field has been trying to obtain a claim in disease modification. One problem is that if someone develops a drug that might have DM effects but has also symptomatic effects, how do we disentangle these two effects and obtain a claim that the regulatory authorities can recognize as disease modifying? Consider, for example, the ADAGIO study of rasagiline. It has been famously debated in the literature. It was conducted under orders of the FDA, and the company followed strictly what the FDA told them to do. The EMA never told the company that a claim for DM could be obtained in this way. We always said: To prove DM, you need to prove that the drug interferes with the pathogenesis of the disease. This was difficult to do just by trial design; you need biomarkers for that. So this was mostly a battle on the U.S. side of the Atlantic. The issue ended up being settled because the higher dose did not reach the primary outcome, so the DM claim was not granted. [Editor’s note: for context, see Sampaio and Ferreira, 2010; Olanow et al., 2009; Pagonabarraga and Kulisevsky, 2010; discussion thread on PD Online Research.]

“I am nevertheless optimistic about future prospects for disease modification, because an understanding of disease pathophysiology is now being revealed. The advances in genetics are very important for that. We are starting from scratch, with α-synuclein and the GWAS results. The field needs time to mature and produce the right targets and drugs.

“The most important unmet medical need in this field is targeting the non-motor features of PD; the depression, the psychosis, the cognitive deficits. Everyone now recognizes this. The number of scientific papers on this is growing, but at the same time, only 44 randomized controlled trials were published addressing different non-motor indications in PD populations. Unfortunately, in the last decade, the trials were of poor methodological quality, and there are still problems in defining the populations. People know how to diagnose PD, but they are unsure how to define the non-motor problems. A further hindrance is that most of the drugs that have been tried are off patent.

“Gene therapy for PD is a very active field. [Editor’s note: see ARF related news story for latest Phase 2 results.] A number of gene therapy trials are ongoing with relatively good success, at least in early phases. So there are at least different approaches, which in most cases try to replace the production of dopamine or deliver growth factors.

“There is a serious hint that there are clinical subtypes of PD. A recent paper clearly demonstrates them (Van Rooden et al., 2011). This confirms what many people have said in many papers, and what we knew from empirical clinical observation. These subtypes must be identified and trials should then be made type-specific.

“The PD field is behind AD because we cannot detect the pre-motor stage. We have a good research concept for it based on enriched populations, but we cannot apply it in clinical trials yet. We have to improve pre-motor detection and biomarker use before we can do better clinical trials. The preclinical window of opportunity is critical. We need a pragmatic way to detect the pre-motor stage.

“In closing, it is urgent that there be an immediate methodological improvement in trials targeting non-motor aspects of PD. We need more trials in PD. And success in disease modification will come as you advance translational science and increase knowledge about disease subtypes.”

Panel Discussion/Q&A:

Jaime Kulisevsky, Hospital de la Santa Creu i Sant Pau, Barcelona: Representing the PD field and non-motor symptoms of PD here, I agree with everything Cristina said. In PD, we are entering a change of paradigm. All past effort has been put on the motor side. Our success there, with good drugs and surgery, has only reinforced our recognition that the disease is more than a motor problem. The cognitive problem emerges as the main unsolved issue. The more drugs we have to treat the motor symptoms, the more it becomes evident that the patients are becoming demented. In PD, I see cognition being the problem of the next few years. We are still defining what is the cognitive deterioration in PD, and this is crucial for good trials. We must share the effort of the AD side to make this happen.

For example, one of the main markers of dementia in PD can be certain haplotypes of the tau gene. [Editor’s note: see MAPT holding place 2 on PDGene Top Results]. We know that, but from a trials point of view, we are still wrestling with defining the cognitive deterioration in PD. In these areas, we can collaborate with our AD colleagues.

Raphael Blesa, Hospital de la Santa Creu i Sant Pau, Barcelona: As a clinician, a problem I have is to translate new diagnostic research criteria into normal clinical practice. So far, we have been relying on the opinion of the families/caregivers. Now, we are moving to base the diagnosis in biomarkers. That is interesting because research specialists will implement changes in the clinic in order to learn how to make the diagnosis of prodromal AD work. But for now, 99 percent of the normal doctors will keep depending on the opinion of relatives in order to establish dementia. That transition is a challenge.

Jean Marc Orgogozo, Université Victor Segalen, Bordeaux, France: I believe the problem is the drugs. Our tools are not that bad. We can distinguish disease-modifying from symptomatic treatments. For example, in migraine we have drugs for both, and we can distinguish their effects quite clearly. That is also true for depression. We can separate the two, even if it is intrinsically difficult to do that for a drug that has both effects, for example, selegiline. Even if we build long-term trials extremely well, they will fail if the drugs do not work.

Bruno Dubois, Salpetrière Hospital, Paris: Clearly, we need better drugs. But we also need to select patients better. In the 1990s, the problem was having the MCI group and trying to determine among those who has AD. We can do that now. Please use the test that can help you identify the specific AD pattern. In this sense, I disagree with the paper by Lon Schneider (Schneider et al., 2010). Even if the biomarkers did not have so much diagnostic accuracy in the ADNI cohort, they have very high negative predictive value. At least for anti-amyloid drug trials, we need to be 100 percent sure that the patients do have AD. The biomarkers can give us that certainty. It is very important to have that homogeneous population.

Christoph Hock, University of Zurich, Neurimmune, Inc., Switzerland: This is a time of great opportunity. We have achieved removing amyloid from the brains of living people. That is a tool we can use now. We can stratify the population we want to treat with anti-amyloid compounds. These are two major achievements, and we should acknowledge them.

There are problems, also. The timing: Can we go in early enough before synapses have died, and how do we do that? The mixed pathologies: amyloid, tau, α-synuclein. Lowering Aβ is not sufficient for all these cases, but it is a start. The right target: What form of amyloid should we remove? That is what academia and industry are working on.

Khalid Iqbal, New York State Institute for Basic Research In Developmental Disabilities, Staten Island: The prodromal phase is very long. Are you thinking of giving preventive drugs to children?

Hock: We have not resolved the timing issue yet.

Iqbal: Another problem I see is that we treat a heterogeneous group of patients with one drug.

Hock: We have to improve diagnostic stratification. We have to measure the tau load, the α-synuclein load, and the Aβ load in patients, and then treat accordingly the pathology we know they do have.

Reisa Sperling, Brigham and Women’s Hospital, Boston: I have been very struck at this conference how the challenges in PD mirror those in AD. In both diseases, we have the imaging agents to detect early. But if we treat early, how can we have outcome measures to know if the treatment works? How do we link the biomarker to the behavior without waiting 10 years? That is the greatest challenge.

Sampaio: That is exactly what we have to learn.

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References

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Paper Citations

Sampaio C, Ferreira JJ. Parkinson disease: ADAGIO trial hints that rasagiline slows disease progression. Nat Rev Neurol. 2010 Mar;6(3):126-8. PubMed.
Olanow CW, Rascol O, Hauser R, Feigin PD, Jankovic J, Lang A, Langston W, Melamed E, Poewe W, Stocchi F, Tolosa E, . A double-blind, delayed-start trial of rasagiline in Parkinson's disease. N Engl J Med. 2009 Sep 24;361(13):1268-78. PubMed.
van Rooden SM, Colas F, Martínez-Martín P, Visser M, Verbaan D, Marinus J, Chaudhuri RK, Kok JN, van Hilten JJ. Clinical subtypes of Parkinson's disease. Mov Disord. 2011 Jan;26(1):51-8. PubMed.
Schneider LS, Kennedy RE, Cutter GR, . Requiring an amyloid-beta1-42 biomarker for prodromal Alzheimer's disease or mild cognitive impairment does not lead to more efficient clinical trials. Alzheimers Dement. 2010 Sep;6(5):367-77. PubMed.

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Barcelona: Taking Parkinson’s Research From Movement to Mind

28 Mar 2011

Parkinson’s disease not only freezes up movement. Scientists are becoming increasingly aware of what the disorder does to the mind as well. A symposium at the 10th International Conference on Alzheimer’s and Parkinson’s Diseases, held 9-13 March 2011 in Barcelona, Spain, showcased researchers’ efforts to get a handle on cognitive impairment in PD. What are its features, how does it progress, and how does it relate to the development of PD dementia (PDD)?

Data from varied speakers displayed a remarkable level of agreement, painting a picture of early brain and cognitive changes characteristic for Parkinson’s pathology. Scientists distinguished between PDD—which denotes the dementia that develops years after a person has been living with diagnosed PD and pathology has spread to the cortex—and early PD-associated cognitive decline. The latter was their prime focus. They largely agreed it selectively affects certain mental abilities, such as visuospatial skills, fluency, and attention. Unlike the cognitive decline that precedes Alzheimer’s disease, it usually spares memory. Intriguingly, the development of posterior-cortical deficits was shown to predict the conversion of cognitive impairment to dementia, and could be a fruitful focus for further research. Speakers noted that cognitive deficits in PD can precede motor symptoms, suggesting they could have prognostic value. Numerous imaging studies bolster neuropsychological test results and provide clues to the biology. As a group, the speakers called for more longitudinal studies to help characterize the factors that lead to dementia in PD, so they can target pathways for future interventions.

How Parkinson’s Progresses
Motor problems are actually an advanced symptom of Parkinson’s disease, said Yoshikuni Mizuno at Kitasato University, Sagamihara, Japan, in an overview talk. The common late-onset variety of PD attacks lower brain regions first, beginning in the medulla oblongata. As Lewy bodies accumulate in the dorsal motor nucleus, digestion slows down. Constipation is often the first sign of PD, appearing up to 15 years before motor symptoms develop, Mizuno said. Other autonomic nervous systems follow, with the second measurable symptom being the loss of sympathetic nerve fibers to the heart. As Lewy bodies spread into the olfactory bulb, a person’s sense of smell deteriorates, occurring about five years before motor symptoms, Mizuno said. The disorder moves into the pons, begetting sleep disorders and depression, and then into the midbrain, where degeneration of the substantia nigra eventually leads to the characteristic motor problems. Much later in the disease, the cortex succumbs, and hallucinations and dementia may develop. Dementia is seen in only about 25 to 30 percent of PD patients, however, Mizuno said.

Where Does Cognitive Impairment Fit In?
Despite the late onset of dementia, the first cognitive deficits can be detected even before motor symptoms manifest themselves, said Kenneth Marek at Yale University in New Haven, Connecticut. He described recent findings from the Parkinson Associated Risk Syndrome (PARS) longitudinal study, which identifies people at high risk of PD and looks for factors that predict who develops the disorder. The researchers screened around 5,000 healthy people over 50; about half were relatives of PD patients and the other half were randomly chosen. To qualify for the study, volunteers needed to score in the lowest 15 percentile on the University of Pennsylvania Smell Identification Test (UPSIT). In addition to a poor sense of smell, 28 percent of the roughly 300 participants also had diminished dopamine transport in the brain as evident by brain imaging. By contrast, only 8 percent of people whose sense of smell was intact had that second early warning sign of PD. The participants with low dopamine also had statistically significant cognitive deficits in trail-making tests (a visuospatial task), semantic fluency, and processing speed. The degree of impairment correlated with the level of both dopamine transport reduction and loss of olfaction, Marek said. This suggests that cognitive losses are an early feature of PD, he said, and that cognitive testing could be used in conjunction with other screens to help predict PD risk.

Scientists have known for some time that mild cognitive impairment (MCI) can be present in PD, with several studies showing a prevalence of around 25 percent (see, e.g., Aarsland et al., 2010). However, this figure may be an underestimate, suggested Roberto Monastero at the University of Palermo, Italy. In a study of some 500 healthy elderly Italians versus 300 with PD, Monastero’s team found that more than half of those with PD had cognitive decline in at least one area and met Petersen’s criteria for MCI (see Winblad et al., 2004). MCI can be classified as amnestic (involving memory problems) or non-amnestic. The researchers found there was no difference in the prevalence of amnestic MCI between people with and without PD, but Parkinson’s patients were twice as likely to have non-amnestic MCI as were controls.

Does Cognitive Decline in PD Lead to Dementia?
A major unanswered question is whether the cognitive impairment in PD inevitably leads to dementia. Perhaps not, suggested Jaime Kulisevsky at the Autonomous University of Barcelona, Spain, who co-hosted AD/PD 2011. Kulisevsky pointed out that the literature shows that cognitive impairment does not greatly worsen over periods of one to three years, and some patients remain non-demented for decades. This is in contrast to dementia with Lewy bodies (DLB), where patients also have both cognitive and motor symptoms, but deteriorate quickly.

To tease apart the factors that contribute to dementia, Kulisevsky’s team designed an assessment, the Parkinson’s Disease Cognitive Rating Scale, that separately scores frontostriatal and posterior-cortical functions (see Pagonabarraga et al., 2008). This scale has been adopted by The Movement Disorder Society for assessing cognition in PD, Kulisevsky wrote to ARF. Kulisevsky’s group validated the test on a prospective cohort of around 90 PD patients and 60 controls. They found that people with PD showed a gradual, steady decline on executive tasks that rely on the frontal cortex and striatum, such as visuospatial tasks and phonemic fluency (the ability to generate words that begin with particular letters). A very different pattern emerged for skills that require the posterior cortex, however. These include copying images (i.e., drawing a clock) and semantic fluency (the ability to generate words that belong to particular categories). On these tasks, Kulisevsky said, about 20 percent of patients exhibited rapidly plummeting abilities, a phenomenon that began roughly three years after PD diagnosis. Notably, semantic fluency and copying ability were the only two tasks where poor performance predicted progression to dementia. These findings have been replicated in a five-year longitudinal study of more than 120 people (see Williams-Gray et al., 2009).

The data suggest a model where, in some people, posterior cortical dysfunction rapidly develops over a background of progressive frontocortical impairment and leads to dementia, Kulisevsky said. He noted that poor posterior cortical function is the primary feature seen in imaging studies of people with PD-MCI. Other people with PD do not progress in this way, even though they also have a cognitive impairment. “Maybe we should not label all cognitive impairment in PD as ‘PD-MCI,’” Kulisevsky proposed. “MCI may be better defined in terms of the posterior-cortical deficit.” This might help distinguish the non-progressive cognitive impairment in PD from the rapidly progressing condition, Kulisevsky suggested. This parallels a prior shift in AD, where MCI was seen for some years as a precursor to AD, until biomarker and more specific cognitive tests enabled the definition within MCI of people who already had very early AD from other people who did not.

Genetics of PD Dementia
The Barcelona team has also unearthed a genetic contributor to dementia. First author Núria Setó-Salvia and colleagues published in Archives of Neurology this month that the H1 haplotype of the MAPT (tau) gene is overrepresented in PD patients, and particularly in PDD. The authors further refined the genetic association. They found a rare sub-haplotype, H1p, that was 20 times more common in people with PDD than in controls, as well as a protective sub-haplotype, H2a, that was twice as common in controls as in PDD. These data support previous findings that MAPT genotype affects the development of dementia, the authors note, seen, for example, in the study by Williams-Gray et al. The latter authors suggest that the dementing posterior-cortical deficits in PD involve tau, while fronto-executive defects have a more dopaminergic basis and evolve independently.

MAPT haplotype has been linked to other forms of Parkinson’s-associated dementia as well, such as frontotemporal dementia with parkinsonism and the atypical parkinsonian syndromes progressive supranuclear palsy and corticobasal degeneration (see ARF related news story). Significantly, MAPT haplotype showed no connection to AD or dementia with Lewy bodies in this study, perhaps suggesting that PDD and DLB involve distinct genetic factors. The tau haplotype had been identified by John Hardy and colleagues many years ago (see ARF St. Moritz story), but, puzzlingly to some, generated only weak signals in AD studies, where tau tangles are a defining pathology (see ARF related news story and Q&A). The gene for tau, MAPT, is not among the AlzGene Top 42, but ranks second place on PDGene Top Results.

Features of Cognitive Impairment in PD
Keith Wesnes at United BioSource Corporation, Gooring on Thames, U.K., took a different approach to dissecting the specific features of cognitive deficits in PD. His team used a computerized test system to measure several aspects of attention and memory. They found the main losses in people with PD were in powers of attention, in working memory capacity, and in pattern separation, which is the ability to discriminate similar pictures. Wesnes said the last suggests a deficit in the dentate gyrus. The loss in attention is dramatic, he said, about twice as much as that seen in a person who has consumed 0.7 g/kg of alcohol. Importantly, decreased power of attention predicted cognitive decline in this study. Wesnes noted that attention deficits can interfere with activities of daily living and could lower scores on standard cognitive tests such as the Mini-Mental State Exam. Attention deficit is a major component of cognitive impairment in PD, Wesnes concluded.

