Vienna: New Genes, Anyone? ICAD Saves Best for Last

21 Jul 2009

In like a lamb, out like a lion. It’s a full season too late for this variation on the March weather proverb, but hopefully our generous readers will forgive their roving reporter for bending it anyway, because starting meekly and finishing with a roar is exactly what the Alzheimer’s Association’s 12th International Conference on Alzheimer’s Disease (ICAD) did when it unfolded in Vienna 11-16 July. And it’s not about changing weather, either. All week, the Austrian capital bathed meeting attendees in sparkling sunshine (which seemed to seduce many into sneaking off for daytrips into this beautiful city as attendance seemed a bit sparse at times, but no names shall be named). No, the scientific program started with humbling reports of the failure—abject and otherwise—of the latest round of clinical trials. The program continued with confirmatory studies on the ability of exercise, a heart-healthy diet, and modest drinking while one is cognitively healthy, to modify one’s risk of cognitive decline somewhat. This is an important life message people should take to heart, but it is not the kind of news close followers of the field are looking for. But then the last morning—when many had departed, the exhibit hall was emptied out, and the press room had closed up shop—featured within the space of three hours arguably the biggest news in AD genetics research in the past decade. Three independent genomewide association studies, one being the largest performed to date on some 4,000 cases, together reported three new genes for late-onset AD. Hence, overall comments about ICAD in Vienna went from a plaintive “I haven’t heard anything really new” at the beginning to a confident “We can declare three new genes for Alzheimer’s” at the end.

Most remarkable from an observer’s perspective was the consensus among independent genetics groups that the new genes would likely hold up to scrutiny in future studies even if their individual effect was small. When is the last time you remember applause and nodding faces, not skepticism or criticism, greeting the first introduction of a new candidate gene at a major AD conference? The genes reported today are ApoJ (aka clusterin), CR1 (which encodes a complement receptor), and PICALM (which encodes an endocytic gene). Together, they point partly to Aβ generation/clearance playing a role in late-onset AD, said Philippe Amouyel of the Institute Pasteur in Lille, France, but more broadly, they also implicate other upstream processes as leading to AD pathology and symptoms. “The GWAS data suggest that innate immunity and cholesterol are key themes in the disease,” said Julie Williams of Cardiff University, U.K. (For more on these presentations, see upcoming ICAD news story.)

Regarding clinical trials, the news in Vienna was mostly dire. Among the trials that reported results, GSK’s Phase 3 rosiglitazone program failed, the ADCS’s valproate and DHA trials were negative, as were the Phase 2b trials of AstraZeneca’s α4β2 nicotinic acetylcholine receptor agonist AZD3480 and of a monthly implant formulation of donepezil called mimopezil. The large anti-amyloid trials are many months to years away from yielding results, and without substantiation the rumors that inevitably fly in such periods of suspended animation are not worth reporting. The presentation by Bengt Winblad of the Karolinska Institute in Stockholm, Sweden, of Novartis’s CAD106 active vaccine, which is currently in Phase 2, dealt mainly with additional Phase 1b efforts to refine the dosing regimen for obtaining stable and sufficient antibody titers, and with expanding the human safety record for this virus-like particle vaccine to a larger sample of patients. Steven Jacobsen’s presentation of Wyeth’s new γ-secretase inhibitor GSI-953, which according to ClinicalTrials.gov has finished three Phase 1 trials, the latest in the summer of 2008, covered mostly preclinical data.

The trials landscape offered some unexpected highlights, though. A new agonist against the α7 nicotinic acetylcholine receptor showed early hints of possible efficacy on top of stable AChE inhibitor treatment in a Phase 1b/2a study (see ARF related conference story). And John Breitner of the University of Washington, Seattle, reported that the ADAPT primary prevention trial of two NSAID drugs in some 2,500 people age 70 and older provided a pleasant surprise at longer-term follow-up. The ADAPT treatments were halted mid-stream in December 2004 amid concerns about the safety of celecoxib and other NSAIDs for long-term use in the elderly (for prior coverage of this topic, see ARF Safety Data news story, ADAPT Trials and Tribulations news story; see also Safety Concerns Halt ADAPT Trial).

ADAPT received funding to follow the trial participants for two more years, and in Vienna Breitner showed results from subgroup analyses. They suggested that people who already had mild cognitive problems at the beginning of the trial indeed worsened if they took NSAIDs, which accelerated their underlying disease. In contrast, people who were cognitively normal at baseline and took naproxen for one to three years appeared to fare better. At the long-term examination, they not only had a lower incidence of AD than did those on placebo, but going along with this apparent clinical protection was a biomarker effect as well. The naproxen group had a favorable, i.e., lower, tau/Abeta42 ratio in their CSF drawn at spinal taps 21-42 months after treatment. “Primary prevention, not disease modification, is the ultimate cure, and timing is everything,” Breitner asserted provocatively. ADAPT has recently received funding to ask the trial cohort back for two further examinations. Breitner suggested that the biomarker results give good hopes to the possibility that one to three years of treatment with naproxen in cognitively intact people may ultimately reduce the incidence of AD for five to 10 years out.

Overall, this ICAD conference was marked by a notable absence of many scientists who tend to be regulars. Citing the growth in the number of conferences in the field, many begged off this time. Some said they are on the road too much to get much else done, and some deplored the cost of trying to bring junior researchers to AD conferences these days. At 3,700, attendance at Vienna’s attractive, light-flooded “Messe” center fell below the 5,400 from Chicago in 2008 and the 5,000 from Madrid in 2006. That said, those who were in Vienna cited a different spirit than in Chicago. Last year, there was palpable tension at ICAD when the Flurizan Phase 3 negative trial and Bapineuzumab’s complicated Phase 2 results failed to meet the expectations that had been building in the months prior. This renewed doubt in the amyloid hypothesis. This time around, researchers expressed a greater sense of calm. In Vienna, Henrik Zetterberg of Sahlgrenska University Hospital in Goteborg, Sweden, spoke for many when he said, “The field has been humbled, and we can now focus on how best to regroup. We can do research to improve the next round of drug testing without the pressure of imminent trial results hanging over our head.”

To an outside meeting observer, two countercurrents seemed to be building simultaneously, though the twain never met for open debate in Vienna. On the one hand, researchers who tend to subscribe to the amyloid hypothesis as the source of the most tangible drug targets at present, drew renewed confidence from results emerging from ADNI and other biomarker studies in Europe and elsewhere. Overall, those results converged around the realization that biomarker signatures of impending AD seen at the MCI and perhaps even earlier stages will make it practical to move the clinical testing of drug candidates to stages of the disease where symptoms are milder—and neurodegeneration less extensive—than at the current “mild to moderate” stage defined by the current NINCDS-ADRDA diagnostic criteria, which are widely seen as being behind the curve of biological research on AD pathogenesis. As a group, these scientists have worried that otherwise respectable drugs may fall by the wayside because they are being tested too late in the disease. “I am coming out of this ICAD meeting with a bump because I think the tools are coming together, from the ADNI study and others, that make it possible to run earlier-stage trials now,” said Reisa Sperling of Brigham and Women’s Hospital in Boston, who oversees AD clinical trials at that institution.

ICAD featured abundant debate about a range of specific biomarker issues, particularly how to diagnose pre-dementia with biomarkers yet avoid false diagnoses. An entire session focused on how the field might be able to implement new diagnostic criteria proposed two years ago by Bruno Dubois at Hôpital de la Salpetrière in Paris and colleagues (see Dubois et al., 2007) and how the stage of “pre-AD” compared with amnestic MCI as defined by Ron Petersen of the Mayo Clinic in Rochester, Minnesota (in short: the two are quite similar; see upcoming story). But all the same, most scientists agreed that at the very least, the more validated ones among the current crop of biomarkers can already serve to ensure that people enrolled in anti-amyloid drug trials indeed have amyloid in their brain, or that people enrolled in AD drugs have hippocampal shrinkage or a pathological Aβ/tau signature in their CSF.

There is no officially anointed “consensus package” of predictive biomarkers yet, if ever there will be one, but some companies are not waiting for it. Word went around in Vienna that Bristol-Myers Squibb, for example, is now recruiting for a 75-center Phase 2 trial of their new γ-secretase inhibitor, BMS-708163, in what that company calls “prodromal AD.” Unusually, that new trial allows study in people as young as 45 and as old as 90, and it uses a combination of a subjective memory complaint plus a defined, low CSF Aβ42 ratio as an inclusion criterion. If people or their partners think their memory is off, and if their Aβ42 is indeed below a set cutoff, then they can ace the MMSE test with a perfect score of 30 and still enter the drug trial. The thinking is that people could be so well educated to begin with that the MMSE would not pick up subtle cognitive changes in them. In addition, a handful of drug companies are already using amyloid imaging either with the original Pittsburgh compound B (PIB) or with Avid Radiopharmaceutical’s AV-45 not as inclusion criteria or primary endpoint, but to gain additional information.

The field of amyloid imaging itself advanced in Vienna with the presentation of new data on four different F18-based amyloid tracers developed by GE Healthcare, Bayer Health Care, Avid Radiopharmaceuticals, and AstraZeneca, respectively. The key news there, in a word, was that they all perform quite similarly save for small technical differences, said Murali Doraiswamy of Duke University Medical Center in Durham, North Carolina. Doraiswamy presented the Phase 2 data of a multicenter trial, in 164 people, of AV-45 (which for a while went by the name florpiramine but now is referred to as AV-45 again, in case anyone got temporarily confused). Called florbetaben, the Bayer compound was tested in a Phase 2 study of 150 people in 18 centers, and the 18F-labelled PIB derivative GE067 was tested in a smaller multicenter study involving 70 patients and controls. Scientists from multiple groups agreed that together, the data to date suggest that all four agents likely will do a serviceable job of detecting whether people have a significant amyloid load in their brains. At the same time, they all have a tad more noise than C11 PIB and may be too crude a tool to quantify sub-threshold amyloid loads at very early stages of deposition, according to Chet Mathis of Pittsburgh University Medical Center, who co-developed PIB. ADNI-2, for its part, will likely use AV-45, and a small study performing a side-by-side comparison of PIB and AV-45, is gearing up to be run at the University of Philadelphia Medical School under the guidance of John Trojanowski.

