San Diego: Newt Gingrich Calls on Neuroscientists to Advocate for Funding

05 Nov 2007

The Alzforum coverage of the 37th annual conference of the Society for Neuroscience, which is currently in full swing at the San Diego Convention Center, kicks off with a story about engaging scientists in a national conversation about the promise and the funding needs of brain science. Former Speaker of the House Newt Gingrich today told some 2,500 scientists that they have a civic duty to talk with their elected representatives. Gingrich called on the best minds of the country to devote time to educating their elected representatives about what they are doing. After his talk, the speaker told this reporter that Alzheimer disease is a priority to him, and that Congress needs to hear a clear vision from stakeholders—including, importantly, the research community. To this end, Gingrich has initiated the Alzheimer's Study Group under the umbrella of the Center for Health Transformation, a leadership group he founded to explore ways of modernizing health systems. The ASG grew out of a bi-partisan Congressional task force on Alzheimer disease. The group is charged with devising a national strategy for dealing with the projected AD epidemic of 12 to 15 million cases by the year 2050. The group is to present its findings in late spring of 2008. This effort provides an opportunity for the intertwined issues of Alzheimer disease research funding, service provision, and drug testing, to get the attention of Congress and lead to new policy.

“We have an excellent opportunity in brain science now that is not being matched by a vision of breakthrough of the scale that is possible, nor by the scale of funding that you need to achieve those breakthroughs,” Gingrich told ARF. In the interview, the former Speaker was well informed about aspects of translational AD research. For example, he said that the FDA operates on rules built on antiquated science. The regulatory framework guiding clinical trials is, in practice, erecting unreasonable hurdles to efforts to test new drugs, he said. When asked about appropriate levels of risk for clinical trials in AD, Gingrich said that an over-reaching risk averseness in AD can prove to be more of an obstacle to progress than a safeguard for vulnerable patients. In particular, families and patients who know full well that they are facing a deadly disease should not be shut out from the process. Gingrich said that early-stage dementia patients, as well as patients with ALS and other neurodegenerative diseases, are effective spokespeople for more and better therapy development, and that he shares their concerns. “We need to take a fresh look at our risk tolerance for these neurologic diseases,” he said. Gingrich also expressed his support for legislation, presently stalled in the Senate, intended to protect people with genetic diseases from insurance and employment discrimination (Genetic Information Nondiscrimination Act). “The Senate should just pass it as is,” he said.

Gingrich’s plenary lecture started with short videos. In one, Patrick Kennedy, U.S. House of Representatives, Rhode Island, spoke openly of his life with bipolar disorder, and how alcohol and prescription drug abuse accompanied his struggle to deal with the periods of depression and anxiety, in particular. Gingrich then addressed the audience, saying that he understands there have been dramatic advances in biomedical science, but not all politicians do. He urged scientists to change that. “If you work in a field of extraordinary importance to humanity, then you have a civic duty to educate your elected representatives. If you devoted a small portion of your time to communicate about biomedical opportunities and priorities, you would move the country forward. The people who know have a moral responsibility to the people who don't know. Your role as a citizen is as important to society as your role as scientists. If you are too busy to talk to your elected representatives, then you have to quit griping about the ignorant decisions they make,” Gingrich said.

Gingrich called the decline in NIH funding “very inappropriate” and supported massive increases in the budgets of NIH and NSF as essential to continue U.S. leadership in science. He encouraged all forms of communication—writing letters to the editor, calling and e-mailing Congress(wo)men, calling in to talk radio, attending town hall meetings—and insisted in response to a skeptical question from the audience that this does indeed create momentum and moves the system.

In a question-and-answer session, Gingrich faced an audience that was at turns friendly and adversarial. When asked about stem cell research, he said that he supports federal funding only for research that does not require destruction of embryos, and that state and private initiatives now fund stem cell research more broadly. When asked about the influence of neo-creationism/intelligent design on science education in the nation’s schools, he said: “I have no problem with creationism being taught as philosophy and every problem with it being taught as science.” Gingrich argued for radical changes to the way math and science are taught in school, claiming that current methods simply are not working. He urged scientists to welcome adolescents into their labs for internships much more frequently. In response to a critical question about current science policy, he merely noted: “I long ago quit trying to explain this administration.”—Gabrielle Strobel.

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San Diego: Society for Neuroscience Conference in Broad Strokes

09 Nov 2007

To a bird’s eyes, the San Diego convention center for the past 5 days would have looked like a mile-long human beehive. A swarm of 32,000 scientists and exhibitors exchanged information on 16,000 presentations, making the main belly of the building hum as discussions resonated across some 60 double rows of posters. The neurodegeneration buzz? Stay tuned as your Alzforum team distills it into news stories over the next few weeks.