Imaging results reinforce many of these cognitive test findings. Irena Rektorova at Masaryk University, Brno, Czech Republic, reviewed a number of recent studies, both structural and functional. On the structural side, a small study showed that cognitive impairment in PD comes with a loss of gray matter in the left frontal lobe and both temporal lobes, a pattern midway between that of PDD and unimpaired PD (see Beyer et al., 2007). Another study showed that cognitive decline in PD was associated with anterior caudate atrophy and posterior ventricular enlargement (see Apostolova et al., 2010). In general, however, structural MRI has not been a useful tool for diagnosing cognitive decline in PD.

Poor cognitive performance also seems to correlate with changes seen by functional MRI, Rektorova said. This includes a decreased functional connectivity between the core regions engaged in the default-mode brain network, and cortical areas responsible for multisensory integration and reorienting of attention. This data dovetails with test findings of reduced attentive power and global cognitive performance. Cognitive problems also go along with weakened connections to occipital areas and disturbances in the resting state visual processing network, Rektorova said. This fits with observed visuospatial and posterior deficits, and with positron emission tomography (PET) markers of cholinergic deficits observed in these brain regions (see Hilker et al., 2005).

PD-MCI also involves metabolic changes. Using fluorodeoxyglucose PET (FDG-PET), one study showed higher activity in brainstem and cerebellum and lower activity in prefrontal and parietal regions in PD-MCI compared to PD alone (see Huang et al., 2008). This finding has been replicated (see Lyoo et al., 2010 and Nobili et al., 2009). Subclinical vascular pathology of brain vessels may be a factor in PD-MCI, Rektorova noted, as work from her group showed that ultrasound markers of both large and small vessel impairment correlated with poor cognitive scores (see Rektor et al., 2009). None of these findings have been broadly replicated and garnered robust consensus yet, however.

One point speakers made repeatedly is that MCI in PD looks different from MCI in AD. For example, Rektorova pointed out that the core default-mode network, which reflects resting activity of the brain and becomes dysfunctional in AD (see ARF related news story), appears normal in PDD. Monastero noted that MCI in PD involves a different pattern of brain atrophy than other forms of MCI (see, e.g., Lee et al., 2010). Non-amnestic MCI predominates in Parkinson’s, further distinguishing it from the memory impairments typical of AD.

What Lies Ahead
The speakers touched only briefly on how medication and treatment might affect cognitive deficits. Wesnes said that attention deficits in PD patients were the same whether the patients were on or off L-dopa, but he noted that treatment with rivastigmine, a cholinesterase inhibitor, gradually improved performance in these tests. Rektorova observed that reduced I-FP-CIT uptake in the caudate nucleus correlates with poor performance on the “Tower of London” task in people with PD (see Rektorova et al., 2008), but that PD patients on L-dopa perform this task normally. ¹²³I-FP-CIT is a SPECT imaging tracer of the dopamine transporter, and reduced uptake denotes dopaminergic degeneration. Rektorova’s team has also shown that repetitive transcranial magnetic stimulation of the inferior frontal cortices can increase processing speed in slowed brain networks (see Baláz et al., 2010). In the future, Rektorova said, a better understanding of PD-associated cognitive impairment will help researchers develop more accurate prognoses and improve selection of people for prevention trials and surgical interventions. The latter, called deep-brain stimulation (DBS), tends to most help those patients who have primarily motor, not cognitive, symptoms (see ARF DBS series).—Madolyn Bowman Rogers

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External Citations

Barcelona: What Lies Beyond Genomewide Association Studies?

29 Mar 2011

The common late-onset forms of Alzheimer’s and Parkinson’s disease are now believed to involve substantial inherited risk. With the use of increasingly large genomewide association studies (GWAS), scientists are making headway dissecting the genetic factors that contribute to these diseases, but GWAS results are merely lists of genetic markers that by themselves say little about the disease. GWAS are also limited to finding common variants, and can miss rare mutations that often carry more risk. At the 10th International Conference on Alzheimer’s and Parkinson’s Diseases, held 9-13 March 2011 in Barcelona, Spain, speakers outlined the field’s answers to these quandaries. They described how second-level analysis can identify disease-causing mutations from GWAS results, and suggested future strategies for finding variants that the current GWAS overlook. Their ultimate goal is to unearth the biology under each genetic association, the speakers emphasized, in order to provide new pathways for therapeutic intervention.

Finding the Disease Mutation
GWAS reveal associations between single nucleotide polymorphisms (SNPs) and disease risk, but they do not automatically point to a particular gene, much less identify the mutation that leads to the disease. “GWAS really identify genomic regions,” Andy Singleton at the National Institutes of Health, Bethesda, Maryland, wrote to ARF. “We associate a gene name with each locus for ease of tracking, but we are really not sure what the affected gene is.” The only way to be sure is to find a disease-causing mutation related to a particular gene. If the mutation is a protein coding (i.e., amino acid) change, then scientists can usually find it by sequencing only about 100 samples, said John Hardy at University College London, U.K. Most GWAS hits, however, involve non-coding changes, and these are much more difficult to spot, Hardy said. For example, neither clusterin (CLU) nor PICALM, two AD risk genes that are currently number 2 and 3 on AlzGene’s Top Results, includes simple coding changes. Non-coding variants typically affect mRNA splicing, expression levels, or stability, Hardy said. They may be caused by changes in gene promoter regions, the RNA tail, or in regulatory antisense or microRNAs. To help find these mutations, scientists at University College and the National Institutes of Health, Bethesda, Maryland, are developing a human brain expression database from more than 400 brains, which should be complete in about nine months, Hardy said. By providing information on normal gene expression levels in 10 brain regions, this database should help scientists spot changes in expression of GWAS hits in AD brains. Another promising technique, Hardy said, is to use transcriptome microarrays to find mRNA changes.

Christine Van Broeckhoven at the University of Antwerp, Belgium, described second-level analysis of several AD-associated GWAS hits. In particular, work published in the March 15 Molecular Psychiatry found that a splice variant in complement receptor 1 (CR1, place 6 on AlzGene) associates with AD risk. CR1 has two common isoforms, CR1-F and CR1-S. The less common CR1-S, with a population frequency of 15 percent, includes an extra copy of a repeat sequence that binds complement proteins C3b and C4b. In a Flanders-Belgian cohort of 1,039 patients and 844 controls, Van Broeckhoven and colleagues found that people with at least one copy of CR1-S had a 30 percent higher risk of AD than those with two copies of CR1-F. The scientists replicated the results in a French cohort, and confirmed by regression analysis that this copy number variation accounted for the original GWAS signal.

Van Broeckhoven pointed out that it is still not clear how CR1-S contributes to the disease. One possibility comes from the fact that when CR1 binds C3b, it inactivates C3b and inhibits the complement cascade. Therefore, the presence of an additional complement binding site on the receptor may dampen complement signaling and reduce Aβ clearance. However, this idea remains to be tested. Last week, a mouse genetics paper by Tony Wyss-Coray’s group at Stanford University implicated complement receptor in adult neurogenesis in the hippocampus (see ARF related news story).

In Barcelona, Van Broeckhoven also described preliminary findings on the GWAS hits CLU and bridging integrator 1 (BIN1). In a large Belgian population, her team found several rare variants that affect the β domain of CLU and were specific to AD patients, suggesting that β chain variations may be important in the disease. The researchers have replicated this result in a French cohort, and are in the process of looking at other samples. For BIN1, a protein involved in endocytosis and trafficking, preliminary results point to a genetic variant in the 5’ end of the protein, Van Broeckhoven said. This could indicate an effect on protein expression levels. The Belgian group also found an association between BIN1-associated SNPs and levels of tau in the cerebrospinal fluid.

In related news, a GWAS study led by Richard Mayeux at Columbia University, New York City, published in the March Archives of Neurology, replicated the original finding of CLU, PICALM, and BIN1 as AD risk factors in a Caribbean Hispanic population, as well as identifying some novel loci associated with late-onset AD.

Pinpointing the Biology
Even after finding a disease-causing mutation, the way in which that variant promotes AD poses the next puzzle. At that point, GWAS hits have to move into in vivo studies to find their biological effects, the speakers said. At AD/PD 2011, Peter St George-Hyslop at the University of Cambridge, U.K., described one such study. He performed genomewide microarrays on the brains of AD TgCRND8 mice at 70 days old, when Aβ levels first start to rise, as well as at 90 and 150 days, and compared gene expression levels with those of control mice. He replicated the result in J20 AD mice.

About 100 genes were dysregulated in AD brains, St George-Hyslop said. The genes clustered into just seven pathways, notably including inflammation, innate immunity, cholesterol, lipid metabolism, and protein trafficking. All of these pathways have been previously implicated in AD, and most GWAS hits also fall into these categories. The results suggest that AD genes act through Aβ-dependent mechanisms, St George-Hyslop said, perhaps by affecting how well the brain deals with Aβ. Aβ therapies may have to target whole pathways rather than particular genes to be successful, he suggested.

Hunting Down Missing Genetic Risk
Alzheimer’s has a strong inherited component, with estimates ranging from 60 to 80 percent, according to Van Broeckhoven. The established AD risk gene ApoE accounts for only a fraction of that variance, said Rudy Tanzi at Massachusetts General Hospital, Boston, and recent GWAS results have not made up the difference. The AlzGene database lists 42 gene candidates with significant meta-analysis results, Tanzi said, but each one has only a small effect on risk. The first-pass GWAS analysis may underestimate risk at each locus, however, said Tanzi, because rarer variants often exist that carry more risk (a point also emphasized by Hardy). As an example of this, Tanzi cited ADAM10, the “good” APP α-secretase. Currently on AlzGene spot 26, this gene was found to have two novel late-onset pro-domain mutations that segregated with the common SNP and increased AD risk (see ARF related news story on Kim et al., 2009).

Several speakers emphasized that GWAS alone cannot discover all disease variants. Large association studies are limited to finding common genetic variants that each contribute little risk. Traditional familial and linkage studies, on the other hand, uncover rare variants of high risk. In between these two extremes probably lie many low-frequency variants that carry moderate disease risk. The best way to find these genes, said many scientists, is to sequence exomes. That reveals rare coding variants. The exome consists of the parts of the genome that are translated into proteins. Since the exome makes up only about 1 percent of the total genome, it is more feasible to sequence exomes than whole genomes, but it is still expensive. Thomas Gasser at the Hertie-Institute of Clinical Brain Research, Tübingen, Germany, said that it currently costs about $5,000 to sequence one person’s exome, and this price may have to drop further for the technique to become widely used.

Exome Sequencing Ferrets Out a Rare PD Gene
Exome sequencing does work, however. In Barcelona, Carles Vilarino-Guell at the University of British Columbia, Vancouver, described the use of this technique to identify a rare PD mutation in a small Swiss family. The family has an autosomal-dominant form of PD, which on average began at age 51. By sequencing the exomes of two affected cousins, researchers identified 69 novel coding variants which the cousins had in common. Most of these variants were absent in other affected relatives or did not segregate with the disease, quickly narrowing the list of candidates to two. The scientists then pinned the disease-causing mutation to the Vps35 gene by comparing their results to a case-control series of more than 4,000 patients, in which the Vps35 mutation was seen only in people with PD, never in controls. In addition, the researchers have now found four different mutations in Vps35, all of which cause PD, in different families, Vilarino-Guell said.

Vps35 is part of the retromer complex, which sorts proteins from endosomes back to the trans-Golgi network for recycling. Vps35 has been implicated in Alzheimer’s disease (see ARF related news story and ARF news story). Sorting proteins have also turned up as culprits in frontotemporal dementia (see ARF related news story) and other neurodegenerative disorders (see ARF related news story). Vilarino-Guell suggested that the retromer complex may play an important role in several neurological diseases, but noted that the mechanism by which Vps35 mutations lead to PD still remains to be identified.

More New Risk Genes for Parkinson’s
Although Parkinson’s was once considered a sporadic disease, the last decade has turned up numerous genes involved in familial and early onset PD, and scientists are now beginning to uncover candidate risk genes for the common late-onset cases as well. Singleton described a recently published meta-analysis of five GWAS conducted by the International Parkinson Disease Genomics Consortium over 18 months. The study included more than 5,000 people with PD and over 12,000 controls, and results were replicated in an independent set of more than 7,000 cases (see also ARF related news story). The data added to the evidence for six known risk gene candidates (SNCA; MAPT; LRRK2; HLA-DRA; GAK; BST1), as well as turning up five novel loci (associated with genes ACMSD; STK39; SYT11; MCCC1/LAMP3; and CCDC62/HIP1R). Interestingly, several of these genes, such as SNCA (α-synuclein) and LRRK2 (leucine-rich repeat kinase 2), also have variants that lead to early onset PD. In AD, too, genetics started out with separate genes for early onset (APP, presenilins) and late-onset cases (ApoE), but later presenilin mutations were found to be able to cause both forms of the disease. Gasser, who is also part of the PD consortium, said that the LRRK2 gene in particular has variants along the whole risk continuum from high to low, and that LRRK2 mutations are quite common, with a frequency up to 30 percent in some populations.

As is common in GWAS studies, the risk from each variant was small, but the cumulative effect was significant. The researchers divided the study population into five equal groups, from those who had the least number of risk variants to those who carried the most. Using risk profile analysis and comparing people with PD to controls, the scientists estimated that people in the highest fifth were 2.5 times more likely to have PD than those in the lowest fifth. Gasser estimates the 11 variants account for about 20 percent of the total genetic variance of PD, with familial PD genes contributing another 5 percent of genetic risk. That leaves a big genetic gap still to be filled by exome sequencing, Gasser noted.

A prominent example of a low-frequency, moderate-risk PD gene is the GBA gene encoding the metabolic enzyme glucocerebrosidase. It was recently discovered to have PD-associated mutations, and has already jumped to place 4 on PDGene Top Results. GBA did not show up in the GWAS results. Gasser said it is the type of gene that slips by GWAS because it has numerous individually rare variants. At AD/PD 2011, Núria Setó-Salvia, Hospital Sant Pau, Barcelona, and Jose Luis Capablo Liesa, University of Zaragoza, Spain, presented independent posters on GBA. They confirmed that sequencing of the whole gene, or at least exome sequencing that included exon-intron boundaries, was necessary to reveal known and new mutations in their respective samples of patients with PD and with dementia with Lewy bodies.—Madolyn Bowman Rogers

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Barcelona: Stocking Biomarker Arsenal for Lewy Body Dementia

30 Mar 2011

Dementia with Lewy bodies (DLB) sits at the interface of several disorders. Sharing features with Parkinson’s, PD with dementia (PDD), and Alzheimer’s disease, DLB challenges the diagnostician mightily. The weapon needed to meet this challenge is an arsenal of good biomarkers, scientists agree, but until quite recently, the stockpile was limited. Part of the problem is that DLB was first recognized only about 20 years ago, and so research in this field has lagged behind that for AD and PD. Two years ago, a workshop in Kassel, Germany, held prior to the 9th International Conference on Alzheimer’s and Parkinson’s Diseases, described some of the early efforts to find DLB biomarkers (see ARF related news story). Scientists provided an update on progress in this area at a symposium at the 10th AD/PD Conference, held 9-13 March 2011 in Barcelona, Spain. They focused particularly on markers that will distinguish between DLB and AD. A repeated theme was that no one biomarker will do the job.

“If you rely on a single biomarker, you’re going to have a lot of people who are misclassified,” said James Galvin at New York University Langone Medical Center, New York City. This is because these dementias overlap in both pathology and clinical symptoms. In particular, many people have features of both AD and DLB. “Combining biomarkers allows you to get fairly strong classification of whether people have pure or mixed forms of dementia,” Galvin told ARF. At the symposium, he showed preliminary data demonstrating how this strategy might work to divide people with AD, DLB, and mixed pathology. The ability to define these groups “could have significant therapeutic implications as we develop more specific disease-modifying medicines,” Galvin said.

Speakers covered a range of biomarker types, including brain imaging, body fluids, and cognitive and neuropsychological testing. “Within each biomarker domain, I think we’re identifying what the best possible markers will be,” David Salmon at the University of California in San Diego told ARF. “We have biomarkers now that are more reliable than we had two to four years ago.” In the future, combining these markers synergistically will improve diagnostic confidence, Salmon predicted.

In a conversation with ARF, Galvin noted another advantage of the boom in biomarkers: It will allow scientists to do more hypothesis-driven Lewy body research, similar to how biomarkers are now driving the AD field.