As for ADNI, ICAD featured several dozen presentations on this 59-center observational biomarker study, as scientists from around the world avail themselves of the publicly available data at the LONI website. Moreover, this initiative is sparking new collaborative efforts to help with international standardization of biomarker use, for example, a budding effort to make surplus CSF from routine clinical practice in Sweden available as a standard against which other groups could eventually normalize the results of their observational or drug studies (see upcoming ICAD story).

And yet, in parallel to this building sense of optimism and expanding cooperation, other well-regarded researchers insisted that it is time to make a greater effort to move beyond the amyloid hypothesis, though they preferred to withhold their name from quotation. “It is becoming increasingly hard to take data year after year that are not only not mildly supportive but actually counter to the hypothesis, and to keep the amyloid hypothesis as the framework for understanding Alzheimer disease,” was how one leading investigator put it. Previous results of early stage immunotherapy trials are often cited in this regard, as are recent studies showing that very old people with dementia frequently do not have the signature pathologies of AD (see ARF related news story). At ICAD in Vienna, more ammo for this view came from puzzling data that Dimebon, the Russian drug currently in Phase 3 trials in the U.S., actually raises the release of Aβ from cells and in mouse models. This could mean several things. One interpretation is that if Dimebon manages to repeat in the U.S. the sizeable drug effect reported from a Russian Phase 2 trial, then there is much to be gained in the way of AD treatment without a focus on lowering Aβ, as do most other experimental drugs in large clinical trials these days. Such a result would boost morale among scientists who have felt disadvantaged in their interests outside the amyloid hypothesis. “If people will acknowledge that it is more complicated than the one-agent theory, then that opens up opportunities for scientists to pursue projects that they have hesitated on because of a perception that they are barking up the wrong tree, or that there is only one tree,” said one scientist. Follow-up studies of the new genes announced in the conference’s last session (see above), may well boost such a broadening of AD research, as they suggested that the risk genes for late-onset AD are functionally separate from the three genes that define familial AD. Those genes, APP and presenilin 1 and 2, fell far below genomewide significance, as did the gene for tau, implying that other problems will help explain at least some people’s genetic propensity to come down with Alzheimer disease late in life.

Another notable trend at ICAD suggested that interest in blood-based biomarkers is on the rise again. In the past decade, efforts to measure Aβ and tau in plasma and detect patterns that would predict AD have led to such inconsistent results between studies that many researchers had expressed a general sense that, at this stage of research at least, markers in plasma do not reflect what is going on in the brain. There was no clear, robust correlation that replicated from study to study. Because cerebrospinal markers do—the CSF is made by the brain’s choroid plexus, after all, and receives a constant influx of interstitial fluid from the brain itself—measurements of that body fluid have taken primacy in the past five years. That seems to be changing again. In Vienna, a number of presentations were testament to renewed efforts toward an eventual blood test for AD. To quote but one example, Jean-Charles Lambert of the Institute Pasteur in Lille, France, reported data from the Three Cities (3C) study in France. In this work, the authors constructed a case-control study from those people among a population-based cohort of almost 10,000 people who had entered this prospective study with a baseline blood test and some of whom then developed incident dementia four years later. At ICAD, Lambert reported that of a range of measures, only the ratio of Aβ42/40 and of truncated Aβn-42/n-40 at baseline were consistently associated with a person’s risk of getting a dementia diagnosis in four years, or, in other words, of having presymptomatic (aka prodromal or incipient) disease at baseline. People with a high ratio had a lower risk for dementia. This was consistent with a similar Dutch study reported previously (van Oijen et al., 2006). Lambert’s presentation focused on the most established biomarkers coming out of the amyloid hypothesis, but other studies are using unbiased approaches to trawl for new markers as well.

Beyond simmering debate about the amyloid hypothesis, the meeting featured several presentations that focused on dementia rates in the oldest old, the fastest growing population segment in some Western societies. One study in particular, by Ugo Lucca of the Istituto Ricerche Farmacologiche Mario Negri in Milan, Italy, punctured the myth that if people manage to dodge cancer or other major diseases in their sixties and seventies, they tend to stay mentally sharp and live surprisingly healthy lives through their eighties, nineties, and even into the 100s. This notion was introduced into the popular imagination by accounts of the centenarian studies in the 1990s, and also by reports of rugged Georgian centenarians who may not have always known their precise birth year but swore by a diet of yoghurt. But this happy notion does not hold up in comprehensive population studies of dementia in the oldest old. Lucca’s study took place in the Varese region north of Milan, Italy, where his colleagues went door to door in eight municipalities, assessed all adults older than 80 and reassessed them after three years. At ICAD, Lucca confirmed previous reports that the prevalence and incidence of dementia doubles relentlessly every five years in old age, to the point where all people in their mid-nineties meet NINDS-ADRDA criteria for dementia. The annual incidence of dementia was 20 percent by this age. Several researchers from France, the U.S., and other countries urged the field at large to pay more attention to AD in the oldest old and to make sure study participants represent the general population. For example, Jean François Dartigues at Bordeaux University, France, emphasized in his talk on the PAQUID prospective study of 3,777 elderly people in the Bordeaux region, that because many more younger AD patients (i.e., in their late sixties and seventies) tend to see doctors and participate in research than do people in their eighties and nineties, much of the available research creates a skewed picture of AD.

In other news, Dessa Sadovnik of the University of British Columbia presented the first report of a presenilin 1 mutation in a large North American aboriginal kindred with early onset familial AD who are living in Canada. Sadovnik emphasized the dilemma of trying to provide counseling, genetic testing, and care services to a population whom Western research understands as being afflicted with a severe form of a genetic disease, but who by tradition view their family’s condition as a spiritual rite of passage into old age that should be respected. Does that mean AD research physicians should leave them be? What kind of contact, support, and information is appropriate and helpful? Other reports at ICAD focused more broadly on the increasing recognition of sporadic AD in indigenous peoples in remote areas in India, Australia, and other countries. A plenary lecture by Gladys Maestre at Columbia University, New York, warned that the dementia prevalence in various Latin American countries is actually higher than in the U.S. for relatively young people, i.e., those in their sixties. This is particularly true among groups with little schooling, who are presumably less able to tap their cognitive reserve and compensate for underlying pathology than are highly literate groups. All these reports highlighted the challenges of furthering dementia awareness and providing care in developing countries. Because life expectancy is rising in many indigenous and developing populations, the number of dementia cases in these groups will inevitably rise, Sadovnik said. For more ICAD stories on specific topics, check this space.—Gabrielle Strobel.

Upper Belvedere Palace, Vienna
Image credit: Marc Aert, Creative Commons Attribution

References

News Citations

Paper Citations

Dubois B, Feldman HH, Jacova C, Dekosky ST, Barberger-Gateau P, Cummings J, Delacourte A, Galasko D, Gauthier S, Jicha G, Meguro K, O'brien J, Pasquier F, Robert P, Rossor M, Salloway S, Stern Y, Visser PJ, Scheltens P. Research criteria for the diagnosis of Alzheimer's disease: revising the NINCDS-ADRDA criteria. Lancet Neurol. 2007 Aug;6(8):734-46. PubMed.
van Oijen M, Hofman A, Soares HD, Koudstaal PJ, Breteler MM. Plasma Abeta(1-40) and Abeta(1-42) and the risk of dementia: a prospective case-cohort study. Lancet Neurol. 2006 Aug;5(8):655-60. PubMed.

External Citations

Vienna: In Genetics, Bigger Is Better—Data Sharing Nets Three New Hits

24 Jul 2009

On the last morning of the International Conference on Alzheimer’s Disease (ICAD), held 11-16 July in Vienna, Austria, investigators from different research groups marked a milestone in AD genetics. They reported results from different genomewide association studies (GWAS) that, thanks to pooling samples by many different laboratories, reached a size where new risk genes of small effect not only showed up with genomewide significance but also, importantly, surfaced repeatedly in several independent GWAS. Not until a gene finding stands up across the board, as ApoE does time and again, does it persuade scientists that it is incontrovertibly true. Last Thursday’s ICAD session breached a barrier of sorts as two groups not only presented data from GWAS that included up to 20,000 samples, but also confirmed each other’s signals. More broadly, the session reflected a shared optimism that growing the sample size for GWAS—both by recruiting more patients and by pooling datasets—has brought them within reach of a list of genes that together account for much of the population’s attributable risk for AD. By upping sample sizes even further past 20,000, some geneticists expect to have a replicated set of genes within the next few years. “Today we saw the best data in a long time. This is an exciting time for complex genetics, which had difficulty this past decade replicating findings. Things are changing and we are beginning to find robust results,” said Julie Williams of Cardiff University in the U.K.

Genetics has been driving AD research ever since it entered the scene. The first wave uncovered rare mutations in APP and presenilins 1 and 2, which account for about 1 percent of cases, plus one common susceptibility gene, ApoE4, which accounts for roughly 15 percent of the population risk, said Philippe Amouyel of Institute Pasteur in Lille, France. Since ApoE’s discovery by Allen Roses at Duke University in 1993, more than 1,200 linkage and association studies have unearthed some 580 additional genetic locations that might contain susceptibility genes. However, initial fanfare about most new proposed genes quickly died down when researchers were unable to replicate a new finding in their own samples. The sample sizes of most everyone’s studies were too small to pick up genes of small effect. To organize and analyze this increasingly unwieldy literature, Lars Bertram, who recently moved from Massachusetts General Hospital to the Max-Planck Institute for Molecular Genetics in Berlin, Germany, developed the science behind, and curates, the publicly available AlzGene database. At ICAD, Bertram noted in his talk that nearly 30 proposed genes show at least one variant with a modestly significant odds ratio when all published studies on them are taken together. AlzGene was the entry point to many genetics talks in Vienna, as groups around the world—besides looking for new genes—are now routinely testing which of the AlzGene Top Results their own studies confirm.