Overall, the 37th annual conference of the Society for Neuroscience, held 3-7 November, showed convergence in some areas of AD research and divergence in others. One trend was a preponderance of presentations on oligomeric forms of the Aβ peptide. Labs are beginning to compare side-by-side the different forms described over the past few years. One group described their isolation of human oligomers from AD brain that were reportedly able to poison synapses, giving a boost to the disease relevance of these protein aggregates. There was a sense of consolidation around the notion that Aβ oligomers likely play a significant role in the cognitive deficits of people with AD, as a growing number of laboratories independently reported data from a variety of angles that were consistent with the central idea. At the human genetics level, there was even a report of a mutation in APP that causes a form of Aβ which rapidly forms oligomers but never forms fibrils at all, yet its carriers reportedly had clinical AD.

More broadly, Aβ and increasingly also tau research this year attempted to drill down into the synaptic biology of AD, picking up with an intense focus the historic observation that synaptic dysfunction, followed by synaptic loss, correlates better with cognitive symptoms than do plaques. The intraneuronal signaling pathways implicated are varied and bewilderingly complex, but Erk, GSK3, Wnt, Akt, Fyn, and calcineurin are a familiar presence in those studies.

By contrast, the emerging field of APP trafficking is at an even younger stage. Scientists agree that the retromer complex doing the trafficking is of central importance in AD, making AD at heart a disease of cell biology. But beyond that the field is presently generating a host of data that diverge in their details as research groups use different cell types and methodology. This field is still wide open and will likely converge again around several major pathways once more is known and labs have established methods to explore the process in the most directly disease-relevant model systems. Progress is also being made, albeit slowly, in figuring out the normal physiological function of APP, something that has so far proven elusive.

The technical wall that for some years had bloodied the noses of drug developers racing to make BACE inhibitors appears to have come down. Compounds that reliably lower levels of Aβ in spinal fluid are being presented, and several pharmaceutical companies are said to be refining their candidates and moving toward human tests. The relationship between Aβ toxicity and elevated BACE activity has also come in for closer scrutiny, and researchers are beginning to dissect the nature of BACE trafficking, stabilization, and transcriptional regulation.

Intriguingly, the tau and even α-synuclein fields are showing signs of following Aβ’s act in the sense that they, too, may prove to be more toxic in their early states of aggregation than in their classic pathological incarnation as tangles and Lewy bodies, respectively. Incidentally, if anyone thinks that tau tangles are unique to humans, they may want to know that a report of a chimp with abundant tangles did away with this dogma.

γ-secretase continued to surprise with a complex range of effects. New observations include a proposed role in mediating the benefits of enriched environments, and a role in controlling calcium streams out of the endoplasmic reticulum at the base of dendritic spines where the synapses sit. Presenilin FAD mutations appear to preclude these benefits and unleash calcium torrents that blunt excitability, respectively.

A growing number of presentations tried to nail down molecular mechanisms for the established overlap among risk for insulin-resistant diabetes mellitus, elevated cholesterol, and risk for dementia. The deplorably small trickle of studies on the biggest genetic risk factor for AD, i.e., ApoE, appeared to be picking up a bit with reports on ApoE’s novel effects on neprilysin- and IDE-mediated aggregation, as well as research of ApoE’s lipidation state, the role of other lipoprotein receptors in amyloidogenic APP processing, and the potential promise of LXR receptors. A number of laboratories are attempting to build integrative models of molecular processes of aging, and the anti-aging gene klotho entered the AD scene. Mitochondrial deficits show up in different guises across a wide range of studies.

A parallel trend in the offing is that many labs are trying to study AD models at ever-earlier stages, well before pathology settles in the brain. This is part of an effort not only to understand oligomers, but as importantly to get a handle on the defense mechanisms that synapses, neurons, and networks are mounting for some time before the disease process overwhelms these attempts to keep the system in balance.

The number of presentations on experimental treatments reported to have effects in mouse models is growing rapidly. Candidates that got attention this year include caffeine, available drugs to lower blood pressure, and the fish oil DHA. Combined with studies on research drugs not fit for human consumption, these data lead to the common frustration that if you are a mouse, scientists know of a host of ways of curing your "AD,” yet none of them have quite translated to humans yet. A more optimistic take might be that never before have drug companies and public drug-testing consortia had such a great number of compounds for AD tests in the clinic. The bottleneck lies not in targets or drug candidates, but rather in ways to diagnose AD earlier, and in biomarker-driven designs that make trials shorter, better, cheaper, and allow more drugs to be tested than is possible now.

Research continues apace on other neurodegenerative diseases that are of interest to AD researchers. We learned of problems and progress in relating pathology to different presentations of primary progressive aphasia, of advances in understanding the function and dysfunction of glia and how they may be exploited to treat spinal cord injury and motor neuron disease, and of the role of kinases in Parkinson disease.