A Spectrum of Dementias
DLB is one of the most common—and some say one of the worst—progressive dementias. It more often afflicts men than women. The disorder is currently diagnosed using a combination of cognitive tests, clinical features, interviews with family members, and positron emission tomography (PET) or single photon emission computed tomography (SPECT) to detect low dopamine transporter levels, said Ian McKeith at Newcastle University, Newcastle upon Tyne, U.K. However, DLB is underdiagnosed. Only about 4 to 5 percent of dementia cases are diagnosed as DLB, McKeith said, whereas 15 percent prove at autopsy to have been DLB. Another way to put this is that about three-quarters of people with DLB initially receive the wrong diagnosis. The disease is particularly tricky to diagnose in the prodromal phase, McKeith said, because early DLB has many non-specific symptoms seen in several neurodegenerative diseases and often shows up at the neurologist’s doorstep with atypical presentations. Better biomarkers are needed to pin down the disease earlier, he said.

DLB is a close cousin of PD and PDD. Its predominant pathology is α-synuclein, which accumulates in Lewy bodies, but amyloid pathology is very common as well. As in PD, dopaminergic neurons in the substantia nigra wither and die. The difference is that DLB brains at autopsy have lost fewer neurons than those with PD or PDD, said Dennis Dickson at the Mayo Clinic in Jacksonville, Florida. Also, in DLB the α-synuclein load in the striatum decreases as the disease progresses, unlike in PD and PDD, where it increases, Dickson said. The main clinical features of DLB include visual hallucinations, movement problems, and fluctuations in alertness, attention, and cognitive function. Sleep disturbances and depression are also common, Dickson said. Many of these features occur in PD as well, although in DLB, motor symptoms are usually milder and hallucinations are more prominent. Like PD, DLB is also marked by loss of the sense of smell, reduced sympathetic innervation of the heart, and low dopamine levels in the brain, Dickson said. The order in which dementia and movement symptoms appear further distinguishes DLB and PDD. When dementia develops several years after Parkinson’s symptoms, it is PDD; when dementia precedes parkinsonism, or arrives within the same year, it is DLB. This is because in Parkinson’s disease, pathology tends to progress from the bottom of the brain up, Dickson said, but in DLB the progression is more top-down, affecting the cortex first.

Distinguishing DLB from AD, especially at early stages, is difficult. People with DLB often suffer from memory problems and cognitive impairments, frequently confusing clinicians. Sometimes the diagnosis depends on whether the patient happens to first see a clinician who specializes in movement disorder or in dementia. McKeith noted that atypical DLB cases, where pathology predominates in the brainstem, pose the greatest puzzle for diagnosis because the clinical presentation is most similar to AD. Amyloid imaging is of little help, as amyloid plaques appear in more than three-quarters of DLB brains, said Galvin. Likewise, about one-third of AD brains contain Lewy bodies, Galvin said, further conflating the two. DLB brains also display neurofibrillary tangles of tau protein, although they are less common than in AD, Dickson said, roughly the equivalent of Braak stage IV. People with DLB lose both dopaminergic neurons as in PD and cholinergic neurons as in AD, Dickson said.

Fluid Biomarkers
What to do when the clinicopathological relationships are such a mess? Scientists are looking at numerous possible biomarkers for distinguishing these disorders. One is cerebrospinal fluid (CSF) α-synuclein, currently the best-validated CSF marker for DLB. Brit Mollenhauer at the Paracelsus-Elena-Klinik, Kassel, Germany, described recently published findings by her team showing that α-synuclein levels are lower in the CSF in DLB and PD patients compared to controls and people with AD (see ARF related news story on Mollenhauer et al., 2011). In a validation cohort of people with parkinsonism, the combination of CSF-tau and α-synuclein measurements allowed researchers to distinguish between synuclein diseases and other disorders with great accuracy, Mollenhauer said. This paper puts to rest prior debate about the strength of CSF findings for α-synuclein.

Other CSF molecules are also being investigated. A poster presented by Malin Wennström and colleagues at Lund University, Malmö, Sweden, reported that people with DLB have lower CSF levels of orexin/hypocretin, a hormone involved in the regulation of hunger and sleep, than healthy people. Orexin-producing neurons in the hypothalamus have been shown to die off in PD, and people with DLB often are excessively sleepy during the day. The authors found that orexin levels correlated with CSF α-synuclein levels, but not with age or cognitive function. CSF orexin has also been studied in other neurologic conditions, including narcolepsy and post-traumatic stress disorder, hence, is most likely not specific to DLB.

Visuospatial Tasks
One promising way to differentiate DLB from AD, said Salmon, is through visuospatial tasks, as impairments in this domain are one of the signature features of DLB. Salmon described a retrospective study that compared neuropsychological test scores of people confirmed at autopsy to have had either DLB or AD. One of the most robust findings was that people with DLB do worse on visuospatial tasks (see Tiraboschi et al., 2006). In a second retrospective study, the researchers found that poor clock-drawing and Block Design performance (both are visuospatial tasks) correlated with faster cognitive decline in DLB patients over a two-year period, but had no predictive value in AD patients (see Hamilton et al., 2008). In addition, more than 60 percent of people with severe visuospatial defects at baseline later suffered from visual hallucinations, compared with about 10 percent of those with mild visuospatial problems, Salmon said.

In light of these findings, Salmon’s team investigated whether they could use a visuospatial task to tell apart people with DLB from those with AD. Visuospatial defects affect the ability to detect both motion and brightness. The scientists used the motion coherence paradigm, in which people look at a screen full of dots in motion. If a large enough fraction of the dots are moving in the same direction, normal people can see the motion. People with AD are as good at detecting the direction of the moving dots as healthy people, but people with DLB just cannot do it, Salmon said. Additionally, normal people are better at this task if the dots moving in the same direction are either more or less bright than the randomly moving dots. This is an example of sensory integration, Salmon said. When the researchers added this luminance clue to the test, both controls and AD patients got better at the task, while people with DLB remained the same. In one small sample, this task provided 100 percent discrimination between people with AD and DLB, Salmon said.

Galvin noted that this test is currently only a research paradigm, but in the future it might be quite useful clinically for differentiating early-stage dementias. Researchers will have to translate the test into a software package that clinicians could use, Galvin said.

Imaging Biomarkers
Imaging markers are another key tool for distinguishing DLB and AD, said John O’Brien at Newcastle University, U.K. So far, the best approach is to use SPECT imaging with the ¹²³I-FP-CIT tracer to measure dopamine transporters in the striatum, O’Brien said. Dopamine levels are normal in people with AD but reduced in DLB. This is the only “category A” biomarker for differentiating these disorders currently recognized by the European Federation of Neurological Societies. It can separate DLB from other dementias with 90 percent specificity and 78 percent sensitivity, O’Brien said (see McKeith et al., 2007). In addition, in diagnostically uncertain cases, an abnormal dopamine scan was highly predictive of DLB (see O’Brien et al., 2009).

Structural MRI also shows promise for distinguishing AD and DLB, O’Brien said. People with AD lose more volume in the hippocampus and medial temporal lobe than do people with either DLB or vascular cognitive impairment. In a study of about 50 people confirmed at autopsy to have had AD, DLB, or vascular cognitive impairment, medial temporal lobe volume diagnosed AD with 90 percent specificity and sensitivity (see Burton et al., 2009). However, another study failed to replicate this finding (see ARF related news story). Galvin pointed out that because people with DLB also have some shrinkage in these brain regions, this measure may be more useful for research than as a diagnostic test.

Another potential approach is to home in on hippocampal subfields using high field strength (3 Tesla) MRI, O’Brien said. Many hospitals now use 3 T magnets, making this feasible for many clinicians. Subiculum and CA1 regions are smaller in AD than in DLB, providing about 80 percent discrimination between the disorders, O’Brien said.

The Combinatorial Approach
The speakers emphasized that no one biomarker will adequately distinguish these dementias, and the best approach will be to use several of them together. This is no surprise because all these tests and markers partly overlap, as do the symptoms and the pathology, after all. Galvin discussed data from several small proof-of-concept studies showing how combos might work. In one study of 45 people, Galvin’s team combined PET imaging with Pittsburgh Compound B, which quantifies amyloid load, along with both cognitive and clinical ratings. For the cognitive assessment, the scientists used composite scores of episodic, semantic, and working memory, as well as visuospatial abilities and global cognitive scores. For the clinical score, Galvin’s team combined four measures—the Unified Parkinson’s Disease Rating Scale, the Mayo Sleep Questionnaire, Mayo Fluctuation Questionnaire, and the Neuropsychiatric Inventory—to create a Lewy Body Risk Score. This biomarker combination divided people into four groups: those predicted to have AD pathology, DLB pathology, mixed pathology, and no pathology. Agreement with the clinical diagnoses was almost 90 percent, Galvin said. All participants are still living, so no pathological confirmations could be made in this study.

In another study of 33 people, Galvin’s team combined two fluid biomarkers, Aβ42 and α-synuclein, and again saw four distinct groups. Four people in this study later died, and autopsy results showed that the biomarkers correctly categorized two people with AD and one with DLB. The biomarkers improved on the clinical diagnosis for one man who was listed as “possible DLB.” CSF results showed normal levels of Aβ and synuclein but elevated tau, and predicted that he had neither AD nor DLB, which turned out to be the case.

Galvin also discussed functional MRI studies that found abnormal connectivity in DLB brains. In a study of 85 people who had DLB, AD, or no dementia, people with DLB had a reversal of the normal connectivity between the default-mode network and other brain regions (Neurology, 2011, in press). The default-mode network reflects the resting activity of the brain and is also perturbed in AD (see ARF related news story). In a separate study, Galvin’s team investigated the basis of the iconic cognitive fluctuations of DLB. “This is an incredibly difficult symptom to get your hands around,” Galvin notes. They picked people who had early dementia with cognitive fluctuations but no other symptoms of DLB and compared the functional connectivity of their brains to that of people who had dementia without fluctuations. The fluctuators had an imbalance between their default and attention networks, Galvin said, suggesting that the “switch” that helps people change from states of daydreaming to attention and back might be faulty in these people (paper in review). “That might lead to people having some odd waxing and waning in their cognitive status,” Galvin speculated to ARF.—Madolyn Bowman Rogers.

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Hamilton JM, Salmon DP, Galasko D, Raman R, Emond J, Hansen LA, Masliah E, Thal LJ. Visuospatial deficits predict rate of cognitive decline in autopsy-verified dementia with Lewy bodies. Neuropsychology. 2008 Nov;22(6):729-37. PubMed.
McKeith I, O'brien J, Walker Z, Tatsch K, Booij J, Darcourt J, Padovani A, Giubbini R, Bonuccelli U, Volterrani D, Holmes C, Kemp P, Tabet N, Meyer I, Reininger C, . Sensitivity and specificity of dopamine transporter imaging with 123I-FP-CIT SPECT in dementia with Lewy bodies: a phase III, multicentre study. Lancet Neurol. 2007 Apr;6(4):305-13. PubMed.
O'Brien JT, McKeith IG, Walker Z, Tatsch K, Booij J, Darcourt J, Marquardt M, Reininger C, . Diagnostic accuracy of 123I-FP-CIT SPECT in possible dementia with Lewy bodies. Br J Psychiatry. 2009 Jan;194(1):34-9. PubMed.
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External Citations

Barcelona: Out of Left Field—Hit to The Eye Kills BACE Inhibitor

31 Mar 2011

Whatever happened to BACE inhibitors? For years after the gene for this APP-cleaving enzyme was cloned (Vassar et al., 1999), excitement was building among scientists who thought they finally had the golden target in hand for rational drug development in Alzheimer’s disease. But the target proved to be surprisingly obstreperous and its luster has dimmed. Clinical trials were a long time in coming, and periodic rumors that company X or company Y did finally have a BACE inhibitor in human tests invariably faded into the silence that means development ended before Phase 2. More recently, the field seems to have developed a second wind, but not without casualties along the way. At the 10th AD/PD International Conference, held 9-13 March 2011 in Barcelona, Martin Citron, told the story of one such setback in some detail (see brief mention in prior ARF related news story). A cautionary tale of the vagaries of drug development, Citron’s talk opened a rare window into the closed world of pharmaceutical company research.

Citron has worked on inhibiting BACE, first at Amgen, now at Eli Lilly and Co., Indianapolis, ever since his lead in cloning the gene. When the protein’s crystal structure came into view (Hong et al., 2000) and the first knockout mice proved viable (Luo et al., 2001; Cai et al., 2001), a field of contenders trying to block the enzyme was off to the races. Pharma companies had been trying to inhibit β-secretase even before 1999, without success, but these papers created more heated momentum behind BACE.

That was a full decade ago. Why so slow? Initially, the reasons were technical. FRET-based assays generated false positives, high-throughput assays failed, and a “myopic focus” on high-potency compounds led scientists astray as they pursued candidates that penetrated the brain poorly or did not clear properly. “I learned that inhibitor development is much more difficult than people think,” Citron said.

He was not alone. Many groups were spinning their wheels, and questions arose about BACE as a drug target in private conversations and in the literature. “Now there is a cloud over the target. I want to show you today that BACE1 is indeed druggable,” Citron said.

How so? The Lilly scientists, including Patrick May, Robert Dean, Stephen Lowe, and others, did eventually find a BACE inhibitor that entered the brain when taken by mouth. It robustly reduced Aβ in humans. They had started fresh, screening non-peptidic small molecules with an eye toward favorable drug properties rather than potency. Then they improved potency with medicinal chemistry. They ended up with a molecule that sits in the S3 pocket of BACE1’s active site and reacts with two catalytic sub-sites of the enzyme. It had a fair potency of 0.25 micromolar in five different cell lines. Called LY 2811376, the molecule behaved itself in vivo. It reduced brain Aβ in PDAPP transgenic mice in dose-dependent fashion. It changed both upstream and downstream CSF and plasma biomarkers in the expected ways in mice and dogs. “With this and a clinical toxicity data package, we were ready to go into the clinic,” Citron said.

There, too, things went passably for a while. A single ascending dose study in healthy volunteers yielded the pharmacokinetic and pharmacodynamic data the scientists wanted to see. Maximal plasma concentration, half-life, time course of Aβ reduction, and dose dependence—all that stuff looked good. Likewise, a 36-hour spinal catheterization study in healthy volunteers showed a decrease in CSF Aβ40 and 42, again with dose dependence, time course, and biomarkers behaving as desired. Adverse events up to this point included headache, palpitations, colds—nothing the data safety monitoring board found concerning.

“We were riding high on the biomarker data,” Citron said.

While they were preparing for Phase 2, the downfall came. Rat toxicology studies showed that a higher dose given for three months ravaged the pigment epithelium of the rat’s eye. This retinal layer had inclusions and extensive damage. Lilly ended dosing and brought people in for eye assessments, which thankfully showed no abnormalities, Citron said.

What happened? The scientists still don’t know what the compound does to the eye. BACE knockout mice do not show this phenotype, but they do develop it when treated with LY 2811376, suggesting to Citron that this is an idiosyncratic effect of this particular compound, not of BACE inhibition. This would imply that the compound is dead for AD (and any other indication, for that matter), but BACE as a target still stands. This toxic effect does not show up with other compounds of this chemical series, either, Citron said.

Asked later what it’s like to get this far only to be bounced by an off-target effect, Citron said dryly: “Welcome to drug development. This happens all the time. It just never gets published.”

So what’s to be learned? LY 2811376 is the first BACE inhibitor with profound CNS effects, and constitutes proof of concept that BACE is druggable, said Citron. He noted that Lilly has another (retina-tested) compound in the clinic, as do CoMentis (see ARF Keystone story), Eisei, Merck/Schering, and TransTech Pharma. At AD/PD 2011, AstraZeneca scientists presented preclinical and biomarker data on a new small-molecule inhibitor on three posters.

Other scientists noted, though, that it remains difficult to find BACE inhibitors that achieve effective exposure levels in the brain without also creating toxic exposure levels in the periphery. Others suggested that, rather than continuing to butt its head against the wall of BACE inhibition, scientists might be well advised to explore BACE modulation. The γ-secretase field appears to be migrating away from inhibition and toward modulation. Likewise, regulation of BACE by modulation, rather than inhibition, could be amenable to manipulation, commented Barbara Tate of Satori, Inc., in Cambridge, Massachusetts. Indeed, the U.S. Patent Office in the past year issued half a dozen patents on compounds claimed to modulate BACE, indicating that some companies are already pursuing this softer route (see, e.g., Albrecht et al., 2011; Malamas et al., 2010).

In addition, little flags about potential safety liabilities with BACE are fluttering on the basic science front. This aspartyl protease cleaves a number of substrates besides APP. Also in Barcelona, Michael Willem presented new data on neuregulin, a physiological substrate of BACE1. Working with Christian Haass at Ludwig-Maximilians-Universität, Munich, Germany, Willem had originally discovered that BACE1 cleaves neuregulin (Willem et al., 2006).