The big push in genetics these days is to conduct GWAS. In these studies, researchers use arrays containing some 20,000 to one million sequence variations called single nucleotide polymorphisms (SNPs). With those they scan a person’s entire DNA for association of any SNPs with disease risk in an unbiased way. In other diseases, for example, diabetes, a trove of new genes were found in large GWAS projects by the Wellcome Trust Case Control Consortium, the Diabetes Genetics Initiative of the Broad Institute/Novartis, and a group coordinated by the National Human Genome Research Institute (Saxena et al., 2007; Zeggini et al., 2007; Scott et al., 2007). In the two years since then, 18 more genes have been published and 31 more are unpublished, according to Williams. These most recent advances came out of meta-analyses and further data merging, which boosted sample size to 55,000 cases. “We need to be thinking about this number of cases to find genes in AD, too,” said Williams.

And the field may be ready to get there. Alison Goate of Washington University, St. Louis, who contributed 800 samples to the British/U.S./German GWAS, put it this way: “We all worked for 10 painful years on our own samples and did not get anywhere. So everyone saw that there was something to be gained from pooling samples.” Earlier this spring, the National Institute on Aging announced that it has awarded nearly $20 million to fund an AD Genetics Consortium. Headed by Jerry Schellenberg at the University of Pennsylvania, Philadelphia, the ADGC is charged with finding the remaining LOAD risk genes in a set of 20,000 case/control samples to which many genetics groups in the U.S. will contribute. “It is important to applaud people who are willing to share their data with other groups to move the overall field forward,” said Williams.

Williams claimed that experience with large GWAS in other diseases has already done away with the myth that these expensive undertakings merely produce a jumble of random genes without meaning. To the contrary, the genes that come up tend to fall into clusters that highlight new biological pathways at play in a given disease. An example from diabetes would be pancreatic β cell regeneration, which came out of the large GWAS. Moreover, this work has also shown that proteins whose genes have merely a small genetic effect can be biologically important, Williams said. An example there is PPARγ. Its odds ratio in diabetes genetics is but 1.14, but its role as the target for some of the insulin-sensitizing drugs prescribed for diabetes leaves no doubt about its biological importance.

In AD research to date, nine GWAS have been published (see AlzGene GWAS page). They are smaller than the new ones reported at ICAD; each group finds ApoE but beyond that mostly weak signals that fail to reach genomewide significance and vary from study to study. This should not be seen as a problem, Williams emphasized. “A smaller GWAS is in no way wasted if it does not find results right away. If we combine these to grow the dataset, we may still nail genes with compelling evidence,” said Williams. Her team’s large GWAS, as well, brought up a smattering of candidate genes below genomewide significance that even larger sample sizes may be able to separate into true hits and false-positives.

On this study, Williams worked with investigators at 11 institutions in the U.K., Ireland, Germany, and the U.S. to pull together the largest GWAS in AD to date (see ARF related news story). In toto, the study included some 20,000 samples. The team screened nearly 5,000 cases and 10,000 aged controls. After quality control, they included some 4,000 cases and 9,000 controls in the first discovery set, which was typed with chips containing some 500,000 SNPs. The top hits from this round were then checked in a separate replication set of 3,000 AD cases and 4,000 controls. Besides ApoE, this study fingered two genes that reached genomewide significance. One is ApoJ/clusterin on chromosome 8. An intron in this gene accounted for 2.3 percent of AD risk in the sample, Williams reported. The other is PICALM, on chromosome 11, and it showed up via a SNP in its 5’ untranslated region. Just below the cutoff for genomewide significance, the researchers spotted 13 variants, many more than the four they had predicted to see by chance. “These probably are false-negatives worth exploring,” said Williams.

One of these runners-up is CR1 on chromosome 1, a gene encoding a complement receptor. CR1 happens to be one of the two major hits of the second-largest GWAS in AD to date, also first presented in Vienna. (The other major hit of that second study was—ApoJ/clusterin.) Presented by Amouyel, this is a European study that included some 2,300 AD cases from the French cities of Lille, Bordeaux, Montpellier, Rouen, Paris, and Dijon, and compared them to 7,000 well-characterized controls from the population-based Three Cities (3C) study. After quality control, about 2,000 cases and 5,300 controls remained for analysis in the discovery sample. The stage 2 replication set included samples from 15 centers in other European countries, including Belgium, Finland, Italy, Spain, and the U.K., and amounted to some 4,000 cases and slightly fewer controls. This study used a chip containing some 600,000 SNPs. “In our studies, no other gene [besides ApoE] has shown an association with AD as strong as CR1 and ApoJ,” Amouyel said in his talk.

Regarding PICALM, Amouyel said that he had heard about it only the night before and that his colleagues were checking for it at present. This gene did, however, pop up in a third, smaller GWAS presented the same morning by Matthias Riemenschneider of the Technical University, Munich. His study used some 1,000 cases and 1.500 controls from Germany for discovery, and 1,300 cases and 1,700 controls from several groups in Australia, Italy, and Sweden for replication. In his talk, Riemenschneider focused on his group’s discovery of TMEM23, a gene encoding a sphingomyelin synthase, and on secondary analyses linking genes to endophenotypes; however, in response to a question from the session chairs about ApoJ and PICALM, Riemenschneider said that his team saw significant associations for those two genes as well.

What do these genes do? Each points to different functions that could fit within the amyloid hypothesis, but could also contribute to LOAD more broadly via lipid metabolism and innate immunity, said Williams. Apolipoprotein J/clusterin is an abundant glycoprotein that occurs in all body fluids; in the brain, astrocytes secrete it. Sometimes called a chaperone, clusterin has been linked to protein clearance in general and Aβ clearance in particular. Genetically, ApoJ/clusterin has been a bit player until now (see ApoJ on AlzGene), but its history in AD research goes back 15 years to its initial description in Blas Frangione’s group (Koudinov et al., 1994). Its role was seen as being intimately tied up with ApoE and with Aβ transport and clearance (e.g., Oda et al., 1995; Zlokovic, 1996; Demattos et al., 2004). Also at ICAD, Madhav Thambisetty from the National Institute on Aging in Baltimore reported on behalf of the London-based AddNeuroMed consortium that ApoJ/clusterin was the only protein that came up as an antecedent plasma biomarker in studies combining plasma proteomics with PIB amyloid imaging in participants of the Baltimore Longitudinal Study of Aging. (This finding still needs replication in independent datasets.) More generally in biomedicine, though, ApoJ is being studied in the context of aging, injury response, and modulation of the innate immune system.

The innate immune system is where CR1 fits in, too. This gene encodes the receptor for proteins of the classical complement pathway, such as C3b/C4b, which mediates clearance of antigen-antibody complexes and other substances through the complement cascade. Complement activation around amyloid plaques has been spotted even during the early wave of interest in inflammation in AD (Eikelenboom and Stam, 1982). It’s not clear yet whether complement activation in AD fuels or slows the disease process. At ICAD, Amouyel said that data gathered to date suggest that CR1 tends to play a protective role via Aβ phagocytosis (e.g., Rogers et al., 2006; Webster et al., 2000). Mouse data, as well, connect complement activation with amyloid deposition (e.g., Maier et al., 2008 and ARF related news story; Wyss-Coray et al., 2002). Beyond that, basic research raises other possibilities for a role of complement in the brain as well. For example, one study has implicated the classical complement cascade in the physiological, activity-dependent pruning of synapses in development, raising the question of whether the loss of synapses in AD could have something to do with complement activation as well (Stevens et al., 2007). For her part, Williams encouraged the audience to take a wider perspective in future efforts at understanding the functional importance of CR1 and the other new genes.

The third new gene, PICALM, is short for “phosphatidylinositol-binding clathrin assembly protein,” and also goes by the alias CALM. It participates in clathrin-mediated endocytosis and intracellular trafficking. This gene and its product are poorly understood, but some previous data hint at a role at synapses. That is because clathrin-mediated endocytosis of APP from the cell membrane affects not only APP’s subsequent cleavage, but also the activity-dependent release of Aβ and Aβ levels in the interstitial fluid (e.g., Cirrito et al., 2008 and ARF related news story; also Rudinskiy et al., 2009). An ongoing collaboration with David Sattelle’s group at the MRC Functional Genomics Unit in Oxford, U.K., at ICAD reported initial findings indicating that PICALM influences plaque deposition in a C. elegans amyloid model, Williams noted.

Readers may wonder where some of the usual suspects fell in the new GWAS data. In the British-international GWAS, APP and the presenilin genes did show up but fell far below genomewide significance. Tau was equally weak, even though it plays a role in the complex genetics of PD (see tau in PDGene; ARF related Prague story). “This is telling us that overproduction of Aβ is not the primary route into LOAD, nor is tau dysregulation,” said Williams. TDP-43 did not show up either, even though it is present upon postmortem pathology in a fraction of AD cases. Ubiquitin was absent as well.

As an aside to the GWAS coverage here, attendees also asked about Tomm40. This gene could not be studied individually in these GWAS, because the Illumina chips lack ApoE itself and scientists actually use Tomm40 as their ApoE marker. Questions about this gene came up repeatedly because earlier in the conference, Allen Roses of Duke University in Durham, North Carolina, had presented data from three separate series of AD patients and controls. The discovery set included 74 patients and 31 controls (with 210 alleles between them); the conformation set included 72 patients and 60 controls (with 264 alleles). For the demonstration of the age of onset of Tomm40 variants connected to ApoE3, Roses used 40 autopsy-confirmed AD patients with the ApoE3/4 genotype whose age of onset had been part of their Duke ADRC record. This work was very different from a GWAS. It used deep sequencing and a phylogenetic analysis of the DNA in the vicinity of ApoE. It set out to explore whether other genes near ApoE could help account for this gene’s extraordinarily large contribution to Alzheimer disease risk and age at onset. Roses reported considerable variation of poly-T variants (long and short forms) of the Tomm40 gene. In people who inherited a long form of Tomm40 along with ApoE3, the age at clinical onset was seven years lower than in people who carried ApoE3 along with less risky short variants of Tomm40. In this way, Tomm40 could help explain why many ApoE3 carriers develop AD, Roses suggested. All Tomm40 variants connected to ApoE4 were long. If confirmed, this finding would mean that combined genotyping of ApoE and Tomm40 might predict a person’s risk and likely age at onset with more precision than do current ApoE tests alone. Roses plans to test this prediction within a diagnostic population-based validation study coupled to a prevention trial for people who are at high risk within the next five to seven years. This project is designed and managed by a startup company called Zinfandel Pharma (see also WSJ story).