On a lighter note, the AD Social proved to be the rowdiest affair of all socials hosted this past Tuesday night. No offense to the rest of neuroscience, but the Synapse Social could have used some theta bursts, the Auditory Signaling Processing Social never revved up the decibels, and the Thalamus Social was a little soporific when compared to the second AD karaoke night. Researchers trilled and warbled with varying levels of talent but with unstinting good humor, and those who did not even know the songs that they were being dragged to perform availed themselves of the lip-synch fake they originally cultivated in fifth-grade chorus. To the paparazzi who took snapshots of our esteemed colleagues, send them to gabrielle@alzforum.org. We will post the pleasantly embarrassing photos (with permission) and keep the truly mortifying ones on file for future arm-twisting purposes.—Gabrielle Strobel and Tom Fagan.

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San Diego: Getting a Grip on Glia, Part 1

14 Nov 2007

This is Part 1 of a two-part meeting report from the 37th annual meeting of the Society for Neuroscience, held 3-7 November, in San Diego. See Part 2.

A mini-symposium at this year’s annual meeting of the Society of Neuroscience devoted attention to the role of glia in brain injury and disease. Chaired by Clive Svendsen, University of Wisconsin, Madison, the symposium covered the gamut from glial cell signaling to therapeutic approaches to Parkinson disease (PD), ALS, and spinal cord injury.

The simple idea that glia are passive bystanders, offering help whenever neurons get into trouble, no longer passes muster. Scientists now know that these cells do a whole lot more. They encourage axon growth, prevent glutamate excitotoxicity, provide trophic and metabolic support, and even help maintain the blood-brain barrier. Glial function and dysfunction has also been implicated in neurologic diseases such as schizophrenia, and neurodegenerative disorders such as amyotrophic lateral sclerosis (see ARF related news story), multiple systems atrophy (see ARF related news story), and Alzheimer disease (see ARF Live Discussion). Glia buffs even go as far as assigning Albert Einstein’s intellectual prowess to the factoid that a major difference between his brain and that of everyone else is reportedly an increase in astrocytic processes (see Colombo et al., 2006). In short, glia are pretty hot these days, even making the cover of this month’s Nature Neuroscience, which devotes a special focus to the role of these cells in disease.

Michael Sofroniew, University of California at Los Angeles, started the symposium, entitled “The Role of Glial Cells in Brain Injury and Disease,” by detailing some of his lab’s recent studies on astrocyte signaling. Sofroniew’s lab is studying the role of astrocytes in response to injury, which is relevant to AD given that astrogliosis is a prominent feature of the disease. There is currently considerable debate about whether reactive astrocytes are beneficial or harmful. Reactive astrocytes form glial scars in response to brain injuries, such as contusions. Do these scars merely impede neuronal regeneration or do they have positive roles, as well? Sofroniew suggested that it would be highly unlikely that such a process would have arisen and been conserved through evolution if it was purely detrimental, and said that his experimental data seem to support that view, as well.

His lab uses transgenic models to study glial scarring. In normal scarring, reactive astrocytes line up in a palisade manner, surrounding a central necrotic lesion that is often packed with inflammatory cells. Sofroniew was interested in what might happen if he disrupted this pattern. Because proliferation seems to be an important step in the astrocyte response, his lab abolished astrocyte cell division. The scientists did this by generating transgenic mice harboring a thymidine kinase (TK) gene driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. In the TK-positive cells, ganciclovir, a thymidine analog and antiviral agent, becomes phosphorylated when the TK gene gets turned on, and the analog then disrupts DNA replication. Astrocytes that are not activated by injury are unaffected.

When the researchers used these mice in a contusion model of brain injury, they found that the glia scar no longer formed properly, nor did the distinct glial boundary around necrotic lesions. The downstream consequences of this change were that the volume of the necrotic lesion increased about threefold and the inflammatory cells—no longer contained—spread much farther from the necrotic site. The astroglial quiescence also had functional repercussions. While normal animals recover locomotive function after a moderate contusion, TK transgenic animals treated with ganciclovir performed poorly in a rotarod test of balance and strength, Sofroniew reported.

What kind of cell signaling controls this glial response, and how might it impact other cells? Sofroniew speculated for the sake of discussion that some of the signals astrocytes send out to limit immune cell responses might also prevent axon regeneration in diseases such as multiple sclerosis. This means that understanding astrocytic signal transduction might offer new ways to stimulate neuronal regeneration, he suggested. Astrocyte signaling, however, is still a bit of a black box. Numerous signal transduction pathways have been suggested to be involved, including those linked to protein kinase C, Notch, Smad, TNFα, and G-protein coupled receptors. For his part, Sofroniew has begun studying the role of the Jak/Stat signaling pathway. His coworkers use the Cre/lox system under the control of the GFAP promoter to make conditional knockouts (cKOs) of the stat3 gene in mouse astrocytes. Data yet to be published show that stat3-negative astrocytes appear normal in healthy animals, but after crush injury their response is compromised. The usual hypertrophy does not set in, and neither does induction of GFAP and vimentin, genes that kick in in wild-type animals in response to injury. The palisade boundary between astrocytes and the necrotic lesion does not form in stat3 cKOs either, and immune cells, as detected by CD45 immunoreactivity, flood out from the lesion site, causing a marked increase in inflammation. These mice also don’t recover function as well as wild-type.