Willem showed a slide of some 16 BACE inhibitor compounds by many companies that have since been abandoned. These make handy tools for his work exploring BACE1 and its targets, both in cell culture and in mice. All BACE inhibitors Willem has used so far, including LY 2811376, slow the turnover of neuregulin-1-b1. Both the peripheral and central nervous system of young and adult mice express this isoform, Willem told the audience. The inhibition causes the unprocessed substrate to accumulate; this, in turn, could slow downstream signaling by neuregulin cleavage products. The biological activity of neuregulin is far from fully understood, but neuregulin-1 is thought to play a role in synaptic transmission and the maintenance of synapses.

“Neuregulin cleavage is dependent on BACE1. There is no redundancy for that in mice. I still do not know what neuregulin really does in the brain. But if you block BACE1, you will block neuregulin, too,” Willem said.—Gabrielle Strobel.

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References

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Paper Citations

Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J, Edenson S, Lile J, Jarosinski MA, Biere AL, Curran E, Burgess T, Louis JC, Collins F, Treanor J, Rogers G, Citron M. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 1999 Oct 22;286(5440):735-41. PubMed.
Hong L, Koelsch G, Lin X, Wu S, Terzyan S, Ghosh AK, Zhang XC, Tang J. Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor. Science. 2000 Oct 6;290(5489):150-3. PubMed.
Luo Y, Bolon B, Kahn S, Bennett BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, Martin L, Louis JC, Yan Q, Richards WG, Citron M, Vassar R. Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci. 2001 Mar;4(3):231-2. PubMed.
Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, Wong PC. BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci. 2001 Mar;4(3):233-4. PubMed.
Willem M, Garratt AN, Novak B, Citron M, Kaufmann S, Rittger A, Destrooper B, Saftig P, Birchmeier C, Haass C. Control of peripheral nerve myelination by the beta-secretase BACE1. Science. 2006 Oct 27;314(5799):664-6. PubMed.

External Citations

Barcelona: Live and Learn—γ-Secretase Inhibitors Fade, Modulators Rise

01 Apr 2011

Is it better to handcuff the bad guy or to reform his ways? The Alzheimer’s research version of this question—whether to inhibit γ-secretase or shift its M.O.—generated a lot of buzz at the 10th International Conference on Alzheimer’s and Parkinson’s Diseases, held 9-13 March in Barcelona, Spain. The question has been around for some time, but AD/PD 2011 galvanized debate about what scientists were to make of the latest thunder clouds gathering on the γ-secretase inhibitor front.

In a nutshell, scientists appear to be losing faith in the concept of Notch-sparing γ-secretase inhibition. That notion had appeared on the scene as the potential savior when Notch first proved to be a particularly troublesome one among the secretase’s many substrates. The enzyme complex is involved in the maturation of some 50 proteins. In his talk, Bart De Strooper of the Flanders Interuniversity Institute of Biotechnology in Leuven, Belgium, made quite clear his view that γ-secretase inhibition may not succeed because the safety margin between lowering Aβ42 and affecting Notch is too small. In his talk, Edward Koo of the University of California, San Diego, essentially concurred. Even some company scientists will say as much. “γ-secretase inhibition is hitting some pretty hard skids,” Dale Schenk of Elan Pharmaceuticals in South San Francisco told this reporter. Instead, scientists said, tweaking the enzyme complex without affecting its total output of Aβ—or that of APP’s C-terminal fragment, for that matter—might be more felicitous. Such compounds would be the γ-secretase modulators, or GSMs for short.

This concept is not exactly new. Koo cautioned that its potential remains theoretical because GSMs have not worked yet or even shown target engagement in humans. Flurizan failed because it may not have engaged its target sufficiently in the brain (see ARF related news story). Even so, excitement about GSMs was palpable at AD/PD 2011. Partly, that’s because scientists are gaining a clearer idea that these might be allosteric modulators and hence fall into an established mechanistic area of enzymology that is amenable to standard medicinal chemistry and drug development. In contrast, exactly how a Notch-sparing inhibitor would preclude processing of one substrate but not another has always remained nebulous. And partly, scientists now have second-generation GSM compounds that penetrate the blood-brain barrier better, and are currently wending their way toward the clinic and through Phase 1 and 2 trials.

What’s Eating γ Inhibitors?
Worry about γ-secretase inhibition could be heard for many years, but the loss of confidence accelerated with the humbling fate of Eli Lilly’s Phase 3 trial of semagacestat. It nosedived last August when the safety monitoring board unblinded the data midway through to discover that people on drug were worsening cognitively and functionally while coming down with unacceptable side effects (see ARF related news story). That trial is still blinded, Lilly’s Patrick May said at AD/PD 2011. Dosing is over, but clinical observation and biomarker measurements are continuing, and only when the data come to analysis this June will the scientists have a chance to figure out what happened.

Reviewing the status of γ-secretase inhibition in a symposium talk, Koo noted the uncertain path ahead. In late September 2010, Script Pipeline Watch reported that Pfizer discontinued development of its γ-secretase inhibitor begacestat after several Phase 1 trials (see ARF related news story). The reasons are unclear. Inherited from Wyeth after the Pfizer merger with Wyeth, this drug had been presented as having a larger therapeutic index than semagacestat, that is, as inhibiting the enzyme’s ability to process APP at much lower doses than are needed to block cleavage of Notch. This difference, the Wyeth/Pfizer scientists had thought, afforded enough space to make γ-secretase inhibition with this compound feasible.

Furthermore, Koo noted that a third compound, which is said to have an even larger window between APP and Notch, may be encountering similar problems. This is the BMS-708163 γ-secretase inhibitor. Bristol-Myers Squibb has completed a Phase 2 trial with this compound in mild to moderate AD, and is currently conducting a Phase 2 trial in prodromal AD. This trial is being closely watched for its novelty. It enriches for patients on the basis of their CSF Aβ42 levels and an objective memory impairment, making it a test case for earlier-stage treatment than the standard mild to moderate trial. According to ClinicalTrials.gov, this trial last October dropped the higher 125 mg dose down to 50 mg. According to a February 17, 2011 article by BioPharm Insight, an industry intelligence company based in Norwood, Massachusetts, dose-related toxicity in the previous mild to moderate trial included skin rash, gastrointestinal symptoms, and a “suggestion of rate of decline not favoring treatment.”

This profile is eerily reminiscent of semagacestat, an earlier-generation γ-secretase inhibitor that does not discriminate well between APP and Notch. BMS-708163 is thought to do so with a much larger margin, and in Barcelona, BMS scientists led by Jere Meredith presented in-vitro data for a selectivity between these two proteins of 193-fold versus 13-fold for semagacestat. Yet when asked about this, scientists at a handful of other biopharma companies and academic labs noted that they had synthesized the BMS compound and discovered that, at least in their hands, it distinguished less strongly between APP and Notch.

While this is true in her lab as well, the field should not put too much weight on in-vitro assays of therapeutic indices of these compounds, commented Barbara Tate of Satori Pharmaceuticals, a Cambridge, Massachusetts-based biotech company. The real answer will come in the clinic as investigators gain experience with drug exposure and deal with the pharmacokinetic and pharmacodynamic balancing act of systemic versus central activity. Human pharmacokinetics could influence dose selection. For instance, the 125 mg dose could have proved too high because the drug accumulates in humans when taken chronically, said Tate, boosting over time the body’s de-facto exposure to the drug. Lowering the dose might solve the problem. Finding the highest tolerable dose, after all, is part of the purpose of Phase 2 trials. Other scientists questioned whether the remaining 50 mg dose in the prodromal BMS trial lowers Aβ enough. Again, if the drug accumulates, both its toxicity and its Aβ-lowering effect would change with its particular human pharmacodynamics and, in theory, both can be brought into range by adjusting the dose, Tate said.

Is Notch the Only Problem?
Recent basic science on the role of Notch signaling in the adult brain has only heightened researchers’ collective awareness that it is important to stay away from this substrate (for a recent review, see Pierfelice et al., 2011). But it’s not just Notch. A poster presented at AD/PD 2011 by Yasuyuki Mitani and colleagues at Astellas Pharma in Tsukuba, Japan, pointed an accusing finger to an intermediate product of APP cleavage that had been the center of debate a decade ago but then faded from view. This is the γ-secretase substrate β-CTF, otherwise known as C99 or C100.

The Japanese group directly compared semagacestat (aka LY 450139), BMS-708163, and GSM-1, a modulator originally made by Mark Shearman and colleagues at Merck, which is not being clinically developed. GSM-1 does not affect Notch cleavage. They fed the three compounds to wild-type and to Tg2576 APP transgenic mice for one day to model acute dosing, and for eight days to model sub-chronic dosing. They measured what each drug did to the mice’s working memory in Y maze tests, and to APP cleavage biochemically. The scientists found that at one day, all three compounds reversed the memory deficits of the Tg2576 mice. By eight days of dosing, however, neither of the two inhibitors helped any longer, whereas the modulator still did. In the wild-type mice, sub-chronic dosing with both inhibitors—but not the modulator—actually impaired working memory. The Lilly and BMS compounds behaved about the same in this experiment. All three compounds lowered Aβ42 in the mice’s hippocampus by a similar amount. Importantly, in the eyes of the Japanese authors, however, only the inhibitors led to the expected dose-dependent rise of the γ-secretase substrate β-CTF. In contrast, with GSM-1, Aβ38 shot up as Aβ42 came down, but β-CTF remained absent.

These scientists concluded that when taken sub-chronically, both γ-secretase inhibitors (GSIs) but not the GSM, worsened cognition. The GSIs affect Notch with different therapeutic indices; both increase β-CTF. Partly for this reason, the Japanese group suggested that the cognitive impairment could be due to the β-CTF. Citing a German study in which Jochen Herms and colleagues had shown that LY 450139 decreases spine density in wild-type but not APP knockout mice (Bittner et al., 2009), the Japanese scientists implied that β-CTF elevation might have contributed to the worsening cognition seen in Lilly’s Phase 3 trial of this discontinued compound.

Debating this study, De Strooper pointed out a limitation. The poster only showed data on Aβ42, 40, and 38, not on other Aβ isoforms such as the shorter Aβ1-16, which has repeatedly been shown to increase in response to γ-secretase inhibition (see Portelius et al., 2010; Mustafiz et al., 2011; Beher et al., 2002) as well. Shearman mentioned a study in which three to seven days of treatment with MRK-560, a GSI originally developed in Shearman’s group at Merck, reversed an LTP deficit in Tg2576 mice (Townsend et al., 2010). Shearman is now at EMD Serono in Cambridge, Massachusetts. Schenk of Elan noted that the β-CTF could be at fault, as could a variety of other factors including other substrates γ-secretase is known to process. Lilly’s May agreed, saying of the Astellas poster, “This is interesting; we certainly see the increase in β-CTF, too.”

β-CTF is the product of BACE cleavage of APP. If this fragment were to turn out to be at fault, some scientists who toiled in the field a decade ago might well say, “Told you!” As early as 1997 and for some years thereafter, Rachael Neve, then at McLean Hospital in Belmont, Massachusetts, published a series of papers claiming that APP’s C-terminal fragment is toxic to neurons (e.g., Neve et al., 1992; Kammesheidt et al., 1992). Neve now studies mood disorders at MIT’s Picower Institute in Cambridge, Massachusetts.

Some scientists suggested that targeting inhibitors to presenilin-1 might be a way to make GSIs work. For example, Santiago Parpal Tamburini and colleagues at AstraZeneca presented on the GSI MRK-560. According to their poster, this inhibitor inhibits presenilin-1 much more potently than its cousin, presenilin-2. The compound caused severe side effects in PS2 knockout mice, but not in wild-type mice, the idea being that the latter deploy PS2 to provide whatever Notch and other substrate cleavage is necessary to stay healthy in the presence of a PS1-specific γ-secretase inhibitor. Other scientists were skeptical of this approach pending further data, and instead gave a nod toward γ-secretase modulation. For AD/PD 2011 reporting on GSMs, see Part 2 of this series.—Gabrielle Strobel.

This is Part 1 of a two-part series. See also Part 2.

References

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Pierfelice T, Alberi L, Gaiano N. Notch in the vertebrate nervous system: an old dog with new tricks. Neuron. 2011 Mar 10;69(5):840-55. PubMed.
Bittner T, Fuhrmann M, Burgold S, Jung CK, Volbracht C, Steiner H, Mitteregger G, Kretzschmar HA, Haass C, Herms J. Gamma-secretase inhibition reduces spine density in vivo via an amyloid precursor protein-dependent pathway. J Neurosci. 2009 Aug 19;29(33):10405-9. PubMed.
Portelius E, Van Broeck B, Andreasson U, Gustavsson MK, Mercken M, Zetterberg H, Borghys H, Blennow K. Acute effect on the Aβ isoform pattern in CSF in response to γ-secretase modulator and inhibitor treatment in dogs. J Alzheimers Dis. 2010;21(3):1005-12. PubMed.
Mustafiz T, Portelius E, Gustavsson MK, Hölttä M, Zetterberg H, Blennow K, Nordberg A, Lithner CU. Characterization of the brain β-amyloid isoform pattern at different ages of Tg2576 mice. Neurodegener Dis. 2011;8(5):352-63. PubMed.
Beher D, Wrigley JD, Owens AP, Shearman MS. Generation of C-terminally truncated amyloid-beta peptides is dependent on gamma-secretase activity. J Neurochem. 2002 Aug;82(3):563-75. PubMed.
Townsend M, Qu Y, Gray A, Wu Z, Seto T, Hutton M, Shearman MS, Middleton RE. Oral treatment with a gamma-secretase inhibitor improves long-term potentiation in a mouse model of Alzheimer's disease. J Pharmacol Exp Ther. 2010 Apr;333(1):110-9. PubMed.
Neve RL, Kammesheidt A, Hohmann CF. Brain transplants of cells expressing the carboxyl-terminal fragment of the Alzheimer amyloid protein precursor cause specific neuropathology in vivo. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3448-52. PubMed.
Kammesheidt A, Boyce FM, Spanoyannis AF, Cummings BJ, Ortegón M, Cotman C, Vaught JL, Neve RL. Deposition of beta/A4 immunoreactivity and neuronal pathology in transgenic mice expressing the carboxyl-terminal fragment of the Alzheimer amyloid precursor in the brain. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10857-61. PubMed.

External Citations

Barcelona: Allosteric γ Modulation Moves Toward Clinic

04 Apr 2011

As the research field’s collective eyes are shifting toward the γ-secretase modulators, what do scientists actually have on that score? At AD/PD 2011, several academic and biopharma groups presented on the topic ranging from fundamental mechanisms to late preclinical data. On the basic science front, researchers have made progress in understanding how γ-secretase modulators might work. For example, Taisuke Tomita and Takeshi Iwatsubo of the University of Tokyo made light-activatable probes with the research compound GSM-1. Using those, and also other methods, the Japanese scientists showed that GSM-1 binds to the N-terminal end of presenilin-1, in particular at the hydrophobic region of transmembrane domain 1. This part of the protein is at a distance from the hydrophilic pore deep in the membrane that harbors the active site of the enzyme complex, indicating that the mode by which GSM-1 tweaks APP cleavage is allosteric. A poster by Hiroyuki Amino and colleagues at the pharma company Eisai showed essentially the same thing for that company’s GSM E2012 (see more on this compound below), and scientists in other labs have independently seen GSM binding to presenilin-1’s N-terminal fragment. Allosteric modulation frequently works through conformational changes at the substrate binding site or the catalytic pocket of an enzyme, and can be quite tractable for drug development (see also Uemura et al., 2009).

On the translational science front, Sascha Weggen of Heinrich Heine University, Düsseldorf, Germany, updated his prior work showing that amino acid sequence matters. Previously, Weggen’s lab had shown that some AD-causing presenilin mutations respond poorly to GSMs (Czirr et al., 2007). Since then, the group has studied this hunch systematically. At AD/PD 2011, Weggen reported that, indeed, GSMs look feeble when tested against almost all familial PS-1 mutations the field has used over the years to model AD in transgenic mice and cell culture. In contrast, the same compounds work fine in models using pathogenic APP mutations. This does not mean GSMs would be ineffective in most patients; after all, the vast majority of AD patients have no presenilin mutation, but it does mean that scientists developing GSMs must choose their models carefully when they screen for such compounds and evaluate them preclinically (see also Hahn et al., 2011). Weggen noted that, in his experiments, the resistance of one presenilin mutation was broken by two recently published GSMs, but not by other second-generation compounds he tested, such as E2012 and BB25, an analog of GSM-1. Harald Steiner of Ludwig-Maximilians-Universität in Munich, Germany, reinforced this point in his talk. He showed that potent compounds such as E2012 and GSM-1 do reduce Aβ42 generation by many mutant forms of presenilin-1, but his group used higher concentrations than Weggen’s. This data appeared online last month (Kretner et al., 2011).

On the preclinical side, Kathy Rogers of EnVivo Pharmaceuticals, a biotech company in Watertown, Massachusetts, told the audience that its GSM, called EVP-0015962, decreased Aβ42 while concomitantly increasing Aβ38, but not the APP-CTFs.