Tomm40 is biologically interesting because it encodes a translocase enzyme that mediates protein transport across the outer membrane of mitochondria, which are drawing intense interest in AD research these days. For example, a study by scientists including Eric Reiman at the Banner Alzheimer Institute in Phoenix, Arizona, who donated samples to Roses study, showed that most genes encoding subunits of the mitochondrial electron transport chain were downregulated in neurons taken from regions of the brain whose glucose metabolism declines early on in AD (Liang et al., 2008).

At ICAD, other geneticists were variously intrigued and skeptical about Roses’ study. Some dismissed the idea of two major AD genes occurring so close together as “intelligent design,” and claimed that sequence-independent variation in ApoE expression explains the finding more parsimoniously. Others noted that the small size of the study made it prone to error, and cautioned that the complexity of this genetic locus would make replication challenging. Yet others, however, said that they would follow up the finding and try to tease apart effects of the ApoE alleles and Tomm40 variants.

Wrapping up the last genetics plenary, Williams encouraged a renewed focus on studying the function of all new genes above and just below genomewide significance in GWAS and Tomm40. Based on an initial pathway analysis of these genes, Williams concluded that cholesterol and sterol factors, as well as innate immunity, are primary to disease development. “That is my crucial take-home message. Now we need to know why cholesterol and inflammation are important. We should make them center stage in AD research,” she said.

A lot of work remains. Scientists are already starting to use these new datasets to probe them for links to the presence of psychosis, depression, or vascular risk factors in AD, or to try to link them to biomarker data. Importantly, scientists agreed they need to study expression, epigenetic modification, and function of the genes in the requisite tissues and understand how the AD-linked SNPs change things away from normal. This is easier said than done. After a talk at ICAD describing his efforts to unravel the details of this process for tau, Richard Wade-Martins of the University of Oxford, U.K., said, “We still don’t understand exactly how the tau H1 haplotype leads to increased risk, even though that association was first described in 1997. I hope we won’t say that about ApoJ, CR1, and PICALM in 12 years’ time.”

When all was said and done, John Hardy, a neurogeneticist at University College London, who chaired the session with Christine van Broeckhoven from VIB/University of Antwerp in Belgium, called into the auditorium, “Can we now declare that we have three new susceptibility genes for Alzheimer disease? Lars Bertram, are you here? You be the referee.” Stepping out of the dark, the AlzGene guru said, “I appreciate the authority. I’ll need to see more data, but based on what I saw today, I’d say we are now as close to declaring three new genes for AD as we have been in a long time.”—Gabrielle Strobel.

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

, Saxena R, Voight BF, Lyssenko V, Burtt NP, de Bakker PI, Chen H, Roix JJ, Kathiresan S, Hirschhorn JN, Daly MJ, Hughes TE, Groop L, Altshuler D, Almgren P, Florez JC, Meyer J, Ardlie K, Bengtsson Boström K, Isomaa B, Lettre G, Lindblad U, Lyon HN, Melander O, Newton-Cheh C, Nilsson P, Orho-Melander M, Råstam L, Speliotes EK, Taskinen MR, Tuomi T, Guiducci C, Berglund A, Carlson J, Gianniny L, Hackett R, Hall L, Holmkvist J, Laurila E, Sjögren M, Sterner M, Surti A, Svensson M, Tewhey R, Blumenstiel B, Parkin M, Defelice M, Barry R, Brodeur W, Camarata J, Chia N, Fava M, Gibbons J, Handsaker B, Healy C, Nguyen K, Gates C, Sougnez C, Gage D, Nizzari M, Gabriel SB, Chirn GW, Ma Q, Parikh H, Richardson D, Ricke D, Purcell S. Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science. 2007 Jun 1;316(5829):1331-6. PubMed.
Zeggini E, Weedon MN, Lindgren CM, Frayling TM, Elliott KS, Lango H, Timpson NJ, Perry JR, Rayner NW, Freathy RM, Barrett JC, Shields B, Morris AP, Ellard S, Groves CJ, Harries LW, Marchini JL, Owen KR, Knight B, Cardon LR, Walker M, Hitman GA, Morris AD, Doney AS, , McCarthy MI, Hattersley AT. Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes. Science. 2007 Jun 1;316(5829):1336-41. PubMed.
Scott LJ, Mohlke KL, Bonnycastle LL, Willer CJ, Li Y, Duren WL, Erdos MR, Stringham HM, Chines PS, Jackson AU, Prokunina-Olsson L, Ding CJ, Swift AJ, Narisu N, Hu T, Pruim R, Xiao R, Li XY, Conneely KN, Riebow NL, Sprau AG, Tong M, White PP, Hetrick KN, Barnhart MW, Bark CW, Goldstein JL, Watkins L, Xiang F, Saramies J, Buchanan TA, Watanabe RM, Valle TT, Kinnunen L, Abecasis GR, Pugh EW, Doheny KF, Bergman RN, Tuomilehto J, Collins FS, Boehnke M. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science. 2007 Jun 1;316(5829):1341-5. PubMed.
Koudinov A, Matsubara E, Frangione B, Ghiso J. The soluble form of Alzheimer's amyloid beta protein is complexed to high density lipoprotein 3 and very high density lipoprotein in normal human plasma. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1164-71. PubMed.
Oda T, Wals P, Osterburg HH, Johnson SA, Pasinetti GM, Morgan TE, Rozovsky I, Stine WB, Snyder SW, Holzman TF. Clusterin (apoJ) alters the aggregation of amyloid beta-peptide (A beta 1-42) and forms slowly sedimenting A beta complexes that cause oxidative stress. Exp Neurol. 1995 Nov;136(1):22-31. PubMed.
Zlokovic BV. Cerebrovascular transport of Alzheimer's amyloid beta and apolipoproteins J and E: possible anti-amyloidogenic role of the blood-brain barrier. Life Sci. 1996;59(18):1483-97. PubMed.
Demattos RB, Cirrito JR, Parsadanian M, May PC, O'Dell MA, Taylor JW, Harmony JA, Aronow BJ, Bales KR, Paul SM, Holtzman DM. ApoE and clusterin cooperatively suppress Abeta levels and deposition: evidence that ApoE regulates extracellular Abeta metabolism in vivo. Neuron. 2004 Jan 22;41(2):193-202. PubMed.
Eikelenboom P, Stam FC. Immunoglobulins and complement factors in senile plaques. An immunoperoxidase study. Acta Neuropathol. 1982;57(2-3):239-42. PubMed.
Rogers J, Li R, Mastroeni D, Grover A, Leonard B, Ahern G, Cao P, Kolody H, Vedders L, Kolb WP, Sabbagh M. Peripheral clearance of amyloid beta peptide by complement C3-dependent adherence to erythrocytes. Neurobiol Aging. 2006 Dec;27(12):1733-9. Epub 2005 Nov 14 PubMed.
Webster SD, Park M, Fonseca MI, Tenner AJ. Structural and functional evidence for microglial expression of C1qR(P), the C1q receptor that enhances phagocytosis. J Leukoc Biol. 2000 Jan;67(1):109-16. PubMed.
Maier M, Peng Y, Jiang L, Seabrook TJ, Carroll MC, Lemere CA. Complement C3 deficiency leads to accelerated amyloid beta plaque deposition and neurodegeneration and modulation of the microglia/macrophage phenotype in amyloid precursor protein transgenic mice. J Neurosci. 2008 Jun 18;28(25):6333-41. PubMed.
Wyss-Coray T, Yan F, Lin AH, Lambris JD, Alexander JJ, Quigg RJ, Masliah E. Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice. Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10837-42. PubMed.
Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N, Micheva KD, Mehalow AK, Huberman AD, Stafford B, Sher A, Litke AM, Lambris JD, Smith SJ, John SW, Barres BA. The classical complement cascade mediates CNS synapse elimination. Cell. 2007 Dec 14;131(6):1164-78. PubMed.
Cirrito JR, Kang JE, Lee J, Stewart FR, Verges DK, Silverio LM, Bu G, Mennerick S, Holtzman DM. Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron. 2008 Apr 10;58(1):42-51. PubMed.
Rudinskiy N, Grishchuk Y, Vaslin A, Puyal J, Delacourte A, Hirling H, Clarke PG, Luthi-Carter R. Calpain hydrolysis of alpha- and beta2-adaptins decreases clathrin-dependent endocytosis and may promote neurodegeneration. J Biol Chem. 2009 May 1;284(18):12447-58. PubMed.
Liang WS, Reiman EM, Valla J, Dunckley T, Beach TG, Grover A, Niedzielko TL, Schneider LE, Mastroeni D, Caselli R, Kukull W, Morris JC, Hulette CM, Schmechel D, Rogers J, Stephan DA. Alzheimer's disease is associated with reduced expression of energy metabolism genes in posterior cingulate neurons. Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4441-6. Epub 2008 Mar 10 PubMed.

External Citations

Vienna: Can a D-Peptide Turn Tiger Into Pussycat?