What relevance do astrocyte responses in general, and their proliferation in particular, have for human disease? To approach this, Sofroniew and colleagues turned to experimental autoimmune encephalomyelitis (EAE), a common model of multiple sclerosis. In a poster session, Scott Peterson from Sofroniew’s lab reported that limiting astrocyte proliferation exacerbated the EAE. When he induced EAE in TK transgenic animals, their clinical severity score rose to ~2.5 after 3 weeks, but in animals also treated with ganciclovir, scores rose to ~4.5. As in animals with contusion injury, the morphology of glial scars was compromised, and there was a statistically significant increase in the number of CD45 positive cells in gray and white matter. Axonal pathology also seemed exacerbated, because levels of neurofilament 200, which drop by about half in EAE, were even further depleted in the anterior and lateral funiculi. It is worth noting that Adriano Aguzzi and colleagues at University Hospital, Zurich, Switzerland, using the very same TK/ganciclovir methodology, found that suppression of microglia actually protected against EAE (see Heppner et al., 2005). Together, the findings indicate that different types of glia can have distinct and apparently contrary roles.

These findings suggest that glial scarring is protective in at least two forms of neuronal injury—trauma and autoimmune disease. But astroglia can clearly contribute to disease as well. Work from Don Cleveland’s group at University of California, San Francisco, has shown, for example, that mutant superoxide dismutase, which causes familial forms of ALS, can lead to motor neuron disease in mice when expressed only in glia (see ARF related news story). How, then, do glia propagate disease? This question captivates Jeffrey Rothstein of Johns Hopkins University, Baltimore, Maryland. Rothstein noted that a major function of astrocytes is to scavenge glutamate, which can otherwise accumulate to neurotoxic levels in synapses. Astrocytes are armed with an array of glutamate transporters, also called the excitatory amino acid transporters (EAAT). But not all astrocytes are equally well endowed, noted Rothstein. His lab found that astrocytes in the cortex and hippocampus have 10-fold higher levels of EAAT2 than do spinal cord astrocytes. Increased thalamic EAAT3 levels have also been linked to schizophrenia. And work from Eliezer Masliah’s lab at the University of San Diego, California, suggests that EAAT2 is downregulated in AD brain and in transgenic mice expressing human APP under control of the Thy1 promoter (see Li et al., 1997 and Masliah et al., 2000). ALS patients have a dramatic loss of EAAT2, said Rothstein. The astrocytes are there, but are not expressing the protein.

To investigate the relevance of EAAT2 changes, Rothstein used bacterial artificial chromosome (BAC) transgenic animals to measure the expression of glutamate transporters. In late-stage motor neuron disease in rodent models, his group found a focal loss of Glt-1, the rodent homolog of EAAT2. The loss seems due to decreased Glt-1 mRNA levels, and Rothstein speculated that some microRNA inhibitors may be at work.

And what about other astrocyte pathways? For example, glia provide neurons with lactate. To facilitate this transfer, both astrocytes and neurons have membrane-bound monocarboxylate transporters (MCTs). The cells have different isoforms, however, with neurons expressing MCT2, while astrocytes express MCT1 and MCT4. Rothstein has looked at MCT expression in rodent models of ALS. He reported that in the spinal cord there is almost a complete loss of MCT1 in diseased animals, while MCT2 and MCT4 remain unchanged. To test whether this is a cause of effect of disease, the scientists used antisense RNA to individually ablate MCT expression in spinal cord cultures, and they found that losing MCT1 leads spinal cord neurons to die. The scientists tested postmortem tissue from ALS patients and found that they, too, have a loss of MCT1. The findings suggest that damage to glia might compromise their crucial metabolic support role, leading to neuronal death.

Rothstein is currently exploring ways to exploit these discoveries for therapeutic purposes. A screen for compounds that elevate EAAT2 turned up mostly beta-lactam antibiotics, which were published to increase expression of Glt-1 and increase survival in ALS mouse models (see ARF related news story). However, Rothstein was challenged in question time by a researcher who noted that this result has been difficult to reproduce. Rothstein suggested that the effect is not robust and that he is currently trying to improve on the EC50 by making structural analogs. He also said that other groups using these antibiotics found improvement in different models of glutamate-related diseases, such as depression, stroke, and epilepsy. Clinical trials of a different antibiotic for ALS have been a disappointment. Rothstein also reminded the audience astrocytes can communicate via gap junctions across long distances. He showed a video of a calcium wave propagating through astrocytes for hundreds of microns. It is possible that one sick astrocyte could spell disaster for many of its neighbors.—Tom Fagan.