The γ-secretase complex generates Aβ peptides of varying length, and the longer ones aggregate, particularly Aβ42. Modulating the enzyme means that it keeps chopping away, but processes APP slightly differently, such that it produces less Aβ42 and more Aβ38, or shorter isoforms but leaves the total amount of Aβ and its cytoplasmic tail AICD unchanged. (Whether the short forms are harmless has not been formally proven, but taking out Aβ42 is widely thought to stop the formation of oligomers and fibrils.)

Rogers showed data to suggest that EVP-0015962 behaves in the desired way, i.e., down with Aβ42, up with Aβ38, no effect on total load, in four different cell types. It leaves AICD generation unchanged, and neither α- nor β-CTF pile up. “We shift the site of cleavage, not the rate of cleavage,” Rogers said. The compound does not affect Notch cleavage. When added to food once, it reduced Aβ42 in the brains of wild-type and Tg2576 mice. Fed to Tg2576 mutant APP-transgenic mice for a year, neither APP’s α nor β-CTF went up. With this chronic exposure, the compound reduced neuroinflammation measured as cortical astrocyte and microglial activation, as well as plaque load in the hippocampus, Rogers showed. A stepwise biochemical extraction protocol showed that the compound reduced Aβ42 in all three pools analyzed: cytosolic, membrane-bound, and aggregated. It also reversed the mice’s memory deficit in the contextual fear-conditioning test. All effects were dose-dependent.

Incidentally, this compound has no effect in wild-type mice; hence, it is not a cognitive enhancer, Rogers said. The compound was well tolerated in mice at the doses tested, Rogers said. How about the all-important pharmacokinetics and pharmacodynamics? Rogers showed a small number of data in rats, where the compound lowers Aβ42 while upping Aβ38 in the CSF, and clears out over the course of some six hours. After the talk, EnVivo scientists said they have calculated a minimally efficacious dose and are currently studying the compound in non-human primates.

Responding to an audience question about whether this compound is an NSAID—those are the anti-inflammatory drugs that originally led scientists on the trail of GSMs years ago—Rogers said the company’s chemists started out with an NSAID and then modified it to where it now has no Cox1 or 2 activity. Another question from the audience touched on intestinal goblet cells, asking whether their numbers were up with EVP-0015962. Nope, said Rogers, they were not.

This question pertains to the intestinal toxicity that arises when γ-secretase inhibitors (GSIs) inhibit Notch. It came up again on a poster by scientists at the pharmaceutical company Eisai Company, a pharmaceutical company that developed donepezil. In Barcelona, Mai Uesugi and colleagues showed how they had compared gene expression in rats treated with either their GSM E2012 or a GSI, looking specifically for changes that would indicate interference with Notch signaling. In theory, a GSM might shift cleavage of Notch as well, and in this way perhaps deprive the cell of certain Notch fragments that are needed for subsequent signaling. On the poster, the scientists showed that the GSI, but not the GSM, reduced expression of target genes downstream of Notch signaling in rat intestine. The GSI, but not GSM, led to increased numbers of goblet cells in the gut as well.

Led by Christa Nagy, Eisai also presented a poster with some single-dose human pk/pd data on 10 different doses ranging from 1 to 400 mg of E2012. The poster suggested that E2012 reduces mostly Aβ42, but also Aβ40 in plasma of healthy volunteers. Eisai had previously tested this drug in Phase 1 but stopped when a high dose group appeared to develop a problem with the lenses of their eyes. Apparently, the FDA permitted resumption of clinical testing for E2012 in 2008, according to an article in FierceBiotech, but no trials with this compound are listed on ClinicalTrials.gov or the IHO clinical trials listing.

At least one other GSM is currently in trials, however. That is Chiesi’s CHF5074 (Lanzillota et al., 2010; Imbimbo et al., 2009). That compound is getting ready for a Phase 2 dose-finding trial in people with MCI to start this month in Italy and New Jersey, U.S. Chiesi, an Italian company, did not present at AD/PD 2011, nor did Satori Pharmaceuticals, a U.S. biotech company that has a preclinical second-generation GSM. Other companies did, though, for example, the Swiss pharma giant Hoffmann-La Roche, which showed data on aminothiazole GSMs.

While everyone is looking for the right GSM, plenty of questions remain. One is when a person would have to start taking it. For example, the Tg2576 mice in the EnVivo study started taking it at five months of age, when Aβ levels are high but before the mice have deposited amyloid. How much would such a drug help in people, who tend to have abundant amyloid deposition in their brains years before they develop symptoms of AD? At AD/PD 2011, Eddie Koo of the University of California, San Diego, spoke for many scientists when he emphasized the need to treat early and to test drugs early. Because scientists think GSMs are safer than GSIs, they hope to use them toward that end. But will a GSM be enough? Joanna Jankowsky at Baylor College of Medicine, Texas, reported long-term findings of her studies treating transgenic mice with GSIs. She cautioned that shutting off Aβ production might not suffice once the brain is riddled with plaques. At that point, a GSM plus immunotherapy together might be required. This kind of treatment is straightforward to model in mice, but not practical in humans yet. In humans, clinical trials are beginning to inch their way back from mild to moderate AD toward prodromal AD, a stage where amyloid pathology is already present. Neither prevention nor combination therapy with GSMs and immunotherapy is a reality yet, but efforts such as the Dominantly Inherited Alzheimer Network (DIAN), API, and ADCS A4 are actively working toward at least secondary prevention trials, where amyloid is present but symptoms are not (for extensive coverage of these efforts, see ARF London conference series; ARF DC series; ARF Webinar).

Another potential hurdle is looming on the horizon. There were whispers at AD/PD and previous conferences that vasogenic edema—a mysterious side effect that first cropped up in Phase 1 trials of passive immunotherapy—might be something all anti-amyloid therapies may have to contend with. Immunotherapy trials already are under orders from the FDA to closely monitor patients for these edemas, and this practice is gradually creating more data about them. One early idea had been that rapid transport of large amounts of amyloid from the parenchyma to the brain’s blood vessels might cause transient fluid retention as capillaries become temporarily more permeable to serum proteins, and that this would subsequently resolve as the brain gradually clears the amyloid. Since last winter, however, some scientists have speculated privately that even γ-secretase inhibitors have caused these fluid shifts in recent trials. In Barcelona, Reisa Sperling of Brigham and Women’s Hospital, Boston, said nothing in her talk about specific trials and declined comment afterward. But she did say that vasogenic edema could potentially be a general complication of all Aβ-lowering strategies. Interestingly, the Chiesi trial of its GSM makes MRI including the FLAIR sequences that visualize vasogenic edemas an inclusion criterion.

Sperling showed data on two women who developed vasogenic edema in immunotherapy trials at her site. One had no symptoms and the edema was gone after three months. The other woman was confused and her MMSE dropped; subsequent imaging procedures showed that she had a microhemorrhage, which resolved on its own. The cases beyond those two examples range from clinically invisible to locally inflammatory and requiring steroids, Sperling said. Vasogenic edema typically shows up after the first or second infusion, and in most cases, patients resume dosing after a while. Thus far, vasogenic edema has not changed patients’ outcomes, she added.

“I believe vasogenic edema has to do with early, rapid shifts in Aβ load,” Sperling said. One possible explanation might be that rapid movement of Aβ plugs up drainage through the perivascular space, though there is also some data for clearance happening right at the vessel and for toxicity to the vessel. “There is an important balance between production and clearance of Aβ. Vasogenic edema is not specific to immunotherapy, but arises when this balance changes at the vessel temporarily,” Sperling said.

ApoE4 genotype and CAA are risk factors; in fact, some people with CAA have those edemas spontaneously even in the absence of dementia or AD. Vascular amyloid somehow is a common underlying pathophysiology, but the exact relationship among vasogenic edema, Aβ, and microbleeds remains elusive at this point (for more on microbleeds, see ARF 2011 HAI conference story).

“Ideally we need to treat 10 years before people get symptoms. We can see that vessel amyloid is associated with vasogenic edema or microbleeds in models even before they are symptomatic. That means we will have to be careful with vasogenic edema even in preclinical trials in the future,” Sperling concluded.—Gabrielle Strobel.

This is Part 2 of a two-part series. See also Part 1.

References

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Uemura K, Lill CM, Li X, Peters JA, Ivanov A, Fan Z, Destrooper B, Bacskai BJ, Hyman BT, Berezovska O. Allosteric modulation of PS1/gamma-secretase conformation correlates with amyloid beta(42/40) ratio. PLoS One. 2009;4(11):e7893. PubMed.
Czirr E, Leuchtenberger S, Dorner-Ciossek C, Schneider A, Jucker M, Koo EH, Pietrzik CU, Baumann K, Weggen S. Insensitivity to Abeta42-lowering nonsteroidal anti-inflammatory drugs and gamma-secretase inhibitors is common among aggressive presenilin-1 mutations. J Biol Chem. 2007 Aug 24;282(34):24504-13. PubMed.
Hahn S, Brüning T, Ness J, Czirr E, Baches S, Gijsen H, Korth C, Pietrzik CU, Bulic B, Weggen S. Presenilin-1 but not amyloid precursor protein mutations present in mouse models of Alzheimer's disease attenuate the response of cultured cells to γ-secretase modulators regardless of their potency and structure. J Neurochem. 2011 Feb;116(3):385-95. PubMed.
Kretner B, Fukumori A, Gutsmiedl A, Page RM, Luebbers T, Galley G, Baumann K, Haass C, Steiner H. Attenuated Abeta42 responses to low potency gamma-secretase modulators can be overcome for many pathogenic presenilin mutants by second-generation compounds. J Biol Chem. 2011 Apr 29;286(17):15240-51. PubMed.
Lanzillotta A, Sarnico I, Benarese M, Branca C, Baiguera C, Hutter-Paier B, Windisch M, Spano P, Imbimbo BP, Pizzi M. The γ-secretase modulator CHF5074 reduces the accumulation of native hyperphosphorylated tau in a transgenic mouse model of Alzheimer's disease. J Mol Neurosci. 2011 Sep;45(1):22-31. PubMed.
Imbimbo BP, Hutter-Paier B, Villetti G, Facchinetti F, Cenacchi V, Volta R, Lanzillotta A, Pizzi M, Windisch M. CHF5074, a novel gamma-secretase modulator, attenuates brain beta-amyloid pathology and learning deficit in a mouse model of Alzheimer's disease. Br J Pharmacol. 2009 Mar;156(6):982-93. PubMed.
Reiman EM, Langbaum JB, Tariot PN. Alzheimer's prevention initiative: a proposal to evaluate presymptomatic treatments as quickly as possible. Biomark Med. 2010 Feb;4(1):3-14. PubMed.

External Citations

Barcelona: Is Parkinson’s Pathology Treatable?

06 Apr 2011

Short answer: Not yet. But scientists are hammering away at the problem, and this series features progress reports on some experimental PD therapies. For Parkinson’s disease, unlike Alzheimer’s, physicians have numerous effective treatments to reduce symptoms and improve their patients’ quality of life. Yet none of these treatments halt the underlying neurodegeneration or cure the disease. Almost 200 years after PD was first described, disease-modifying treatments remain an elusive goal. Indeed, the majority of PD treatments in the development pipeline are symptomatic, said Cristina Sampaio of the European Medicines Agency, speaking at the 10th International Conference on Alzheimer’s and Parkinson’s Diseases, held 9-13 March 2011 in Barcelona, Spain (see ARF related news story). The approaches that have been tried, such as cell replacement and gene therapy, have yielded, at best, mixed results so far.

Why has PD pathology been such a difficult target? One barrier has been the lack of good biomarkers to prove that a therapy alters pathology, Sampaio said, a point echoed by other scientists. Another issue is that, as in AD, available animal models for PD do not model the full complexity of the disease, which may cause problems for translating therapies from the bench to the clinic. Manuel Buttini at Elan Pharmaceuticals, South San Francisco, California, pointed out that the classic PD animal models, which are produced by lesioning the striatum with toxins, are particularly limited in the disease features they model. Newer transgenic mouse models, such as Richfield mice, Line 61, and other strains, express familial PD genes in the substantia nigra and are more versatile, Buttini said. Their brain pathology more closely resembles that of PD patients, and they reveal subtle motor and cognitive defects similar to those in people. Localized, moderate α-synuclein overexpression most closely mirrors human PD, Buttini said, while higher levels of expression produce greater neurodegeneration. He noted that there is still no ideal model, and the best choice of animal depends on the particular research questions being asked.

Despite these obstacles, numerous academic scientists and pharmaceutical companies continue to plug away at the problem of disease-modifying treatments, and AD/PD 2011 featured a number of progress reports. Scientists provided more data on why cell replacement has failed to cure the disease, held out hope for gene therapy, and discussed early, preclinical efforts to directly attack the underlying pathology by blocking α-synuclein deposits or transmission.

Fetal Grafts: A Checkered History
The motor symptoms of PD begin after more than half of the dopamine-producing neurons in the substantia nigra have died. Replacing these lost cells, therefore, was one of the earliest disease-modifying treatments tried. Many of the approximately 350 patients who received grafts of fetal dopaminergic neurons initially did well, moving more easily and with better control. One- to two-year-old transplants examined at autopsy looked “fabulous,” said Jeffrey Kordower at Rush University Medical Center, Chicago, Illinois. Kordower is also a founder of Ceregene, Inc., a biotech company in San Diego, California, that is developing gene therapy for AD and PD. Kordower said the transplanted neurons not only survived, but also innervated the host tissue and robustly expressed tyrosine hydroxylase, the enzyme that produces the precursor to dopamine.

Over time, however, problems cropped up. In some groups, about half the people who received grafts developed dyskinesias, or involuntary movements. The reason for this is controversial, with some researchers suggesting it is because the grafts produce too much dopamine. Other scientists say the quality of the grafted tissue is a major factor. Some studies indicate that solid-tissue grafts trigger more inflammation, leading to dyskinesias (see Kirik and Björklund, 2005), or that contaminating serotonergic neurons cause the uncontrolled movements (see ARF related news story). Additionally, some PD symptoms, such as gait problems and falls, do not respond to dopamine. These problems worsened several years after surgery, Kordower told ARF.

Perhaps most disturbingly, the young grafts succumbed to PD pathology within 10 years after transplant, developing Lewy bodies and Lewy neurites (see ARF related news story). First reported in 2008, Kordower said this has now been shown to be a widespread phenomenon. Clinicians including Patrik Brundin at Lund University, Sweden; William Langston at The Parkinson’s Institute, Sunnyvale, California; and Curt Freed at the University of Colorado, Aurora, have all reported the same thing, Kordower said (see, e.g., Li et al., 2010 and Brundin et al., 2010). About 6 to 8 percent of the grafted neurons develop Lewy bodies, comparable to the percentage in host tissue. Levels of dopamine transporter and tyrosine hydroxylase also drop off. This pathology is specific to the PD process and not just a general inflammatory response, Kordower said, because fetal grafts in Huntington’s patients show inflammation and degeneration, but no Lewy bodies. “Whatever is causing Parkinson’s disease is still there,” Kordower concluded. Cell replacement does not change the underlying disease. Experiments in mice have now shown that virally overexpressed α-synuclein in the striatum moves into grafted neurons, implying that the same mechanism could be at work in people (see Hansen et al., 2011).

What’s Next for Cell Replacement?
Even so, Kordower told ARF that he believes fetal grafts are still a viable therapy, because the majority of the transplanted cells remain healthy and patients often experience years of motor improvement. Brit Mollenhauer at the Paracelsus-Elena-Klinik, Kassel, Germany, told ARF that, because the initial damage in PD is so localized, grafts might still be a reasonable treatment strategy if the malignancies surrounding this therapy could be gotten under control. At present, however, deep-brain stimulation (DBS) is a much better therapy, these clinicians agreed. DBS, in essence, has set a standard that grafts would have to surpass (see ARF DBS series).

TRANSEURO, a European research consortium coordinated by Roger Barker at Cambridge University, U.K., is working to improve the efficacy of fetal cell replacement, using lessons from past efforts. The group plans to start a new round of clinical trials, paying careful attention to how tissue is prepared and delivered, and how patients are selected. Also, the researchers will suppress patients’ immune systems after surgery to avoid inflammation that might lead to dyskinesias or graft rejection. Kordower, who consults for the consortium, said that in the future, TRANSEURO may switch from using fetal neurons to cultured stem cells. Embryonic or adult stem cells are more readily available than fetal tissue, and could be coaxed to form purer populations of dopaminergic neurons.