04 Aug 2009

A variety of approaches are beginning to home in on Aβ oligomers as drug targets, and some of them were showcased at the International Conference on Alzheimer’s Disease held 11-16 July in Vienna, Austria. Take Dieter Willbold’s D3 peptide, for example. Willbold is heading institutes at the University of Düsseldorf and the Forschungszentrum Jülich, near Düsseldorf, Germany (see ARF companion story) on oligomer/aggregate detection). In Vienna, he introduced the field to a stable peptide that turns toxic oligomers into an apparently innocuous non-amyloid material and appears to work in mouse models.

Called D3, the peptide came out of a method called mirror-image phage display. In essence, this is an elegant way of obtaining D-enantiomeric peptides that bind to a target of choice, in this case Aβ oligomers. D-peptides are chiral opposites of the more common L-peptides. They appeal to drug developers because unlike L-peptides, they withstand protease degradation in the blood, stomach, and intestine. With mirror-image phage display, scientists screen a large library of phages displaying L-peptides with the D- version of their favorite drug target, and then synthesizes a mirror image of the screen’s best hit. That nets a D-peptide sticking to the L version of the target (for a detailed description of this method and other examples of its use in medical research, see free review).

Willbold’s institutional website features a concise description of two of the peptides obtained in this way. At ICAD, Willbold focused on the 12-mer D3. He first showed using a thioflavin T assay that D3 inhibits Aβ42 fibril formation in vitro. Then he showed that increasing doses of it restored cell viability in the presence of otherwise toxic Aβ42 using an MTT toxicity assay (Shearman et al., 1994). In mice expressing both the APPswe and PS1DE9 mutations, D3 infused directly into the brain with a micropump, or dripped into the drinking water, reduced amyloid deposits in the brain (van Groen et al., 2008) and restored a water maze learning impairment in these mice.

Willbold wondered, How might this work? To address this question, the Jülich team then performed size exclusion chromatography as described previously by Lars Lannfelt and colleagues at Uppsala University in Sweden (Johannson et al., 2006). Lannfelt’s group has poured much effort into optimizing biochemical methods to separate different forms of Aβ in human samples. When Willbold’s team compared the fractions that came off a size exclusion column loaded with either an Aβ sample or the same Aβ sample with D3, the scientists noticed that in the latter, the protofibril fraction was gone. But the monomer fraction was no bigger. “So where was the Aβ? We checked the centrifugation pellet, and there it was,” Willbold told a reporter. The D3 peptide had precipitated the oligomers/protofibrils. With density gradient centrifugation, the result was similar. This method separates Aβ species in a sample by size, and they can then be lined up on SDS PAGE. On the PAGE, the Aβ/D3 sample was missing the middle fractions representing oligomers/protofibrils; most of the Aβ showed up in the highest-molecular-weight fractions.

Precipitation of Aβ in the brain would sound alarming to anyone even vaguely aware of AD research, but this material is different, Willbold emphasized. It is not an amyloid. Besides being non-toxic to cells and the mice, at least, it does not bind thioflavin T, nor does it form fibers or fibrils in the electron microscope, Willbold reported. In a dynamic light-scattering experiment (a biophysical method to measure the size distribution of particles in a given sample), Aβ stayed at one constant size, whereas the Aβ/D3 sample separated after about seven minutes into larger particles of varying sizes. Aileen Funke, a group leader in Willbold's lab, added that in an in-vitro seeding assay, the Aβ-D3 complex was unable to seed fibrillization. In light of these findings, the D3 peptide cannot be considered an example of what were previously dubbed “plaque busters,” Willbold said. Some of these compounds have given rise to products that are themselves either toxic and/or amyloidogenic. “We do not know exactly yet what D3 does to the protofibrils, but the resulting complex is neither toxic nor amyloidogenic,” Willbold said.

This is not the only study targeting oligomers with the help of D-amino acids. At the AD/PD conference last March in Prague, Anat Frydman-Marom won a Young Investigator Award for her work on a β-sheet breaking di-peptide that does much the same thing to Aβ oligomers in vitro and in an APP transgenic mouse. According to a recent article, this di-peptide comprises a D-isomer and a non-chiral amino acid, and behaves like a safe, orally available small-molecule drug (see Frydman-Marom et al., 2009).—Gabrielle Strobel.

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

Shearman MS, Ragan CI, Iversen LL. Inhibition of PC12 cell redox activity is a specific, early indicator of the mechanism of beta-amyloid-mediated cell death. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1470-4. PubMed.
van Groen T, Wiesehan K, Funke SA, Kadish I, Nagel-Steger L, Willbold D. Reduction of Alzheimer's disease amyloid plaque load in transgenic mice by D3, A D-enantiomeric peptide identified by mirror image phage display. ChemMedChem. 2008 Dec;3(12):1848-52. PubMed.
Johansson AS, Berglind-Dehlin F, Karlsson G, Edwards K, Gellerfors P, Lannfelt L. Physiochemical characterization of the Alzheimer's disease-related peptides A beta 1-42Arctic and A beta 1-42wt. FEBS J. 2006 Jun;273(12):2618-30. PubMed.
Frydman-Marom A, Rechter M, Shefler I, Bram Y, Shalev DE, Gazit E. Cognitive-performance recovery of Alzheimer's disease model mice by modulation of early soluble amyloidal assemblies. Angew Chem Int Ed Engl. 2009;48(11):1981-6. PubMed.

External Citations

Vienna: New Shoot Among Ashes of Drug Trials

29 Jul 2009

At the International Conference on Alzheimer’s Disease (ICAD) held 11-16 July in Vienna, Austria, news on the drug trials landscape painted a mostly bleak picture. On Sunday afternoon in particular, as a discouraging string of one negative trial presentation after another unfolded in the main lecture hall, attendees shared, at least at a gut level, a sense of “Ugh, nothing is working.” But of course, that’s not the whole story. For one thing, a much larger number of trials are recruiting, hence, aren’t ready to present data. For another, even amid the rubble of completed trials, ICAD’s truffle seekers sniffed out efficacy signals of new drug candidates at the conference. Consider, for example, a new α7 nicotinic acetylcholine receptor (nAChR) agonist called EVP-6124. A Hot Topics poster on a Phase 1b/2a study with this compound was not listed in the program addendum, but avid conference-goers found it anyway, and a crowd in front of this poster kept attracting people, including a reporter. Here’s a summary.

The poster was the first publication on safety, tolerability, and some early hints of efficacy, for this compound in people with Alzheimer disease. The candidate drug is a selective small-molecule agonist made by the biotech company EnVivo Pharmaceuticals of Watertown, Massachusetts. In recent years, the α7 nAChR has been enjoying renewed interest as a target—either for agonists or antagonists, depending on whom you ask—in AD (see ARF related news story for background). In an interview, coauthor Gerhard Koenig of EnVivo Pharmaceuticals said that their team had previously established efficacy and potency of this compound in various animal models. Led by Dana Hilt of EnVivo, four different Phase 1 studies in some 125 healthy volunteers preceded the trial presented at ICAD. According to the Phase 1 studies, those pesky pharmacokinetic and dynamic details that don’t interest basic scientists too much yet are the downfall of so many a candidate drug appear to bode well for this one. The compound is bioavailable in the brain when taken by mouth, capsules of it can be taken once a day, and it shows no gender, age, or food effects; in other words, people need not plan their capsules around their meals. In normal volunteers, the compound was safe and well tolerated, and even then gave some hints of enhancing cognition, Koenig said.

Moreover, this compound has survived a Phase 1b trial in 20 people with schizophrenia, in whom it partially normalized evoked potentials. This is not a clinically relevant test—rather, it is an electrophysiological biomarker to indicate that the drug is active in the brain. Study volunteers have fitted to their heads electrodes which record brain activity in response to certain stimuli, for example, spaced tapping noises. People with schizophrenia have a characteristic deficit in their sensory gating that can be measured with evoked potentials, and EVP-6124 appeared to ameliorate that. The company hopes that this drug might one day treat certain cognitive deficits in schizophrenia and Alzheimer disease (see biotech newsletter story).

On the ICAD poster, then, were new data on a trial of 48 people aged 50 to 80 with mild to moderate AD, who were randomized to take either placebo or one of three doses of EVP-6124 for 28 days. The scientists first observed patients for three days in an inpatient unit, then kept them under observation for the first few days on drug/placebo before letting them finish the drug course in an outpatient setting. All trial participants had been taking stable doses of the acetylcholinesterase inhibitor (AChEI) donepezil or rivastigmine for at least four months before joining the trial. The scientists emphasized this point, because a frequent criticism of α7 agonists holds that their effects are likely to be so similar to current AChEIs that they will only work in people who do not already take these drugs. “All effects seen here are on top of stable AChE inhibition,” Koenig said. For comparison, a second Phase 1b/2a trial of this compound in AD patients who do not take AChE inhibitor is being wrapped up at present, Koenig added.

In the trial presented in Vienna, primary endpoints were safety, secondary endpoints were pharmacokinetic measures of EVP-6124 and AChE drug to check for drug-drug interaction, and cognition tests made for tertiary endpoints. On safety, Hilt and colleagues report that there were no specific adverse events they interpreted as being related to treatment or dose, nor any serious adverse events. The researchers found no evidence of drug interaction. On efficacy, the trial recorded positive effects on some of the tasks contained in either the CogState or NTB batteries used in this trial. In particular, the identification and detection tasks of CogState, as well as the COWAT, CNT, and Trail Making Tasks of the NTB appeared to show a dose-dependent response, the scientists report on the poster. These tasks measure attention, verbal fluency, and executive function. The effect grew over the course of treatment. The scientists don’t know if this short trial reached the maximum drug effect, nor whether the effect would track over time like that of other symptomatic AD drugs. For that, longer trials will be necessary. CogState (which is similar to CANTAB) is a commercial, computerized cognitive test battery; the NTB is a set of paper-and-pencil tests developed largely by scientists at Elan/Wyeth Pharmaceuticals, who claim these tests are particularly sensitive for measuring cognition in mild AD (Harrison et al., 2007). AD clinicians increasingly use these measures in the belief that they pick up slight changes faster than does the ADAS-cog.