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References

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Colombo JA, Reisin HD, Miguel-Hidalgo JJ, Rajkowska G. Cerebral cortex astroglia and the brain of a genius: a propos of A. Einstein's. Brain Res Rev. 2006 Sep;52(2):257-63. PubMed.
Heppner FL, Greter M, Marino D, Falsig J, Raivich G, Hövelmeyer N, Waisman A, Rülicke T, Prinz M, Priller J, Becher B, Aguzzi A. Experimental autoimmune encephalomyelitis repressed by microglial paralysis. Nat Med. 2005 Feb;11(2):146-52. PubMed.
Li S, Mallory M, Alford M, Tanaka S, Masliah E. Glutamate transporter alterations in Alzheimer disease are possibly associated with abnormal APP expression. J Neuropathol Exp Neurol. 1997 Aug;56(8):901-11. PubMed.
Masliah E, Alford M, Mallory M, Rockenstein E, Moechars D, Van Leuven F. Abnormal glutamate transport function in mutant amyloid precursor protein transgenic mice. Exp Neurol. 2000 Jun;163(2):381-7. PubMed.

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San Diego: Getting a Grip on Glia, Part 2

15 Nov 2007

This is Part 2 of a two-part meeting report from the 37th annual meeting of the Society for Neuroscience, held 3-7 November, in San Diego. See Part 1.

Cell-based therapies have the potential for treating a variety of diseases including diabetes, leukemia, and even neurodegenerative diseases such as Parkinson and Alzheimer disease. But while replacing worn out pancreatic or hematopoietic cells may be relatively straightforward, getting new neurons to seamlessly slip into pre-existing circuitry may be a bit like sending an understudy into the last act of a play without one ever having read the lines. Might there be a better way to keep the show on the road? At this year’s Society for Neuroscience annual meeting, Clive Svendsen, University of Wisconsin, Madison, suggested that for neurodegenerative diseases such as ALS and Parkinson disease (PD), it may be unnecessary to replace the lost neurons if the neurons that are still there can be protected. Svendsen was chair of a mini-symposium entitled “The Role of Glial Cells in Brain Injury and Disease.”

There have been several attempts at shoring up neurons in humans, but none were successful. A recent trial for GDNF in Parkinson disease was perhaps the most disappointing because initial signs had been promising (see ARF related news story). Svendsen avoids the complications that plague some delivery systems, such as pumps and live viruses, by using ex-vivo techniques to turn glial cells into mini-protein factories. Other advantages of this approach are that the glial cells alone may be therapeutic and the delivery system can be tested in vitro before implantation. With Patrick Aebischer, at the Swiss Federal Institute of Technology in Lausanne, Svendsen has developed a way to differentiate human neural precursor cells into predominantly astroglia and engineer them to release GDNF. The researchers tried this system in rodent and primate models of PD and have seen some improvement in function (see Behrstock et al., 2006). They have also tried the same approach in ALS models but were less successful, Svendsen said. Even though the cell transplants survived, spread into areas of degeneration, and almost completely protected against neuronal loss, the animals’ function did not improve (see Suzuki et al., 2007). The outcome was poor probably because the treatment failed to protect the neuromuscular junction. Svendsen showed how α-bungarotoxin staining revealed loss of the muscle endplate, and he is now working on strategies to deliver a cocktail of GDNF, IGF-1, VEGF, and BDNF via mesenchymal cells to the neuromuscular junctions. So far this has protected about 50 percent of the junctions, and improved function. Svendsen noted that cell-based strategies may work better in a slowly progressing disease, such as PD, than in more rapidly deteriorating diseases such as ALS. This is because the transplanted glia take time to mature. Even though cells injected into the brain diffused into the striatum in the PD models, it took 120 days before GFAP showed up there. In the case of ALS, targeting both the spinal cord and the muscle may achieve the best results.

One other potential use of cell therapy lies in spinal cord injury repair. Mary Bunge, University of Miami, Florida, described how transplanted Schwann cells, under varying conditions, can support spinal cord repair. The idea is that when axons die back after injury, they leave behind a tunnel of Schwann cells. If the axons can be coaxed to grow back, they may re-establish their previous connections. Since Schwann cells provide not only myelin to ensheath axons, but also a variety of neurotrophic factors, matrix proteins, and cell adhesion molecules that promote axon growth, transplanting these cells to sites of injury could prove beneficial. Strategies based on a single therapeutic approach, i.e., transplanting Schwann cells alone, have proven unsuccessful. At present, the scientists are experimenting with combinations of multiple approaches to get better results. These include providing Schwann cells in a growth-supportive matrix, or matrigel, limiting the formation of scar tissue by delivering chondroitinase to the site of injury, coaxing axons to travel through the site of injury by adding olfactory ensheathing glia (OEG) to the distal side (these cells promote axon elongation), and activating neurons by transfecting with transgenes or Schwann cell-derived neurotrophins.

Bunge reported that the combination of Schwann cells, OEG, and chondroitinase led to an increase in myelinated nerve fibers within grafts at the site of spinal cord transection, and to significant improvement in locomotion. Her lab also found that axon growth into the injury site can be stimulated by transfecting the spinal cord with AAV virus carrying MEK and ERK kinase constructs for expression in neurons.