Other groups are also interested in the potential of stem cells to replace lost brain cells. At AD/PD, Shimon Slavin at Bar-Ilan University, Ramat-Gan, Israel, described a Phase 2 clinical trial in which mesenchymal stem cells from a patient’s bone marrow are injected into the brain or bloodstream. The stem cells migrate to inflammatory sites and differentiate into neural cells, Slavin said. His group has now treated more than 100 patients, most of whom had multiple sclerosis (MS) or amyotrophic lateral sclerosis, and seen no serious safety issues (see ARF related news story on Karussis et al., 2010). MS patients had the best response, Slavin said, with about 60 percent of them showing improvement so far. In some patients, the results were “spectacular,” Slavin said, noting the case of a man who was confined to a wheelchair before treatment, and now bikes and plays golf. However, this outcome is not typical, and in a difficult field riddled with setbacks, Lazarus stories from Phase 1 tend to provoke skepticism and some concern about inspiring false hopes that are later dashed in Phase 2 or 3. In the meantime, the researchers are trying to find ways to get a more consistent treatment response, Slavin said, for example, by differentiating the stem cells before transplanting them. A similar mesenchymal stem cell approach is currently in a single-center clinical trial for PD patients in India.

Even if these stem cell therapies work, they will have to circumvent the problems that cropped up with fetal grafts. For example, “We have to make sure that stem cells don’t cause off-medication dyskinesias,” Kordower said, adding that this will be hard to test because there are no good animal models for dyskinesia. For a discussion of other treatments in the works, see Part 2.—Madolyn Bowman Rogers.

This is Part 1 of a two-part series. See also Part 2.

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References

News Citations

Paper Citations

Kirik D, Björklund A. Histological analysis of fetal dopamine cell suspension grafts in two patients with Parkinson's disease gives promising results. Brain. 2005 Jul;128(Pt 7):1478-9. PubMed.
Li JY, Englund E, Widner H, Rehncrona S, Björklund A, Lindvall O, Brundin P. Characterization of Lewy body pathology in 12- and 16-year-old intrastriatal mesencephalic grafts surviving in a patient with Parkinson's disease. Mov Disord. 2010 Jun 15;25(8):1091-6. PubMed.
Brundin P, Barker RA, Parmar M. Neural grafting in Parkinson's disease Problems and possibilities. Prog Brain Res. 2010;184:265-94. PubMed.
Hansen C, Angot E, Bergström AL, Steiner JA, Pieri L, Paul G, Outeiro TF, Melki R, Kallunki P, Fog K, Li JY, Brundin P. α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells. J Clin Invest. 2011 Feb 1;121(2):715-25. PubMed.
Karussis D, Karageorgiou C, Vaknin-Dembinsky A, Gowda-Kurkalli B, Gomori JM, Kassis I, Bulte JW, Petrou P, Ben-Hur T, Abramsky O, Slavin S. Safety and immunological effects of mesenchymal stem cell transplantation in patients with multiple sclerosis and amyotrophic lateral sclerosis. Arch Neurol. 2010 Oct;67(10):1187-94. PubMed.

External Citations

Barcelona: Parkinson’s Treatments on the Horizon

07 Apr 2011

With effective symptomatic treatments in hand, many Parkinson’s researchers have now set their sights on loftier goals. Experimental treatments aim to restore lost brain function and arrest disease progression. Researchers are exploring diverse strategies, from cell replacement (see Part 1 of this series), to gene therapy, to small molecules that could counteract pathological processes. Scientists at the 10th International Conference on Alzheimer’s and Parkinson’s Diseases, held 9-13 March 2011 in Barcelona, Spain, unveiled a number of approaches, most of them still preclinical.

Growth Factor Therapy
An alternative to replacing lost cells is providing growth factors that will protect or even restore neurons. One way to do this is to engineer stem cells to pump out growth factors. Eldad Melamed at Tel Aviv University, Israel, described how his team converted mesenchymal stem cells to astrocytes in vitro. These cells then secrete glial-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). The researchers injected these newly generated astrocytes into two different rodent models produced by lesioning the brain with toxins. In both cases, rodents who received astrocytes walked better than those who received undifferentiated mesenchymal stem cells, Melamed said. The researchers obtained similar results in mouse models of multiple sclerosis (see Barhum et al., 2010). The approach has been approved for clinical trials with ALS patients in Israel, Melamed said, and an upcoming small Phase 1/2 trial will be conducted in collaboration with startup company Brainstorm Cell Therapeutics in Petach Tikva, Israel.

Scientists can also deliver growth factors through viral vectors. The gene therapy approach recently saw its first Phase 2 success for PD, with the enzyme glutamic acid decarboxylase (see ARF related news story). At AD/PD, Raymond Bartus at Ceregene, Inc., San Diego, California, updated the crowd on CERE-120, which uses an adenovirus to deliver the growth factor neurturin. Initial Phase 2 trials failed to show benefit (see ARF related news story on Marks et al., 2010). By looking more closely at a monkey model, Bartus said, his team discovered that the growth factor was not reaching the substantia nigra (see Bartus et al., 2011). Ceregene developed a new dosing paradigm, adding injections directly into the substantia nigra, and has now completed Phase 1 testing of this method. Toxicity results were good, and the trial is proceeding into Phase 2, Bartus said.

In a slightly different approach, researchers led by John Forsayeth at the University of California in San Francisco are interested in whether high levels of GDNF would restore brains ravaged by PD. They used adenovirus to deliver GDNF into the putamen of rhesus monkeys whose brains they had lesioned with the toxin MPTP six months earlier. The monkeys’ clinical rating improved and has stayed stable out to two years, Forsayeth said. Improved dopamine turnover and innervation of the putamen indicates a partial restoration of the dopamine system, Forsayeth said, although the treatment did not reconstruct the lesioned regions. A Phase 1 safety trial with 24 participants will start soon, Forsayeth said, adding that, to deliver the virus, the scientists will employ the same MRI-guided stereotactic system approved by the FDA for electrode implantation. This system targets the injection with remarkable accuracy, Forsayeth said, allowing genes to be delivered in a reproducible way without off-target effects or leakage into the CSF.

Blocking α-Synuclein Deposits
Scientists believe that α-synuclein pathology causes both motor and non-motor symptoms of PD. One way to directly attack the disease, therefore, would be to block or break up α-synuclein aggregates. Though not yet in clinical trials, many scientists are pursuing this strategy. “I think this should receive high priority,” Jeffrey Kordower at Rush University Medical Center, Chicago, Illinois, told ARF. Kordower is also one of the founders of Ceregene. Brit Mollenhauer at the Paracelsus-Elena-Klinik, Kassel, Germany, concurs, saying, “This is a good focus for research. I put a lot of hope in it.” Kordower pointed out, however, “Since α-synuclein is all over the brain by end-stage PD, the ability to get widespread delivery of some agent will be a challenge.”

Luke Esposito at ProteoTech, Inc. in Kirkland, Washington, described the screening process his company went through to find their lead anti-aggregant candidate, dubbed Synuclere, which evokes a French dessert perhaps as much as the desired allusion to “clear.” The researchers developed a library of novel synthetic organic molecules, which they screened on cultured cells and in one-year-old transgenic α-synuclein mice to find their lead compound. The compound reversed α-synuclein aggregation in a dose-dependent manner, inhibited β-sheet structures, and worked at a concentration one-tenth that of the aggregated target, Esposito said. It also shows good drug-like properties and no toxicity. After six months of 50 mg/kg/day treatment with this compound, the mice had 80 percent less accumulated α-synuclein in the cortex and substantia nigra, 70 percent less α-synuclein oligomers, and were better able to walk on a beam than untreated mice were, Esposito said. A backup compound gave similar results, he added.

Armin Giese at Ludwig-Maximilians-Universität, München, Germany, discussed a preclinical candidate with broad anti-aggregation effects. His team searched for compounds that inhibit prion aggregates as well as α-synuclein. They identified a class of 3,5-diphenyl-pyrazole derivatives active at concentrations below one micromolar in cell culture. The lead compound, ANLE138B, stopped propagation of all prion strains tested in vitro, Giese said, indicating it is not strain-specific. ANLE138B also gets into the brain well, Giese said, reaching three times the concentration in brain as in blood. When prion-infected mice late in the incubation period were fed 5 mg/day of ANLE138B, the compound blocked prion deposition, prevented cell death, and prolonged survival up to 10 weeks. Mice survived even longer when the treatment was started earlier. The compound is also active against α-synuclein, preventing oligomers from forming. When transgenic mice that express human A30P α-synuclein ate ANLE138B, the animals had less α-synuclein deposition at 16 months, lived up to 10 weeks longer, and had nearly normal motor abilities, Giese said. The compound lessened dopaminergic neuron death in lesioned rodent brains. The results support the ideas that different kinds of protein aggregates have common structural features and that inhibiting aggregation can be therapeutic, Giese said.

Other Small-Molecule Approaches
Kevin Barnham at the University of Melbourne, Australia, described a different tactic for inhibiting harmful α-synuclein aggregates. Drawing on data showing that nitrated α-synuclein is more toxic than un-nitrated forms (see Yu et al., 2010), Barnham’s team looked for small-molecule inhibitors of nitration. They found that an organic molecule containing copper, Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone), or Cu-ATSM, blocked both nitration and oligomerization of α-synuclein. Barnham’s team tested the molecule in four animal models of PD, including one transgenic line and three toxin models. In all four models, the copper-containing molecule prevented dopaminergic neuron death and improved dopamine metabolism, Barnham said, and the mice recovered normal motor skills and memory. Nitration may be a factor in other neurodegenerative diseases, Barnham said, noting that the drug also extends lifespan and reduces inflammation in a SOD1 ALS mouse model. Cu-ATSM accumulates in the brain, but the researchers have not yet analyzed the pharmacokinetics, Barnham said. Thus, it remains to be seen whether the molecule will make a good drug in people.

In Alzheimer’s disease, agonists of the M1 muscarinic acetylcholine receptor have been shown to reverse cognitive problems and amyloid and tau pathologies in mice (see ARF related news story). The same strategy works in PD mice, said Abraham Fisher at the Israel Institute for Biological Research in Ness-Ziona. Fisher is a co-organizer of AD/PD. In a collaboration, Eliezer Masliah at the University of California in San Diego, Manfred Windisch at JSW Life Sciences, Grambach, Austria, and Fisher discovered that the M1 selective muscarinic agonist AF102B clears α-synuclein pathology. When transgenic α-synuclein mice were given 2.5 mg/kg/day of the drug by intraperitoneal injection for three months, α-synuclein deposits and inflammation decreased and dopaminergic neurons survived. Fisher noted that the data have been reproduced by both labs in the U.S. and Austria. Additionally, Windisch saw the same results after chronic treatment with another M1 agonist, AF267B, Fisher said. The effects appear to be mediated by the M1 receptor, since chronic treatment with the relatively selective M1 antagonist, dicyclomine, elevated α-synuclein deposits in the transgenic mice. Developing additional low-molecular-weight compounds that target the M1 receptor should be a priority, Fisher suggested.

Immunotherapy
Immunotherapy approaches, which harness the body’s immune system to clear harmful deposits, are less advanced for PD than for AD. One reason for this, Kordower told ARF, is because α-synuclein aggregates are intracellular, not extracellular, as are Aβ deposits. This heightens the challenge for clearance. However, some evidence suggests that α-synuclein does not just remain hidden inside cells, but gets released and even propagates from cell to cell, Masliah said (see, e.g., Lee et al., 2010). For example, the protein accumulates at pre-synaptic sites and can be found in the extracellular space, Masliah said, suggesting it may be released from synapses. Trans-synaptic transmission has been demonstrated in cell culture, and other data indicate that cells can disgorge α-synuclein through exocytosis. The findings fit with observations from both mice and people that grafted neurons in the striatum become peppered with α-synuclein from the host tissue.

Antibodies might be able to block α-synuclein transfer, Masliah suggested. His team tested this idea by injecting different anti-synuclein antibodies into transgenic PD model mice for six months. Treated mice had less α-synuclein in their neuropil and learned better in a water maze. Antibodies against the C-terminus of α-synuclein gave the best response, Masliah said. These antibodies facilitated the phagocytosis and autophagy of α-synuclein. To show that the antibodies were specifically blocking synaptic transmission, Masliah’s group used a culture model containing neurons in two adjoining chambers. They found an antibody, 1H7, that dose-dependently lowered the amount of α-synuclein transferred between chambers. They then tested the antibody in vivo, using an α-synuclein-null mouse. By injecting a lentivirus expressing α-synuclein into a specific brain region, the researchers could observe trans-synaptic propagation through the brain. Antibody 1H7 reduced this transmission and improved axonal stability, Masliah said. As before, treated mice showed better memory in a water maze test. Antibody treatment shows potential for arresting the spread of Parkinson’s pathology, Masliah concluded.—Madolyn Bowman Rogers.

This is Part 2 of a two-part series. See also Part 1.

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References

News Citations

Paper Citations

Barhum Y, Gai-Castro S, Bahat-Stromza M, Barzilay R, Melamed E, Offen D. Intracerebroventricular transplantation of human mesenchymal stem cells induced to secrete neurotrophic factors attenuates clinical symptoms in a mouse model of multiple sclerosis. J Mol Neurosci. 2010 May;41(1):129-37. PubMed.
Marks WJ, Bartus RT, Siffert J, Davis CS, Lozano A, Boulis N, Vitek J, Stacy M, Turner D, Verhagen L, Bakay R, Watts R, Guthrie B, Jankovic J, Simpson R, Tagliati M, Alterman R, Stern M, Baltuch G, Starr PA, Larson PS, Ostrem JL, Nutt J, Kieburtz K, Kordower JH, Olanow CW. Gene delivery of AAV2-neurturin for Parkinson's disease: a double-blind, randomised, controlled trial. Lancet Neurol. 2010 Dec;9(12):1164-72. PubMed.
Bartus RT, Herzog CD, Chu Y, Wilson A, Brown L, Siffert J, Johnson EM, Olanow CW, Mufson EJ, Kordower JH. Bioactivity of AAV2-neurturin gene therapy (CERE-120): differences between Parkinson's disease and nonhuman primate brains. Mov Disord. 2011 Jan;26(1):27-36. PubMed.
Yu Z, Xu X, Xiang Z, Zhou J, Zhang Z, Hu C, He C. Nitrated alpha-synuclein induces the loss of dopaminergic neurons in the substantia nigra of rats. PLoS One. 2010;5(4):e9956. PubMed.
Lee SJ, Desplats P, Sigurdson C, Tsigelny I, Masliah E. Cell-to-cell transmission of non-prion protein aggregates. Nat Rev Neurol. 2010 Dec;6(12):702-6. PubMed.

External Citations

Barcelona: Antibody to Sweep Up Aβ Protofibrils in Human Brain

14 Apr 2011

The only approved treatments for Alzheimer’s disease are symptomatic, doing nothing to stem the disease’s inexorable progression. Better treatments are urgently needed, and a decade of setbacks in the clinic has left researchers searching for new ideas. The focus is broadening from amyloid plaques to blocking Aβ oligomers, now widely believed to be the most toxic form of the peptide. At the 10th International Conference on Alzheimer’s and Parkinson’s Diseases, held 9-13 March 2011 in Barcelona, Spain, scientists discussed numerous approaches for tackling the disease, from the preclinical to Phase 1. Many talks centered on ways to target oligomeric or protofibrillar species, from antibodies to small molecules. Other strategies roamed further afield, looking at the role of metals and the potential of a recently discovered anti-aging gene.

Immunotherapy strategies for AD are at all stages of clinical trials, though success has been mixed so far. Past trials showed that antibodies cleared amyloid plaques, but have not so far slowed mild to moderate disease (see ARF related news story). Some participants suffered encephalitis, while others develop a poorly understood side effect called vasogenic brain edema (see ARF related AD/PD story). Even so, researchers remain optimistic about the potential of antibodies to mop up Aβ and promote its clearance from the brain (see ARF related news story and ARF related SfN story). Current clinical trials employ heightened vigilance in hopes to avoid and learn about side effects, said Norman Relkin at Weill Cornell Medical College, New York City, and new trials are starting up.

Most current antibodies target sequence-based antigens of Aβ; hence, they bind peripheral Aβ. In contrast, conformation-specific antibodies against oligomers/protofibrils would be more like “guided missiles,” Relkin said, targeting what is thought to be the most toxic form of Aβ. At AD/PD, Kaj Blennow of Sahlgrenska Academy at Göteborg University, Molndal, Sweden, noted that this strategy draws its most recent support from assays measuring oligomeric Aβ in human cerebrospinal fluid (CSF). These were originally reported by Japanese scientists (see Fukumoto et al., 2010), but have now been developed in several other labs independently, including Blennow’s. All show an increase of large-sized oligomers in human CSF from people with AD compared to controls, Blennow noted.