“These are first signs of a pro-cognitive response,” said Koenig. “But it’s important to be cautious. This was a small, first trial in AD patients. We do not yet know if and how these effects will translate into ADAS-cog or clinically relevant endpoints,” he added.—Gabrielle Strobel.

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

External Citations

Vienna: New Tack to See Amyloid Oligomers in Body Fluids

03 Aug 2009

It’s not just about dimers, dodecamers, and protofibrils anymore. With its wide range of presentations on the topic of small aggregates of Aβ (and α-synuclein, for that matter), the International Conference on Alzheimer’s Disease made it plainly obvious that the interest in these tiny culprits has branched out beyond the initial debates about whether they even exist and which forms are important. Held 11-16 July, ICAD showed that, in particular, the twin challenges of detecting small aggregates in human body fluids and of detoxifying them (see ARF companion story) are drawing new labs and new techniques to the field. Here is a summary of some selected examples; as always, the Alzforum welcomes reader comments on similar approaches.

Several groups are working on detecting oligomers of Aβ in body fluids. Some of those are developing methods other than ELISA, which in the past has been the staple of Aβ measurement. To quantify Aβ as a clinical biomarker, Xmap multiplex technology is increasingly replacing conventional ELISAs, which have long been dogged by great variability between labs. But to pinpoint oligomeric Aβ specifically, here’s something completely different.

At ICAD, Susanne Aileen Funke, working with Dieter Willbold at the Forschungszentrum Jülich, near Düsseldorf, Germany, presented a method that detects only Aβ aggregates, not its monomers. What’s more, it does so with a sensitivity down to a single aggregate, Funke said. In clinical AD research, it’s by now widely accepted that CSF Aβ42 levels plummet before a person develops AD. This biomarker has entered diagnostic practice in some centers, and has even become an inclusion criterion in a clinical trial of a new γ-secretase inhibitor. This concerns the Aβ monomer, however. Many scientists believe its drop-off reflects amyloid deposition in the brain, whereas others suspect that it might reflect a corresponding increase of aggregated Aβ in CSF (Englund et al., 2009). This implies that other forms of Aβ could be found in CSF besides the monomer if only scientists had a robust search method.

This is where Funke comes in. She is applying to Aβ detection a method called Surface-FIDA (short for fluorescence-intensity-distribution-analysis), which was originally developed for prion aggregate detection by fluorescence correlation spectroscopy (FCS). With this method, Funke immobilizes the aggregates contained in a few microliters of CSF onto a glass surface coated with an anti-Aβ capture antibody. Then she decorates them with two separate monoclonal antibodies, and lets a confocal microscope beam scan the surface to count the resulting fluorescence bursts. The trick is that only aggregates attract enough antibodies to give rise to a fluorescent burst; the monomers that are also present in the CSF sample do not. To preclude more than one antibody binding to monomer, the two different decoration antibodies target overlapping epitopes on Aβ.

“We can detect a single aggregate. That can never be done by ELISA. With ELISA, you detect a summarized signal of all aggregates in one well,” said Funke. In her talk, Funke showed three ways of validating that monomeric Aβ does not emit fluorescence bursts. She cross-correlated the signals from each decoration antibody, showed a linear dose response correlation, and eliminated the fluorescence bursts from an aggregate-spiked Aβ sample by adding the denaturing agent SDS.

In a first attempt to test the raw assay on clinical samples, CSF from three AD patients indeed appeared to contain more aggregates than control CSF (Funke et al., 2007). Since then, Funke and Willbold have improved the assay. It was also adapted to confocal imaging with laser scanning microscopes, which are more abundant than FCS devices. Moreover, this new version of the assay is able to determine the composition of each aggregate (e.g., Aβ40 and Aβ42) and robustly measures in the picogram range, Funke showed.

How does FIDA compare to ELISA? There is no publication on this yet, but Funke said that in a side-by-side experiment of the same clinical CSF samples, the ELISA test showed a decrease of Aβ42 in AD whereas FIDA showed an increase in oligomers/aggregates. In a separate ICAD presentation, Seong An at Kyungwon University, working with SangYun Kim at Seoul National University, both in the Republic of Korea, reported a similar approach using multiple antibodies whose epitopes overlap in order to distinguish oligomers from monomers. This group does not use FIDA, but they, too, reported seeing an increase of Aβ aggregates in CSF of AD patients. This group is actively investigating Aβ oligomer detection in blood.

In terms of moving the FIDA system toward clinical application, Funke said the next steps are to optimize the assay for CSF and for blood. Once that’s done, her team needs to test it on larger numbers of well-characterized clinical samples. She also wants to check which kinds of aggregates occur in CSF and blood of healthy people and patients—are they made of Aβ40, 42, or perhaps the pyroglutamated Aβ3-42? The scientists plan to see if the size, number, or composition of such aggregates changes with age and as people approach disease, and whether this test can predict that a person will develop AD. For a detailed description of Surface-FIDA and a publication list, see the institute’s website.

Funke adapted this detection method to Alzheimer disease after learning it initially from Eva Birkmann at the same institute (see institute website). Birkmann had developed Surface-FIDA for the detection of single prion particles. Her laboratory is presently adapting it to do so in CSF and plasma of cattle and sheep, as well as in the human prion disease Creutzfeld-Jakob disease. Most of currently available tests for the diagnosis of prion diseases in animals exploit the proteinase K resistance of PrPSc. In an e-mail to ARF, Birkmann wrote that her lab has not yet compared that established test and Surface-FIDA side by side. The hope is that, eventually, Surface-FIDA might provide a more accurate tool for human and veterinary diagnosis.

Regarding new collaborations, Birkmann recently teamed up with Brit Mollenhauer at the Paracelsus-Elena Klinic in Kassel, who has co-developed an ELISA to quantify monomeric α-synuclein in CSF of patients with PD and DLB. Their goal is to see if Surface-FIDA can detect oligomeric α-synuclein in this fluid, too. That such oligomers are there seems likely not only by analogy to AD. Prior hints exist as well. For one, Omar el-Agnaf at United Arab Emirates University in Al Ain, UAE, has reported seeing them with an ELISA both in brain extracts and CSF of PD (see ARF related Prague conference story). For another, Walter Schulz-Schaeffer at the University of Goettingen at ICAD presented an update to his previously published PET blot method (Kramer et al., 2007). This simple method reveals an overwhelming abundance of small α-synuclein aggregates in brain sections of people who had Parkinson’s or DLB, whereas neighboring brain sections stained conventionally may reveal at best a few scattered Lewy bodies or Lewy neurites. Other clinically relevant brain areas contain the small aggregates but no Lewy pathology at all. According to Schulz-Schaeffer, small α-synuclein aggregates are the species to watch. If you can see them.—Gabrielle Strobel.

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

Paper Citations

Englund H, Degerman Gunnarsson M, Brundin RM, Hedlund M, Kilander L, Lannfelt L, Pettersson FE. Oligomerization partially explains the lowering of Abeta42 in Alzheimer's disease cerebrospinal fluid. Neurodegener Dis. 2009;6(4):139-47. PubMed.
Funke SA, Birkmann E, Henke F, Görtz P, Lange-Asschenfeldt C, Riesner D, Willbold D. Single particle detection of Abeta aggregates associated with Alzheimer's disease. Biochem Biophys Res Commun. 2007 Dec 28;364(4):902-7. PubMed.
Kramer ML, Schulz-Schaeffer WJ. Presynaptic alpha-synuclein aggregates, not Lewy bodies, cause neurodegeneration in dementia with Lewy bodies. J Neurosci. 2007 Feb 7;27(6):1405-10. PubMed.

External Citations

Vienna (and Burkina Faso): What's New With Methylene Blue?

30 Jul 2009

The old drug methylene blue generated instant curiosity among AD researchers when it burst on the scene as RemberTM with Phase 2 trial results at last year's International Conference on Alzheimer's Disease (ICAD) in Chicago, and was subsequently featured on CNN and other national media (for background, see ARF related news story). Many scientists took it as the first hopeful signal that—maybe, just maybe—inhibiting tau aggregation might actually work in people with Alzheimer disease. So what has happened with methylene blue research in the year since then? This story presents a reporter's update from this year's ICAD conference, held 11-16 July in Vienna, Austria, together with an extended Q&A with the developer of RemberTM, Claude Wischik of the University of Aberdeen, U.K., and the biotech company TauRx. Alongside this update, the Alzforum is pleased to bring to the table an outside perspective from an expert on the biochemistry and pharmacology of methylene blue, and its use as a treatment for other indications. Heiner Schirmer is professor at the biochemistry center at Heidelberg University in Germany. Besides having coauthored a leading textbook on protein structure, Schirmer has published extensively on the structure and biochemistry of parasitic flavoenzymes, some of which bind methylene blue. Schirmer is presently in Burkina Faso. There he is working on a field study sponsored by the German government on using methylene blue as an inexpensive and readily available treatment for malaria in rural Africa (for a recent report, see Meissner et al., 2006). The Alzforum editors would like to thank Schirmer, as well as Eckhard Mandelkow at the Max-Planck Unit for Structural Molecular Biology in Hamburg, who invited Schirmer to brief Alzheimer disease researchers on methylene blue's 120-year history as a drug. See Schirmer essay.

Top image: Green crystals of glutathione reductase (a yellow enzyme) with bound methylene blue. Bottom image: Crystal structure of same. Image credit: Heiner Schirmer

At ICAD in Vienna, Wischik discussed further analysis of the same Phase 2 trial of RemberTM, his biotech company's patented formulation of methylene blue. Wischik noted in his talk that the company had patented a new form called leuco-methylthioninium or LMT, which is no longer blue and renders the drug more bioavailable and less toxic at higher doses. For their part, Schirmer's group had characterized a reduced white version of methylene blue (called leucoMB or methylene white) as a possibly superior form of this drug for use in a colorless drug syrup to treat malaria (see Buchholz et al., 2008 and Schirmer essay). TauRx's new formulation is presently undergoing preclinical studies. For a detailed first-person account of what happened with the high dose of RemberTM in the Phase 2 trial and related topics, see Q&A with Wischik below.