Spinal contusion is a more common form of injury in humans than complete transection. Contusions in animals are usually followed about 12 weeks later by formation of a cyst at the site of injury, but it can be reduced, and function improved by transplanting Schwann cells 1 week after injury. To build on this, Bunge’s lab has genetically modified Schwann cells to produce a bi-functional neurotrophin called D15A. This is a mutant form of the human neurotrophin NT-3. Substituting aspartic acid 15 with alanine confers both TrkB and TrkC receptor binding, so the protein effectively mimics both NT-3 and BDNF. Six weeks after transplantation of D15A-expressing Schwann cells into rat spinal cord, the graft volume was about fivefold bigger than that elicited by untransformed cells, and there were more myelinated axons and the serotonergic and sensory fibers were longer. Unfortunately, the strategy did not improve locomotion, probably because the axons failed to migrate through the graft to the distal side. One strategy to overcome this problem might be as simple as inducing cAMP signaling, Bunge said. Infusing cAMP and the phosphodiesterase inhibitor rolipram into the site of damage along with Schwann cells was better than cell therapy alone. The numbers of myelinated fibers increased, serotonergic fiber grew into and beyond the graft, and that correlated with improved function.

Glia are not the first things that come to mind when one thinks of Alzheimer disease; nevertheless, understanding their contributions to both the healthy and diseased central nervous systems could lead to a better grasp of pathogenesis and, potentially, novel therapeutic approaches. The November issue of Nature Neuroscience features additional review articles on the role of glial cells in demyelination, ischemic toxicity, regulation of the microvasculature, and neuropathology—all areas that impinge in some way on age-related dementia. So do microglia and the brain’s oligodendroglia, but these cell types unfortunately got little attention at this symposium.—Tom Fagan.

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Behrstock S, Ebert A, McHugh J, Vosberg S, Moore J, Schneider B, Capowski E, Hei D, Kordower J, Aebischer P, Svendsen CN. Human neural progenitors deliver glial cell line-derived neurotrophic factor to parkinsonian rodents and aged primates. Gene Ther. 2006 Mar;13(5):379-88. PubMed.
Suzuki M, McHugh J, Tork C, Shelley B, Klein SM, Aebischer P, Svendsen CN. GDNF secreting human neural progenitor cells protect dying motor neurons, but not their projection to muscle, in a rat model of familial ALS. PLoS One. 2007;2(8):e689. PubMed.

San Diego: Oligomers Live Up to Bad Reputation, Part 1

16 Nov 2007

Over the past decade, researchers have shifted away from a literal interpretation of Alois Alzheimer’s groundbreaking discovery of plaques and tangles as the likely cause of Alzheimer disease. After years of argument—mostly in the 1990s—about whether plaques or tangles were the culprit, the answer appears to be, “Both and neither.” How can that be true? Scientists have recognized that both constituent proteins of those hallmark pathologies—the amyloid-β (Aβ) peptide and tau—play essential roles in the development of the disease, relegating the Baptist-Tauist divide solidly to the past. That’s the “both” part. But scientists also increasingly agree that the microscopically visible protein deposits are not the worst offenders: hence, the “neither.” Instead, they blame smaller, oligomeric forms of the Aβ peptide that they believe exist in a complex equilibrium with higher-order protofibrils along a path to aggregation. These, they say, damage synapses and interfere with cognitive function. In short, they say plaques are bad, but oligomers are worse. For tau, this story isn’t nearly as far along, but trends suggest that it may well develop along similar lines. (And ditto for α-synuclein.)

The Society for Neuroscience conference, held 3-7 November in San Diego, was a testament to how deeply the science of Aβ oligomers has taken hold in the field. There were some 35 presentations about Aβ species variably called oligomers, ADDLs, AβOs, or protofibrils. Speakers increasingly cited the “Amyloid Oligomer Hypothesis” rather than the “Amyloid Hypothesis” in the introductory slide of their talk. Indeed, a range of presentations from a diverse group of labs reported data largely concurrent with its essential tenet that AD begins with synaptic dysfunction caused by soluble Aβ species. Here are selected highlights.

Perhaps the most direct support came from Ganesh Shankar, an M.D.-Ph.D. student working with a team of colleagues in Dennis Selkoe’s laboratory and Cindy Lemere at Brigham and Women’s Hospital, Boston, Dominic Walsh’s group and Ciaran Regan’s group, both at University College in Dublin, Ireland, and with Bernardo Sabatini at Harvard Medical School. In a sparsely attended slide session on the last afternoon of the conference, Shankar expanded on what a poster presented by Shaomin Li from the same team had foreshadowed days before. The scientists isolated soluble Aβ species from cortex of human AD brain, and report that oligomers as small as a dimer recapitulated the synaptotoxic effect the scientists had previously published for similar small oligomers secreted by cultured cells.