Lars Lannfelt at Uppsala University, Sweden, with Hans Basun and other colleagues, developed an antibody, mAb158, specifically directed against such large-sized oligomers, aka protofibrils. A biotech company Lannfelt co-founded, BioArctic Neuroscience AB in Stockholm, collaborated with Eisai Co., Ltd., to develop a humanized version, dubbed BAN2401, which Eisai is now testing clinically. Andrew Satlin at Eisai told the AD/PD audience about an ongoing Phase 1 trial and another imminent one. The antibody has a thousand times greater affinity for protofibrils than for monomers, Satlin said. In cell culture, BAN2401 lowered the binding of Aβ protofibrils to hippocampal neurons and neutralized their damaging effect on those cells.

The human trials will assess safety, tolerability, and pharmacokinetics, Satlin said. The trials include a single-ascending dose phase, in which the researchers will test six doses from 0.1 up to 15 mg/kg given intravenously, followed by a multiple-ascending dose phase in which four doses will be given one month apart. The four doses in the multiple phase range from 0.3 to 10 mg/kg, Satlin said. The multiple ascending dose phase will only begin with its lowest dose after a higher dose has passed the safety muster in the single dose phase, Satlin said. This design shows that the scientists are watching out for potential safety concerns, which, according to Satlin, include vasogenic edema, microhemorrhage, hypersensitivity reactions, hypotension, formation of anti-BAN2401 antibodies, or inflammation. All this will need to be monitored closely, Satlin said.

In response to a question from immunologist Michael Agadjanyan at the Institute for Molecular Medicine in Huntington Beach, California, Satlin acknowledged that the doses at the upper end of the spectrum are high, hence, the stepwise design. Satlin’s talk prompted discussion about how to find the right dose for immunotherapy trials. Satlin said he expects the 0.3 mg/kg dose to provide roughly the amount of drug that had effects in experiments in AD transgenic mice. He also said that he expects some 0.5 to 1 percent of the antibody to penetrate the brain and work there, not through a peripheral mechanism. He hopes one of the lower doses will achieve effective exposure in the brain that is nonetheless safe in plasma.

The researchers will evaluate changes in Aβ and tau CSF and plasma biomarkers, and will look for interaction with ApoE genotype on those exploratory measures. Effects on neuroimaging markers and cognition are also part of the trial’s exploratory readouts, and will hopefully help in the dose setting and design of further trials, Satlin said. People 50 or older with clinically mild AD will be eligible (i.e., those who have a score between 16 and 28 on the Mini-Mental State Exam); the trial will exclude anyone with a history of microhemorrhages (microhemorrhages are not the same as vasogenic edema, but some scientists fear they may be connected, and more research is needed). Single ascending dose trials are ongoing at eight sites in the U.S., and a multiple ascending dose trial will start in May 2011 in the U.S. and later expand to sites in Sweden, including one led by Martin Ingelsson at Uppsala University.

The clinical trial program follows results of animal tests of mAb158, which Lannfelt discussed at AD/PD. Lannfelt developed the antibody after his discovery of the Arctic mutation in APP. AD caused by this mutation is particularly aggressive, and is driven by Aβ protofibrils, not plaques. Lannfelt said that mAb158 selectively binds Aβ protofibrils, not normal monomeric Aβ or other amyloids such as those made by α-synuclein (see Englund et al., 2007). When mAb158 was given to 10-month-old transgenic AD mice, levels of soluble Aβ dropped by three-quarters, but existing plaques remained undisturbed, Lannfelt said. In young mice, the treatment prevented plaque formation and again lowered soluble Aβ (see Lord et al., 2009). Lower levels of Aβ protofibrils correlated with better cognition, Lannfelt said.

On the heels of this clinical anti-protofibril antibody comes a different, preclinical one that supposedly also recognizes Aβ “aggregates” much more strongly than monomers, though no structural information about the particular antigen is available. At AD/PD, Christoph Hock of Neurimmune, a biotech company based in Schlieren, Switzerland, introduced BIIB037, a monoclonal antibody that Neurimmune’s larger U.S. partner Biogen Idec of Cambridge, Massachusetts, will push into human trials this year, according to Hock. This antibody is unusual in that it is not a humanized form of a mouse antibody but was isolated directly from humans in what Hock called “reverse translational medicine.” This means that the Swiss scientists started with living older people who appear resistant to AD, and tried to exploit their immune response. “We speculate that natural generation of antibodies protects some people,” Hock said.

How would this work? Using a donor cohort of 265 research volunteers in their seventies, the scientists screened the B cells of those people who were cognitively stable over a period of three years, recovered from mild cognitive impairment, or whose AD barely progressed. The scientists generated recombinant versions of naturally occurring antibodies from sequence information, and selected for development an IgG1 that binds to aggregated Aβ with an affinity below one nanomolar. With repeated intraperitoneal injection into Tg2576 mice, the antibody crossed the blood-brain barrier, accumulated on amyloid plaques, and persisted in brain over the measurement period of 2 weeks, Hock said. Repeated injection to mimic chronic treatment of chimeric versions of the human-derived antibody dose-dependently reduced soluble and insoluble Aβ40 and 42, as well as plaque load in hippocampus and cortex. “This human antibody dramatically lowered all Aβ species where deposits can be measured by biochemistry and immunohistochemistry,” Hock said.

At the same doses (3 to 30 mg/kg), microglial activation went up around plaques; amyloid angiopathy or frequency of microhemorrhages stayed unchanged, Hock added. He presented data suggesting that the antibody appears to protect the shape and survival of adult-born neurons in APP/PS1 transgenic mice as well as dendritic spines in cultured neurons (Biscaro et al., 2009). Fielding a question about memory tests in those mice, Hock noted that the antibody improves their performance in contextual fear conditioning and other tests. In response to a second question about exactly what BIIB037 binds to, Hock demurred, divulging only that binding to monomer is many fold weaker than to aggregates. “We are not disclosing the epitope,” Hock said.

As the field at large develops the basic science and translational knowledge base for further anti-oligomer or anti-protofibril approaches, investigators would be well advised to become much more precise in how they prepare, study, and name those potential targets, Dominic Walsh of Brigham and Women’s Hospital in Boston told the audience in Barcelona. “I don’t like the term ‘oligomers’ because it is nebulous. The chemical definition of oligomer as a ‘low-N mer’ tells us nothing about structure,” Walsh said. There are studies about Aβ56*, about ADDLs, about dimers extracted from human brain, the globulomer, protofibrils, and various other synthetic species. “We cannot all dump them into the same category, call them ‘oligomers’ and essentially consider them the same. We need to be much more specific,” Walsh said.

“I do think the protofibrils are one of the mediators of toxicity. The stable dimer boosts protofibril formation and may be the fundamental building block of synaptotoxic protofibrils. Going forward, I prefer that we talk about biophysically defined species,” Walsh went on. Indeed, a strongly worded editorial published in the current Nature Neuroscience makes the same point. Charging that a multitude of different methods and vague language “muddle the field,” the journal calls for scientists to state exactly where the particular form of Aβ used in their experiment comes from, to characterize its aggregation state rigorously, and to discuss its physiological relevance.

Notwithstanding the precise antigen, a slew of vaccine-based approaches are wending their way through trials. Relkin surveyed these studies, in particular noting three passive vaccines that are now in Phase 3: Eli Lilly and Company, Indianapolis, has solanezumab in trials (EXPEDITION and EXPEDITION2); Janssen Alzheimer Immunotherapy, South San Francisco, California, has bapineuzumab in trials (ApoE4 Carrier and ApoE4 Non-Carrier); and Baxter Healthcare Corporation, Deerfield, Illinois, has intravenous immunoglobulin in trials. Results from these studies should come out in 2012 and 2013, Relkin said, at which point the field can reassess what works and what does not. In addition, Merck, Genentech/Roche, Novartis, Affiris, and other companies have antibodies in Phases 2 and 1, and many labs are working on yet more preclinical ones.

With all these immunotherapies, does the field really need another? Indeed it does, Agadjanyan told Alzforum. First, multiple approved vaccines will be needed, because some people will respond to one vaccine but not another. Secondly, the field of AD immunotherapy is so young—and in Agadjanyan’s view, still a bit thinly staffed with card-carrying immunologists—that many of the vaccines that are currently in development have not been deliberately optimized for an elderly population. Vaccines for older people should be specifically designed to tap and reactivate the patient’s pre-existing memory T helper cells. That’s because in older people, those constitute by far the larger pool of T helper cells than naïve ones, which predominate in younger people. In other words, an AD vaccine could rouse an older patient’s memory T helper cells if it contained as its carrier protein a foreign T helper epitope from a conventional public health vaccine against which large numbers of older people had already mounted a T cell response earlier in life. Those could be epitopes that proved their mettle in childhood vaccines, such as from the diphtheria or tetanus toxins, or in influenza vaccines widely used in the elderly. When coupled with multiple copies of the right self-epitope from Aβ’s N-terminus, such heterologous vaccines would rally memory T helper cells to ignite a robust immune response, whereas the actual antibodies the AD patient’s B cells churn out would target Aβ. “If these vaccines prove safe, they may help especially older people overcome immune senescence,” Agadjanyan told the audience.

His group experiments with various peptide-based epitope vaccines along these lines. In Barcelona, he told the audience that some of these generate a strong response in mouse models and rabbits, whereby the T helper cell reaction was specific to the vaccine’s T cell epitope and the humoral response to Aβ. One vaccine is ready for tests in rhesus monkeys; another one, which boosts pre-existing memory T helper cells specific to the tetanus toxin epitope P30, reduced cored and diffuse amyloid plaques in Tg2576 mice. Another category of vaccine use DNA, not peptides. For those, the challenge lies in strengthening the body’s immune response, which tends to be feeble. Commercial vaccine research is developing ways to do that, for example, adjuvant patches that stimulate Langerhans cells in the skin or hand-held electroporation devices, Agadjanyan said. In addition, his group is working out prime-boost regimens that enhance both humoral and T helper cell responses with successive injections of DNA and protein (Davtyan et al., 2010).—Madolyn Bowman Rogers and Gabrielle Strobel.

This is Part 1 of a two-part summary of experimental therapies. For a discussion of small-molecule, metal-targeting, and other AD therapeutic approaches, see Part 2.

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Fukumoto H, Tokuda T, Kasai T, Ishigami N, Hidaka H, Kondo M, Allsop D, Nakagawa M. High-molecular-weight beta-amyloid oligomers are elevated in cerebrospinal fluid of Alzheimer patients. FASEB J. 2010 Aug;24(8):2716-26. PubMed.
Englund H, Sehlin D, Johansson AS, Nilsson LN, Gellerfors P, Paulie S, Lannfelt L, Pettersson FE. Sensitive ELISA detection of amyloid-beta protofibrils in biological samples. J Neurochem. 2007 Oct;103(1):334-45. PubMed.
Lord A, Gumucio A, Englund H, Sehlin D, Sundquist VS, Söderberg L, Möller C, Gellerfors P, Lannfelt L, Pettersson FE, Nilsson LN. An amyloid-beta protofibril-selective antibody prevents amyloid formation in a mouse model of Alzheimer's disease. Neurobiol Dis. 2009 Dec;36(3):425-34. PubMed.
Biscaro B, Lindvall O, Hock C, Ekdahl CT, Nitsch RM. Abeta immunotherapy protects morphology and survival of adult-born neurons in doubly transgenic APP/PS1 mice. J Neurosci. 2009 Nov 11;29(45):14108-19. PubMed.
Davtyan H, Mkrtichyan M, Movsesyan N, Petrushina I, Mamikonyan G, Cribbs DH, Agadjanyan MG, Ghochikyan A. DNA prime-protein boost increased the titer, avidity and persistence of anti-Abeta antibodies in wild-type mice. Gene Ther. 2010 Feb;17(2):261-71. PubMed.

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Barcelona: A Fistful of Schemes for Tackling Alzheimer’s

15 Apr 2011

As scientists continue to pursue disease-modifying treatments for Alzheimer’s disease, various forms of oligomers have become the target du jour, and another way to disrupt them is with small molecules. JoAnne McLaurin at the University of Toronto, Ontario, updated AD/PD attendants on scyllo-inositol, which she said is gearing up for a Phase 3 trial after mixed results in Phase 2 (see ARF related news story). Previous studies suggested that scyllo-inositol works by binding Aβ42 oligomers and preventing large aggregates (see ARF related news story). In Barcelona, McLaurin presented new data indicating that the drug binding to Aβ42 promotes autophagy in TgCRND8 mice. Autophagy works poorly in these mice, as seen by the accumulation of large autophagic vesicles. Treatment shrank those enlarged vesicles and increased the amount of Aβ in microglia, suggesting the peptide is being cleared. It also reduced the amount of Aβ in brain blood vessels. McLaurin said her current hypothesis is that scyllo-inositol binds oligomers and helps them get phagocytosed. Autophagy has recently generated intense interest in both mechanistic and therapeutic AD research (e.g., see ARF related news story; ARF news story). Additionally, McLaurin performed microarray studies that showed 275 genes changed expression in the hippocampus after scyllo-inositol treatment. Of these genes, almost half have previously been implicated in AD.

What about other small molecules believed to inhibit aggregation? Christofer Lendel at the Swedish University of Agricultural Sciences, Uppsala, presented the results of testing two such compounds, the dyes Congo red and lacmoid. Both have been reported to interfere with oligomerization and have some similarities to methylene blue, which is currently under investigation as an anti-tau aggregant (see ARF related news story and ARF update). When the researchers looked closer at the biophysics, however, they found that Congo red actually promoted the assembly of Aβ into β-sheets (see Lendel et al., 2010). Lacmoid, on the other hand, preserves the random coil structure of Aβ and prevents its assembly, and thus is a true anti-aggregant. This illustrates the importance of characterizing a molecule’s mechanism of action with structure-based studies, Lendel noted.

Aging and Myelination
Age is the single greatest risk factor for AD, prompting a growing number of AD researchers to study what changes in the aging brain. For example, in monkeys, rats, and mice, levels of a transmembrane protein called Klotho drop with age, said Carmela Abraham of Boston University (see ARF related news story on Chen et al., 2007; Duce et al., 2008). In particular, Klotho levels decline in the brain’s white matter, and this correlates with age-related myelin deterioration, Abraham said. Establishing Klotho’s status as a longevity gene, Klotho knockout mice age prematurely and show hallmarks of human aging, whereas Klotho overexpressors live about one-quarter longer than normal mice (see Kuro-O et al., 1997; Kurosu et al., 2005). APP and presenilin double-transgenic AD mice have little Klotho in their brains, Abraham said. APP fragments have been shown to regulate Klotho expression (see ARF related news story), suggesting a connection between Klotho and AD. Exactly what Klotho does, however, besides suspected roles in insulin signaling and antioxidant activity, is still unclear.

To characterize Klotho’s role in white matter, Abraham’s team added the protein to cultured oligodendrocytes, which are the cells that form myelin. Klotho sped up the rate of the cells’ maturation, Abraham said. She noted that in normal mice, expression levels of Klotho and myelin proteins rise together shortly after birth, suggesting they are closely linked. Additionally, adult Klotho knockout mice have less myelin protein. Electron microscopy reveals that their optic nerve is only about 10 percent myelinated, versus 90 percent in control mice, Abraham said. Young knockouts have normal myelination, however, suggesting that Klotho plays a part in maintaining myelin with age but not in development. The gene might be a therapeutic target in multiple sclerosis (MS), a disease marked by myelin degeneration, Abraham said. She plans to cross MS model mice with mice overexpressing Klotho to see if the protein can promote myelin repair.

Abraham also investigated what lies upstream of Klotho to figure out why its levels decline with age. She found that in the white matter of aging monkeys, the Klotho promoter acquires more methyl groups, which tend to silence genes. Abraham’s team screened 150,000 compounds to find drugs that enhance Klotho expression. The high-throughput screen produced two leading candidates. Both increase Klotho expression in cultured opossum kidney cells and in choroid plexus cells, tissues that normally secrete the protein. These compounds could have promise as treatments for AD, MS, and potentially even in normal aging, Abraham said. It is not yet certain if Klotho levels decline in aging human brains, but since it is decreased in three other species, it is likely to decline in humans as well. However, Abraham told ARF that Klotho is secreted into the cerebrospinal fluid, where she plans to compare its levels in healthy people and those with AD.