Intrigued by the apparent effect in humans, a growing number of research groups are now exploring methylene blue, a phenothiazine compound, in various model systems. Of those, few are ready to report at meetings quite yet, but here is a summary of what's public so far. In Vienna, Christian Haass of the Ludwig Maximilians University in Munich, Germany, presented data straight off the bench from his group's new transgenic zebrafish model system for neurodegeneration. As reported this past March (see ARF related Prague story, play neuron death clip [Acridine uptake and death of P301L-expressing tau neuron] from end of story, and see Paquet et al., 2009), fish embryos expressing mutant human tau rapidly develop signs of neurodegeneration. In this model, Dominic Paquet and Bettina Schmid in Haass' group characterized a series of readouts that range early on from abnormal (PHF1) tau phosphorylation followed by defective neurite outgrowth, a motor phenotype, widespread neuronal death, and tangle pathology. First signs of toxicity show up 32 hours after fertilization, individual tau-laden neurons die by 60 hours after fertilization, and extensive neuron loss is visible after six days. The researchers found tangles later, by five weeks of age. At ICAD, Haass described tests of methylene blue in this model.

Frauke van Bebber in the Haass lab treated the embryos from four hours to 20 hours after fertilization with non-toxic concentrations of 10 to 100 micromolar methylene blue, some of which ends up being absorbed by the fish. The fish turned blue, hence the study was not blinded. “Any investigator studying methylene blue knows precisely what he or she is analyzing,” Haass noted in his talk. In this study, the drug did not alter the abnormal PHF1 phosphorylation of mutant human tau that occurs in the untreated transgenic fish. The scientists take PHF1 phosphorylation as an indirect measure of aggregation and tangle formation, in part because testing for tangles directly with Gallyas staining in older fish is too late and elaborate for drug studies, Haass noted by e-mail. The German scientists next checked whether methylene blue rescued either tau-induced cell death, stunted neurite growth, or the sluggish swimming of the transgenic fish. It did not, Haass said. The model itself is responsive to drug effects, however. For example, some of a series of new GSK3β inhibitors developed by AstraZeneca do reduce abnormal PHF1 phosphorylation by up to 70 percent, Haass reported.

“At this point we wondered whether our methylene blue preparation was inactive,” Haass said in his talk, so the team next tested the drug on a different zebrafish strain, one that models Huntington disease. These fish express fluorescent-coupled huntingtin with an expanded polyglutamine repeat and accumulate aggregates of the transgene in their neuronal nuclei. When treated with methylene blue, the aggregates did not form. “Methylene blue wiped out aggregate formation of httQ102GFP,” Haass said. Next, the scientists looked for effects on toxicity, as this particular transgene is highly toxic, killing the fish. Methylene blue did not block the toxicity of httQ102GFP.

Haass interpreted these findings to mean that methylene blue does inhibit aggregation of tau, as published (Wischik et al., 1996), but that this inhibition is unable to prevent tau toxicity. “I believe that soluble oligomers are the toxic species of tau and huntingtin. It appears that this drug affects species that show pathology but may not be the toxic ones,” Haass said. This finding taps into a fluid area of tau research. Some in-vivo multiphoton imaging studies in transgenic mutant tau mouse models point in a similar direction by suggesting that tau tangles are not what cause neurons to die (e.g., de Calignon et al., 2009; for a recent review on tau and neurodegeneration, see Spires-Jones et al., 2009), but tau aggregation inhibitors are nonetheless drawing renewed interest in academia and drug development (e.g., see Bulic et al., 2009).

Another presentation at ICAD took methylene blue research in a different direction. On a poster, Takashi Nonaka of the Tokyo Institute of Psychiatry, Japan, presented his group's latest data on TDP-43. Working with Haruhiko Akiyama and Masoto Hasegawa, Nonaka has been establishing cell-based models of TDP-43 aggregation. This protein has rapidly moved front and center of molecular studies on amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD), because intracellular aggregates of TDP-43 mark the pathology of these diseases and TDP-43 mutations have turned up in familial ALS and mixed ALS with FTLD-U. Few TDP-43 animal models are quite ready for primetime just yet. In the interim, the Japanese scientists devised two neuroblastoma lines that express either a TDP-43 deletion mutant missing its nuclear localization signal or an aggregation-prone, fluorescent-tagged C-terminal fragment. Both cell lines form intracytoplasmic aggregates that resemble aggregates seen in patients with these diseases in that they are insoluble to detergent and abnormally phosphorylated. The scientists used these cells to screen for drug effects, and they came up with a combination of none other than Dimebon and methylene blue. These are both available drugs whose safety record could accelerate their use in trials in ALS or FTLD-U. (For frontotemporal dementia, ClinicalTrials.gov at present lists active trials for only one drug, memantine, whose effect is known to be modest, at least in AD.)

Nonaka and his colleague Makiko Yamashita found that treatment with 50 nanomolar methylene blue reduced the number of cellular inclusions by about half compared to untreated cultures. (Dimebon had about the same effect at 5 micromolar.) When given together, both drugs reduced aggregates by 80 percent. The effect was concentration-dependent and occurred in both cell lines. Two other phenothiazine compounds, chlorpromazine and perphenazine, which do not affect tau aggregation, did not affect TDP-43 aggregation in these cell lines, either. The transgenic cells do not appear to die from the TDP-43 inclusions, though TDP-43 is abnormally phosphorylated and its normal, unphosphorylated form is somewhat depleted in the nucleus. Each drug, but especially both of them together, reduces this abnormal phosphorylation, Akiyama wrote in an e-mail to ARF. The scientists suggest that a combination of these drugs be considered for clinical testing in ALS and FTLD and have begun early efforts in this direction (Yamashita et al., 2009; Nonaka et al., 2009; Nonaka et al., 2009).

Together, then, current research suggests that methylene blue blocks aggregation of several different proteins involved in neurodegeneration—tau, TDP-43, polyQ huntingtin, and previous research has shown at least in-vitro effects on Aβ and α-synuclein aggregation as well (e.g., Necula et al., 2007). The Japanese drug study measured aggregation, not toxicity or functional outcomes in vivo, hence gives no clues as to whether blocking TDP-43 aggregation might be beneficial in humans. A rat model of TDP-43, and mouse, fly, or zebrafish models, may be amenable to this question. Several labs in the field are buckling down on developing TDP-43 animal models; for the latest status, see ARF related London story. The Haass lab has a zebrafish line expressing TDP-43 at physiological levels, but because this line does not so far form aggregates, the scientists have not tried methylene blue in it, Haass wrote by e-mail.

With methylene blue, it's not all about aggregation, either. As a drug, it is “dirty.” In pharmacology, this is not nearly as iffy a label as in gastronomy. It simply means a drug may bind to many different targets (see Schirmer essay), not all of which scientists necessarily understand equally well. Dimebon, too, falls into this category, as do some NSAIDs. In the past year, research groups interested in oxidative damage and mitochondrial protection have taken an interest in methylene blue and tested it in various animal models. These scientists did not present at ICAD, but briefly, a Brazilian group reported in the journal Neurochemistry International that injecting methylene blue into the striatum protected rats against induced seizures (Furian et al., 2007). And this year, researchers led by Francisco Gonzalez-Lima at the University of Texas, Austin, reported that methylene blue protected the rat striatum and the retina against injections of the Parkinson disease-related neurotoxin rotenone, possibly by way of propping up mitochondrial energy metabolism (Rojas et al., 2009; Rojas et al., 2009).—Gabrielle Strobel.

Q&A with Claude Wischik. Questions by Gabrielle Strobel.

Q: Are people giving methylene blue to their loved ones?
A: Yes, I am worried that this has the potential to become a serious problem. We have received numerous requests for advice regarding the use of methylene blue. People seem to think that if they find a supplier of methylene blue, they could in principle get a hospital or pharmacy to formulate it for them and then administer it either alone or with the help of a physician. We have heard of a case like this in the U.K., and we understand that the police had to be called in to deal with a report arising from the local health authority. But we have also heard of examples in the U.S.

Q: What's the worry?
A: This is not an approved product. People taking it into their hands to give it to their loved ones are acting irresponsibly. The due regulatory processes have been set up for good reasons, to ensure safety and efficacy of pharmaceuticals, and these apply very much in the case of methylene blue for reasons I will now discuss.

Methylene blue is a relatively crude dyestuff which has various industrial applications. The actual pharmacopoeial substance is methylthioninium chloride (MTC), the chloride salt of the methylthioninium moiety. MTC is not available in a pure pharmaceutical grade anywhere for treatment of AD and related disorders apart from what we have produced for clinical trials. There are several distributors of a form of methylene blue who claim to meet U.S. pharmaceutical standards. However, the standard they quote is very old and was defined by 1970s technology, which would not generally be considered appropriate for a pharmaceutical substance today. The batches of this material that we have tested are impure in regard to MTC itself. They contain manufacturing contaminants, and high levels of heavy metals. We therefore do not advocate their use as a pharmaceutical product, however desperate people may be.

TauRx has gone to great lengths to develop processes whereby MTC can be produced at greater than 99 percent purity. We have obviously patented these processes, which are published as WO06/032879. The material produced by us has very low levels of heavy metal contaminants and meets modern standards for a pharmaceutical product. We believe that the material produced by us would be acceptable to regulatory authorities for Phase 3. I believe the commonly available form of methylene blue is unlikely to satisfy current regulatory standards that apply in the EU or U.S. for administration of pharmaceutical substances to humans.

The next issue concerns the formulation. We found in our Phase 2 clinical trial that unless the formulation is right, even high doses of MTC (e.g., 100 mg 3x/day), whilst having little efficacy, can produce side effects, particularly on red blood cells and diarrhea. I spoke at the recent ICAD in Vienna about our analysis of this data. We now understand why the 30 mg and 60 mg doses showed a simple dose-response relationship in terms of efficacy, but the 100 mg dose did not.