Prior studies from several laboratories have consistently found synaptotoxic effects for various forms on Aβ oligomers (e.g., Walsh et al., 2002—from conditioned media of 7PA2 Chinese hamster ovary cells; Lambert et al., 1998—from synthetic Aβ42; Lesne et al., 2006—from Tg2576 mouse brain). Yet these studies begged the question of how relevant to human Alzheimer disease all this can be until human Aβ oligomers are in hand. To address this question, Shankar and colleagues obtained postmortem cortical tissue from several patients with late-onset AD (one of whom had had no clinical AD but pathological AD upon autopsy). As controls, the scientists used cortex from patients with Lewy body dementia (LBD—they get parkinsonism and dementia at about the same time and are thought to have mixed pathologies), Down syndrome (who have typical AD-type amyloid pathology), and frontotemporal and multi-infarct dementia (who do not). Readily detectable soluble Aβ showed up in cortex from all clinically demented AD patients but not in one cognitively normal person who had the plaque pathology. It also showed up in the Down brain, and to a much smaller extent in the LBD brain. Curiously, soluble extracts from normal control brains appeared to contain very little or no soluble monomeric Aβ by this immunoprecipitation/Western blot assay, even though the brain presumably produces some all the time.

These AD cortical extracts were made merely in TBS buffer without detergent, and they showed primarily monomer at a weight of 4 kDa and dimer at 8 kDa. Extracts made in parallel with detergent also had monomer and dimer in them. Shankar showed experiments suggesting that besides the dimer, soluble Aβ extract from human AD brain also contains complexes having a larger molecular weight—either Aβ aggregated with itself or bound to other proteins—but that these fall apart upon treatment with detergent. This is a technical difference with studies on ADDLs and Aβ*56, both of which are reported to be SDS-stable. Shankar said that his colleagues and he searched for SDS-stable species in the human extracts but so far have been unable to find any that are larger than trimer. Shankar and colleagues used detection by two antibodies that detect the free N- and C-terminus of Aβ, respectively, and also used mass spectrometry, to ascertain that the dimers contained true Aβ, and to exclude any other Aβ-containing APP cleavage fragments that might be contained in the extracts.

Next, the scientists applied their preparations to tests of LTP and spine integrity that they had developed previously. The TBS extracts from AD brains blocked LTP induction, whereas extracts from the other diseases and age-matched controls did not, Shankar reported. (The Down’s extract was not tested.) Immunodepletion of Aβ restored LTP, meaning the effect was specific to Aβ. The effect was potent, acting in the picomolar range. By enriching Aβ through immunoprecipitation, eluting with SDS buffer and then running on size exclusion chromatography, only the fraction enriched for Aβ dimers inhibited LTP significantly; the monomer had no effect. The soluble AD brain extract also facilitated long-term depression, reducing neuronal excitability after a period of stimulation. The main point, Shankar said, is that Aβ dimer extracted from human AD brain is sufficient to disrupt the molecular basis of learning and memory. It is not the only form, but the smallest form that can be toxic.

Various anti-Aβ antibodies are in clinical trials at present, and one debate in the field revolves around which type of antibody might be most potent. Shankar and colleagues indirectly addressed this debate by testing which of the three classes of anti-Aβ antibody used in those trials—N-terminal, mid-region, C-terminal—was best able to rescue the detrimental effect on LTD of the human AD extract. In a subtraction experiment, where the investigators selectively depleted the extract with only one kind of antibody, N-terminal antibodies best protected LTP, Shankar reported. This electrophysiology result concurred with associated biochemistry, in that the N-terminal antibodies also captured the most Aβ from the extract. (Not all immunotherapy clinical trials, however, are based on the premise of directly counteracting Aβ oligomers in brain; some aim to draw down Aβ from the periphery, or target Aβ more generally.)

Beyond LTP and LTD, do these human oligomers really matter to the structure of synapses? There is strong consensus in the field that synapses in AD-relevant brain areas gradually decrease in number early on as people develop cognitive symptoms (Davies et al., 1987; Scheff et al., 2007). At Neuroscience, Shankar showed evidence that the human AD oligomers reduced the density of dendritic spines in cultured brain slices in much the same way as cell-secreted oligomers do (Shankar et al., 2007). Furthermore, Shankar showed data on a rat behavioral test. The human AD extract impaired learning in a passive avoidance paradigm. It did so when infused 3 hours after the rats had initially learned, the period that other studies have identified as the time when synapses undergo remodeling following learning.