Where Do Metals Come In?
Other scientists are taking a different approach to AD treatment, looking at the role of metals in the disease (see ARF story on Top 13 Trends in 2010). Iron has been repeatedly linked to AD, as iron levels in the brain go up with aging (see ARF related news story) and APP has been shown to export iron (see ARF related news story). Moussa Youdim at Technion Israel Institute of Technology in Haifa discussed an iron chelator, M30, which shows therapeutic potential. M30 has a chimeric structure and contains a piece from rasagiline, a PD drug, said Orly Weinreb at Technion. Youdim said M30 has multimodal effects, not just binding iron but also inhibiting monoamine oxidase and affecting the levels of several neurotransmitters such as serotonin, dopamine, and acetylcholine. Ten-month-old APPswe/PS1 mice treated with M30 showed better memory and had less plaque pathology, soluble Aβ, and phosphorylated tau, Youdim said, adding that the drug increased the expression of growth factors and protected the mice from neurodegeneration. Because M30 is lipophilic, it enters the brain well, Youdim said, and is not toxic at effective doses in mice. M30 itself is not a candidate for human development, however, and Youdim said his group is currently developing a more stable variant for Phase 1 studies. M30 may also help Parkinson’s patients (see Gal et al., 2006) and people with amyotrophic lateral sclerosis (ALS). The drug improved survival in a mouse model of ALS, Weinreb said, adding that a Phase 1 trial for ALS patients will start soon. To date, no drug that was reported to extend survival in an ALS mouse model has subsequently worked in human trials (see Alzforum Webinar).

Zinc has also been linked to AD. Zinc is normally released during neurotransmission and affects NMDA receptors involved in long-term potentiation. Researchers at the University of Melbourne found that mice that lack zinc in their synapses develop AD-like symptoms (see ARF related news story). Amyloid plaques sequester zinc and copper, said Rudy Tanzi at Massachusetts General Hospital, Boston, and this could drive some of the synaptic and memory defects of the disease. In addition, zinc and copper promote Aβ aggregation, creating a vicious circle. Tanzi is a co-founder and shareholder in Prana Biotechnology Ltd., Parkville, Australia, a biotech company developing a zinc/copper ligand called PBT2. PBT2 had beneficial effects in AD mice (see ARF related news story) and in a short Phase 2 trial, where it improved some cognitive measures in people with mild AD (see ARF related news story). More people improved on the 250 mg dose than on the 50 mg dose, indicating a dose-dependent response (see Faux et al., 2010). PBT2 was recently shown to increase the number of dendritic spines in the hippocampus of AD mice and restore levels of NMDA receptor subunits to normal levels (see Adlard et al., 2011). After some attempts to secure industry financing for further trials of PBT2, Prana recently announced that the Alzheimer’s Drug Discovery Foundation in New York City will fund a one-year Phase 2 trial to begin in late 2011 in Australia (see press release). The trial will enroll 40 patients who have mild AD and amyloid deposition in the brain as seen by positron emission tomography with Pittsburgh Compound B. Scientists will look for changes in amyloid burden as the primary outcome.—Madolyn Bowman Rogers.

This is Part 2 of a two-part series. See also Part 1.

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References

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Lendel C, Bolognesi B, Wahlström A, Dobson CM, Gräslund A. Detergent-like interaction of Congo red with the amyloid beta peptide. Biochemistry. 2010 Feb 23;49(7):1358-60. PubMed.
Chen CD, Podvin S, Gillespie E, Leeman SE, Abraham CR. Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17. Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):19796-801. PubMed.
Duce JA, Podvin S, Hollander W, Kipling D, Rosene DL, Abraham CR. Gene profile analysis implicates Klotho as an important contributor to aging changes in brain white matter of the rhesus monkey. Glia. 2008 Jan 1;56(1):106-17. PubMed.
Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997 Nov 6;390(6655):45-51. PubMed.
Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn CR, Rosenblatt KP, Kuro-O M. Suppression of aging in mice by the hormone Klotho. Science. 2005 Sep 16;309(5742):1829-33. PubMed.
Gal S, Fridkin M, Amit T, Zheng H, Youdim MB. M30, a novel multifunctional neuroprotective drug with potent iron chelating and brain selective monoamine oxidase-ab inhibitory activity for Parkinson's disease. J Neural Transm Suppl. 2006;(70):447-56. PubMed.
Faux NG, Ritchie CW, Gunn A, Rembach A, Tsatsanis A, Bedo J, Harrison J, Lannfelt L, Blennow K, Zetterberg H, Ingelsson M, Masters CL, Tanzi RE, Cummings JL, Herd CM, Bush AI. PBT2 rapidly improves cognition in Alzheimer's Disease: additional phase II analyses. J Alzheimers Dis. 2010;20(2):509-16. PubMed.
Adlard PA, Bica L, White AR, Nurjono M, Filiz G, Crouch PJ, Donnelly PS, Cappai R, Finkelstein DI, Bush AI. Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease. PLoS One. 2011;6(3):e17669. PubMed.

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Barcelona: Inflammation—That Two-Faced Beast

19 Apr 2011

Inflammation is common in neurodegenerative diseases, but scientists are not yet sure how it relates to pathology. Is inflammation a harmful driver of disease, a protective response, or something in between? All of the above, and it all depends on the cell subtypes and signaling proteins involved, suggested scientists at the 10th International Conference on Alzheimer’s and Parkinson’s Diseases, held 9-13 March 2011 in Barcelona, Spain. Researchers are beginning to tease apart what factors make a microglial cell a helpful vacuum cleaner or a dangerous toxin-spewing machine. At AD/PD, speakers described specific cytokines, markers, and signaling pathways involved in both beneficial and injurious inflammation. Many talks focused on AD, but some speakers highlighted the similarities and differences of inflammatory processes in prion diseases, Parkinson’s, and tauopathies. Below are some excerpts. This story is far from comprehensive, and all inflammation aficionados who don’t find their presentations included here are cordially invited to round it out with a comment about their own work on the subject.

Inflammation’s Dark Side
Taking the broad view, Piet Eikelenboom at VU University Medical Center, Amsterdam, The Netherlands, discussed the close links among aging, inflammation, and sporadic AD. Eikelenboom’s group was the first to describe inflammation and the complement system in Alzheimer’s disease in the 1980s (see ARF related news story), and he sees a resurgence of interest around the topic now. Eikelenboom argued that inflammation precedes AD. Eikelenboom pointed out that the diffuse amyloid plaques commonly found in non-demented elderly people contain numerous inflammation-related molecules, such as activated complement proteins, ApoE, clusterin, and α1-antichymotrypsin (see Eikelenboom et al., 2006; Eikelenboom et al., 2011). Most of these proteins have since been genetically tied to AD (see AlzGene top results), and scientists increasingly voice a suspicion that a person’s particular set of risk alleles might exert their effect in young and middle adult life, before the actual disease process begins. People whose parents had AD are more susceptible to inflammation in midlife, producing greater quantities of pro-inflammatory cytokines when their systems are challenged (see van Exel et al., 2009). This suggests that genetic risk factors for AD go hand-in-hand with inflammation-prone systems.

As AD develops, fibrillar Aβ deposits accumulate, appearing around Braak stage III or IV and before any clinical symptoms. Activated microglia and astrocytes surround these plaques and pump out cytokines that promote inflammation, Eikelenboom said. Thus, fibrillar plaques may play a key role in triggering an inflammatory reaction, an idea discussed by several presenters. Also, several studies have found that systemic infections worsen AD symptoms and accelerate degeneration and cognitive decline (see Perry et al., 2007; Holmes and Cotterell, 2009; Holmes et al., 2009). As Eikelenboom described it, infections can put “inflammatory pressure on the brain.”

Eikelenboom suggested that the process of aging itself may put the body into a low-grade inflammatory state. This would prime microglia and allow a late-life infection or injury to activate these cells and precipitate rapid presentation of serious disease. For example, something as simple as a hip fracture can cause delirium in elderly people due to a neuroinflammatory response involving acute-phase proteins, Eikelenboom said (see Eikelenboom et al., 2002). The general scheme is not limited to AD, Eikelenboom believes. An elderly person at risk for type 2 diabetes can rapidly develop diabetes after a late-life infection or injury, while somebody at risk for AD would come down with this disease after a late-life challenge.

Activated microglia also damage the cholinergic system, Eikelenboom noted. This system normally inhibits the inflammatory activation of microglia (see Hwang et al., 2010). Microglia thus escape cholinergic control and ramp up further, causing more neurodegeneration and more damage to cholinergic neurons (see van Gool et al., 2010). In this model, then, inflammatory processes are not just a response to neurodegeneration, but play a key role in driving late-onset AD.

Pathology indicates, too, that inflammation is not limited to AD. Neuroinflammation is an early feature of almost all neurodegenerative diseases, said Annemieke Rozemuller, also at VU University. She presented postmortem data from studies comparing patients with AD to others who had various prion diseases such as Creutzfeldt Jakob and Gerstmann Straussler Scheinker's disease. In both AD and prion diseases, she said, amyloid plaques in brain and blood vessels contain activated complement proteins and α1-antichymotrypsin. Activated microglia cluster around prion plaques, and Rozemuller saw deposits of tau and ubiquitin in prion-riddled brains. Rozemuller concluded that inflammatory changes are related to the process of amyloid deposition, regardless of the type of amyloid.

Good Inflammation, Bad Inflammation—Ways to Tell Them Apart
Despite all the data showing the dark side of inflammation, it can be protective, too. Stimulated microglia can devour Aβ, helping keep plaques in check (see ARF related news story and ARF news story). Researchers have just begun to tease apart what factors make microglia harmful or helpful, and there is not a clear consensus yet. Researchers recently discovered that knocking out the microglial-expressed immune phosphatase CD45 caused microglia to secrete more pro-inflammatory cytokines and fail to chew up Aβ, and mice lost more neurons (see ARF related news story).

At AD/PD, Todd Golde at the University of Florida, Gainesville, described data that go against conventional wisdom, showing that pro-inflammatory cytokines can promote phagocytosis and reduce plaques. His team uses viral vectors to express various immune factors in the brains of TgCRND8 AD model mice, creating “somatic brain transgenic” animals. This method is quicker and cheaper than breeding traditional transgenic mice, Golde noted. Injection at different ages produced different expression patterns. Injection into newborns yielded the broadest expression, throughout midbrain and forebrain. The scientists first tested the pro-inflammatory cytokine IL-6, which is elevated in AD brains. They injected newborns and examined brains at five months. Instead of seeing the expected worsening of pathology, they saw less Aβ deposition (see Chakrabarty et al., 2010). They also saw widespread activation of astrocytes and microglia, more microglia associated with plaques, and more phagocytosis of Aβ. Overall levels of APP or Aβ were unchanged. The researchers saw similar effects with the pro-inflammatory cytokines IFNγ and TNFα (see Chakrabarty et al., 2010; Chakrabarty et al., 2011). In contrast, preliminary studies show the anti-inflammatory cytokines IL-10 and IL-4 both enhanced Aβ deposition, resulting in more and/or larger plaques. Although the results suggest that some inflammation can help limit Aβ deposits, Golde noted that it is unclear if this actually protects the brain. Inflammation could be causing problems, too, and more research is needed to determine if the animals fare better overall. Nor is it clear how microglial subtypes contribute to this picture, Golde said.

Golde described another mouse model to illustrate the point that the effects of a particular cytokine depend on where and when it acts. When Golde’s team expressed IFNγ primarily in mouse choroid, the mice developed calcium deposits in the basal ganglia reminiscent of human Fahr’s syndrome. This syndrome includes Parkinson’s-like symptoms, and, indeed, the mice showed parkinsonism as well as profound progressive degeneration of the nigrostriatal tract. Being presented with a parkinsonian model after merely expressing IFNγ in the choroid was totally unexpected, Golde said. Since IFNγ levels rise in response to viral infections, this may explain why Fahr’s syndrome can develop after viral infection of the brain, Golde noted. IFNγ has been reported to be elevated in Parkinson’s disease (see, e.g., Mount et al., 2007), suggesting that this mechanism could be important in general PD as well, Golde said. He added that this mouse model places specific types of inflammation and inflammatory mediators on the pathway toward neurodegeneration (Chakrabarty et al., in press). The effect of IFNγ contrasts with that of IL-6, which does not drive neurodegeneration despite causing massive gliosis.

To determine whether inflammation is good or bad in particular models, scientists may have to dissect the specific signaling pathways involved. Saskia van der Vies at VU University described one. Her team discovered that interleukin-1 receptor-associated kinase 4 (IRAK-4) and its substrate, the kinase IRAK-1, were elevated in late-stage AD brains. These kinases are expressed in microglia and astrocytes, van der Vies said, where they mediate signaling through the interleukin-1 (IL-1) receptor and through toll-like receptors. Downstream, the kinases activate the transcription factor NF-κB, which itself increases the production of other cytokines including monocyte chemotactic protein-1 (MCP-1). MCP-1 goes up with age in people (see ARF related news story) and is elevated in AD (see Galimberti et al., 2006). It has been fingered as a culprit in chronic inflammation (see Sokolova et al., 2008). Van der Vies’s team stimulated microglia and astrocytes while inhibiting IRAK-4 activity, and found that the glia were unable to make MCP-1 but continued to gobble up Aβ. This indicates that IRAK-4 activity is essential for MCP-1 production in both astrocytes and microglia, van der Vies said. Since IRAK-4 inhibition stops production of a harmful cytokine while leaving presumably helpful phagocytosis intact, inhibiting this kinase could be promising therapeutically, van der Vies suggested.

Makoto Higuchi at the National Institute of Radiological Sciences, Chiba, Japan, presented another way to separate good and bad microglial functions. He also points an accusing finger at MCP-1. The harmful subset of microglia express the 18-kDa translocator protein (TSPO; also known as peripheral benzodiazepine receptor), Higuchi said. Conveniently for scientists, TSPO radioligands exist to visualize this protein in the living brain using positron emission tomography (PET). This method reveals TSPO-positive microglia almost exclusively around plaques in the AD brain in both mice and humans (see Yasuno et al., 2008).

To characterize the effects of this microglial subtype, Higuchi’s team prepared a microglial cell line clone that expressed high levels of TSPO, as well as one that expressed very little TSPO. When the researchers implanted the low TSPO clone into the brains of APP transgenic mice, they saw less Aβ and higher levels of brain-derived neurotrophic factor, whereas injection of high TSPO-expressors seemed to prevent neighboring glia from mopping up Aβ and to worsen plaques.

To investigate what makes the TSPO-expressors harmful, Higuchi’s team analyzed their cytokine profile and found that they make high levels of MCP-1. Higuchi noted that the enzyme glutaminyl cyclase (QC) converts MCP-1 to a more stable form (see ARF related news story and ARF AD/PD story). The same enzyme creates a stable, pyroglutamate form of Aβ that has been proposed to initiate the formation of Aβ plaques (see, e.g., ARF related SfN story). This makes QC a good therapeutic target, Higuchi noted, since QC seems to promote both amyloid deposition and harmful inflammation.

In mouse experiments, anti-Aβ therapy reduces amyloid deposits, but also increases TSPO, Higuchi said. He concluded that antibodies stimulate both protective microglia that phagocytose Aβ as well as harmful microglia that secrete MCP-1. Since MCP-1 seems to mediate the harmful effects of TSPO-expressing microglia, Higuchi’s team combined anti-Aβ therapy with anti-MCP-1 antibodies in APP transgenic mice. The combination provided long-lasting removal of Aβ without any increase in TSPO-positive microglia, Higuchi said.

The Flip Side: Microglia and Tau
If you think you are beginning to understand microglial subtypes and their respective effects on pathology, consider experiments looking at the effect of microglia on tau. In the PS19 mutant human tau-overexpressing mice, TSPO-positive microgliosis occurs as well, Higuchi said. MicroPET TSPO signals were actually higher in tauopathy mice than in amyloid mice, and they preceded neuronal loss, Higuchi said, suggesting that a TSPO signal could eventually serve as a “toxicity alarm” in AD brains (see Maeda et al., 2011).

David Morgan at the University of South Florida, Tampa, also presented evidence that inflammation interacts quite differently with tau tangles than it does with amyloid plaques. It’s known that in APP transgenic mice, stimulation of microglia with the generic inflammatory agent lipopolysaccharide (LPS) activates phagocytosis and reduces amyloid. More recently, Morgan’s team looked at what happens in the Tg4510 tauopathy model mouse. This strain seems to model a slightly later stage of AD, with more neurodegeneration, brain shrinkage, and poor memory, than do amyloid models, which reflect early, preclinical AD (see Dickey et al., 2009). Tauopathy mice normally have less microglial activation than do amyloid mice, and it shows up later, Morgan said in Barcelona, but in the Tg4510 mice, microglial stimulation with LPS was harmful, increasing the amount of phosphorylated tau (see Lee et al., 2010). LPS injections dramatically increased the numbers of CD45-positive, activated microglia, which is the subtype that has beneficial effects in amyloid mice (see ARF related news story).

Since many scientists believe that tau mediates the harmful effects of Aβ (see, e.g., ARF related news story and ARF news story), a treatment that decreases one while increasing the other might not help the patient. “LPS injection enhances degradation of amyloid deposits in APP mouse brain but exacerbates pathology in tau-depositing mice. That makes it hard to decide what you want to do therapeutically with microglia,” Morgan said.—Madolyn Bowman Rogers.

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