Q: In brief, why?
A: The formulation of MTC we used in the RemberTM Phase 2 trial was a semi-solid gelucire suspension in a gelatin capsule in which different dosage strengths were produced by adjusting the fill-weight during manufacture. For the 100 mg dose, the capsules were near to maximum fill, whereas the 30 mg and 60 mg doses had free head space when the suspension set on cooling in the capsule. MTC was found to cross-link gelatin in the region of contact over time. For the 30 mg and 60 mg doses, this cross-linking did not matter because the capsules could be readily breached via regions of the capsule not in contact with MTC. But breach of the 100 mg capsule was delayed, particularly in simulated gastric fluid, but there was an adequate dissolution profile in simulated intestinal fluid. This led to differential dissolution and absorption according to dose. In effect, the 100 mg capsule was a delayed-release formulation, whereas the 30 mg and 60 mg doses were nearer to rapid release formulations.

We assumed naively going into the trial that it did not matter whether the MT moiety was absorbed via the stomach or the small intestine. In light of the trial data, we developed a simple mathematical model which appears to explain both the beneficial cognitive effect and the adverse hematological effect on the basis of differential absorption of two species according to dissolution time in vitro. This two-species model fits all of the available clinical data with a correlation coefficient of 0.997. The model suggests that absorption of the MT moiety in the reduced form is the main determinant of clinical efficacy, and that this in turn depends on rapid dissolution. However, delayed breach of the 100 mg dose appears to lead to preferential absorption of the MT moiety in the oxidized form from the small intestine, possibly as an uncharged dimer, which forms readily in vitro at high concentration. This species has low clinical efficacy and a high tendency to oxidize hemoglobin.

Q: You also reported at ICAD that you have developed a new, colorless formulation of MTC?
A: Yes. This follows directly from the mathematical analysis I described above. The formulation is critical as the absorption of MT is a complex process, something we had to learn the hard way from the RemberTM Phase 2 trial. This is fully explained in our published patent WO07/110627. When administered as MTC, the MT moiety is presented as the chloride salt of the oxidized form of MT. In order for MT to be absorbed as the beneficial monomer, it has first to be reduced to the uncharged form (i.e., leuco-MT, or LMT), which is able to cross the gut lining and enter the blood. We believe this reduction process is probably enzyme-mediated, and occurs most favorably at the low pH of the stomach. We believe from our experimental work and analysis of our Phase 2 data that it is the LMT form which carries the activity required to retard the progression of AD. The oxidized form is the predominant form absorbed if the formulation is not right (as, e.g., with our 100 mg capsule), and it has the potential to give low efficacy coupled with increased side effects.

We have discovered a process to produce a pure LMT form that can be administered as a tablet. Yes, it is substantially colorless (actually, a light yellowy-green). We have found that when administered orally to animals, this LMT form enhances availability of the active moiety in the brain while reducing toxicity.

Q: Is there going to be a Phase 3 trial?
A: In the near future, we should be ready to conduct Phase 3 with either of the two pure forms of MT that we have developed, but most likely the new LMT form. The final decision as to which form of MT is to be adopted has to await completion of ongoing studies and further discussions with the relevant regulatory authorities.

TauRx is also planning studies in other closely related neurodegenerative disorders which have similar tau pathology to AD, i.e., progressive supranuclear palsy and corticobasal degeneration. We have also had some preliminary evidence of efficacy in a case of frontotemporal dementia with a primary tau mutation, so a clinical trial in FTD is also a likely option.

Q: When and where?
A: In addition to the ongoing PK studies, the further timeline critical issue is completion of relevant higher species toxicology, and regulatory views on this can differ according to jurisdiction. Studies are ongoing with both forms of MT. We plan to start Phase 3 about mid-2010 after these studies have been completed and reviewed by the regulatory authorities. We are planning to undertake Phase 3 in the EU and U.S. with either form. We are also in the midst of a financing round to support Phase 3 and welcome investment enquiries.

Q: Is a manuscript of the full Phase 2 data in preparation/submitted/in press at a peer-reviewed journal?
A: We do plan to submit the RemberTM Phase 2 trial data for peer-reviewed publication in due course. As I discussed at the recent ICAD in Vienna, we have conducted a relatively large disease-modifying trial by Phase 2 standards, and this provides many useful insights into the problems that are inherent in the conduct of such trials in AD. However, we also have to be aware that the overriding imperative is to complete due regulatory process required for us to be able to conduct a Phase 3 trial to confirm our Phase 2 findings. This trial will necessarily require some subjects to receive either placebo or a relatively ineffective dose. Our expert advice is that early peer-reviewed publication of data, which might appear to support off-label use of an impure and inappropriately formulated methylene blue, could work against our ability to conduct such a trial. As I explained above, this is already a potential problem even after a simple conference presentation. This is an important concern and may require us to delay publication until well into Phase 3. Myriad and Medivation both appear to have followed a similar course. We don't rule out earlier publication, and the matter is under constant review.

There is a high level of skepticism about Phase 2 trial data in general in AD irrespective of publication, and there is a further layer of skepticism towards a tau-based approach arising from widely held β amyloid preconceptions. What the field is really waiting for is robust Phase 3 confirmation of any promising Phase 2 efficacy signal for a new disease- modifying approach. The fundamental TauRx objective is to confirm at Phase 3 that a treatment based on targeting the tau aggregation cascade with RemberTM will achieve control over disease progression in AD.

References

News Citations

Paper Citations

Meissner PE, Mandi G, Coulibaly B, Witte S, Tapsoba T, Mansmann U, Rengelshausen J, Schiek W, Jahn A, Walter-Sack I, Mikus G, Burhenne J, Riedel KD, Schirmer RH, Kouyaté B, Müller O. Methylene blue for malaria in Africa: results from a dose-finding study in combination with chloroquine. Malar J. 2006;5:84. PubMed.
Buchholz K, Schirmer RH, Eubel JK, Akoachere MB, Dandekar T, Becker K, Gromer S. Interactions of methylene blue with human disulfide reductases and their orthologues from Plasmodium falciparum. Antimicrob Agents Chemother. 2008 Jan;52(1):183-91. PubMed.
Paquet D, Bhat R, Sydow A, Mandelkow EM, Berg S, Hellberg S, Fälting J, Distel M, Köster RW, Schmid B, Haass C. A zebrafish model of tauopathy allows in vivo imaging of neuronal cell death and drug evaluation. J Clin Invest. 2009 May;119(5):1382-95. PubMed.
Wischik CM, Edwards PC, Lai RY, Roth M, Harrington CR. Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):11213-8. PubMed.
de Calignon A, Spires-Jones TL, Pitstick R, Carlson GA, Hyman BT. Tangle-bearing neurons survive despite disruption of membrane integrity in a mouse model of tauopathy. J Neuropathol Exp Neurol. 2009 Jul;68(7):757-61. PubMed.
Spires-Jones TL, Stoothoff WH, de Calignon A, Jones PB, Hyman BT. Tau pathophysiology in neurodegeneration: a tangled issue. Trends Neurosci. 2009 Mar;32(3):150-9. PubMed.
Bulic B, Pickhardt M, Schmidt B, Mandelkow EM, Waldmann H, Mandelkow E. Development of tau aggregation inhibitors for Alzheimer's disease. Angew Chem Int Ed Engl. 2009;48(10):1740-52. PubMed.
Yamashita M, Nonaka T, Arai T, Kametani F, Buchman VL, Ninkina N, Bachurin SO, Akiyama H, Goedert M, Hasegawa M. Methylene blue and dimebon inhibit aggregation of TDP-43 in cellular models. FEBS Lett. 2009 Jul 21;583(14):2419-24. PubMed.
Nonaka T, Kametani F, Arai T, Akiyama H, Hasegawa M. Truncation and pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43. Hum Mol Genet. 2009 Sep 15;18(18):3353-64. PubMed.
Nonaka T, Arai T, Buratti E, Baralle FE, Akiyama H, Hasegawa M. Phosphorylated and ubiquitinated TDP-43 pathological inclusions in ALS and FTLD-U are recapitulated in SH-SY5Y cells. FEBS Lett. 2009 Jan 22;583(2):394-400. PubMed.
Necula M, Kayed R, Milton S, Glabe CG. Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem. 2007 Apr 6;282(14):10311-24. PubMed.
Furian AF, Fighera MR, Oliveira MS, Ferreira AP, Fiorenza NG, de Carvalho Myskiw J, Petry JC, Coelho RC, Mello CF, Royes LF. Methylene blue prevents methylmalonate-induced seizures and oxidative damage in rat striatum. Neurochem Int. 2007 Jan;50(1):164-71. PubMed.
Rojas JC, Simola N, Kermath BA, Kane JR, Schallert T, Gonzalez-Lima F. Striatal neuroprotection with methylene blue. Neuroscience. 2009 Oct 20;163(3):877-89. PubMed.
Rojas JC, John JM, Lee J, Gonzalez-Lima F. Methylene blue provides behavioral and metabolic neuroprotection against optic neuropathy. Neurotox Res. 2009 Apr;15(3):260-73. PubMed.

Vienna: New Genes, Anyone? ICAD Saves Best for Last

References

News Citations

Paper Citations

External Citations

Further Reading

News

Vienna: In Genetics, Bigger Is Better—Data Sharing Nets Three New Hits

Comments

Make a Comment

References

News Citations

Paper Citations

External Citations

Further Reading

News

Vienna: Can a D-Peptide Turn Tiger Into Pussycat?

Comments

Make a Comment

References

News Citations

Paper Citations

External Citations

Further Reading

News

Vienna: New Shoot Among Ashes of Drug Trials

Comments

Make a Comment

References

News Citations

Paper Citations

External Citations

Further Reading

News

Vienna: New Tack to See Amyloid Oligomers in Body Fluids

Comments

Make a Comment

References

News Citations

Paper Citations

External Citations

Further Reading

News

Vienna (and Burkina Faso): What's New With Methylene Blue?

References

News Citations

Paper Citations

Other Citations

External Citations

Further Reading

News