Finally, the researchers reported taking a hard crack at the Aβ dimers. Acting on a hunch that the dimers might represent a seed for plaque formation, the team isolated mature, cored plaques and removed as many associated components from them as possible by repeated washes in detergent and TBS buffer. This left behind insoluble, microscopic cores that stained with Congo red. These cores did not inhibit LTP. They were very hardy, but when the scientists blasted them apart with highly concentrated formic acid, Aβ dimers were released, and those did inhibit LTP. Taken together, these investigators interpret their data to mean that soluble Aβ oligomers from typical AD patients, starting with dimers, disrupt synaptic function in humans, and that insoluble cores sequester these species. As to plaques, they represent a reservoir of soluble Aβ in a given brain region, Shankar said. For their part, the dimers would seem to be a relevant substrate for both research into the molecular pathways of synaptic impairment, and also for testing prospective therapeutic agents preclinically, Shankar added.

Other labs need to replicate these findings. When asked whether the recipe for isolating the human oligomers was technically difficult, he replied: “No, it’s pretty much standard biochemistry. But rigorous clinical and histopathological information on the patients should be available before attempting it, so close interaction with a brain bank is key.”—Gabrielle Strobel.

This is Part 1 of a three-part story. See Parts 2 and 3.

References

News Citations

Paper Citations

Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 2002 Apr 4;416(6880):535-9. PubMed.
Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6448-53. PubMed.
Lesné S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH. A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006 Mar 16;440(7082):352-7. PubMed. RETRACTED
Davies CA, Mann DM, Sumpter PQ, Yates PO. A quantitative morphometric analysis of the neuronal and synaptic content of the frontal and temporal cortex in patients with Alzheimer's disease. J Neurol Sci. 1987 Apr;78(2):151-64. PubMed.
Scheff SW, Price DA, Schmitt FA, Dekosky ST, Mufson EJ. Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. Neurology. 2007 May 1;68(18):1501-8. PubMed.
Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, Sabatini BL. Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci. 2007 Mar 14;27(11):2866-75. PubMed.

San Diego: What Goes Around Comes Around—Cell Cycle in Postmitotic Neurons

16 Nov 2007

Could the failure of cell cycle regulation be an underlying cause of neurodegenerative disorders, including Alzheimer disease? High school biology teaches that the cell cycle, in which one mature cell divides into two daughter cells, is essential for the replication of eukaryotic cells. So how might cell cycle proteins influence neurons? After all, these long-lived cells are considered to be amitotic. On Saturday, 3 November, scientists convened at the 37th annual meeting of the Society for Neuroscience in San Diego, California, to address the diverse roles of cell cycle molecules and to discuss their impact on postmitotic neurons in a symposium dedicated to this topic. Beginning in the mid-1990s, an influx of research highlighted cell cycle reactivation in the pathogenesis of Alzheimer disease (AD), suggesting that the atypical re-entry of neurons into the cell cycle contributes to the neuronal loss seen early in its course (see ARF related news story and ARF news story). Until recently, many of the details of this process have remained a mystery.

Karl Herrup of Rutgers University, Piscataway, New Jersey, co-chaired the symposium with Lloyd Greene (see below). Herrup discussed the role of cyclin-dependent kinase 5 (Cdk5) in both normal and stressed neurons. Cdk5 has previously been implicated in the development of AD pathology at numerous levels. This kinase acts to phosphorylate the microtubule-associated protein tau, whose hyperphosphorylation results in the accumulation of neurofibrillary tangles (see ARF related news story). However, Cdk5 is generally portrayed as having no role in cell cycle regulation. Instead, the regulation of Cdk5 is reported to control both neuronal outgrowth and development. In an effort to better understand these mechanisms, Herrup examined the brains of Cdk5 knockout mice. The Cdk5 knockout mice demonstrated the unique characteristics of neurons out of position, neurons lacking differentiation markers, and neurons displaying embryonic markers, such as Nestin. Unexpectedly, in regions that should otherwise be postmitotic, such as the embryonic cortex, cells re-engaged in the cell cycle and then ultimately went on to die. Cdk5 appears to predominantly interact with cell cycle proteins in the nucleus. However, under conditions of stress, Cdk5 emigrates from the nucleus to the cytoplasm, acting as a signal that the cell should move along the pathway to division.

In a similar vein, Ruth Slack, University of Ottawa, Ontario, discussed new roles for the retinoblastoma (Rb) family of proteins in both neuronal differentiation and migration. Though the Rb protein (pRb) is known to be a key regulator of the cell cycle (acting to control entry into S phase), Slack wanted to determine whether pRb possesses an alternate function. The absence of pRb from the telencephalic region of the brain results in impaired development of nerve cells, in addition to abnormal cortical development. This suggests that, like Cdk5, pRb may play a role in the regulation of differentiation. Recent studies have demonstrated that the Rb/E2F pathway can act to coordinate novel functions beyond proliferation (Höglinger et al., 2007; McClellan and Slack, 2006). To examine this further, Slack compared the roles of p107 and pRb, proteins present in neuronal precursor cells and postmitotic neurons, respectively. Using BrdU incorporation as a cell cycle marker, Slack observed that p107 regulates both self-renewal of neural precursors and the rate of commitment to a neuronal fate. Once the precursor cell commits to a neuronal lineage, p107 exits, making way for the appearance of pRb. The interaction of E2F2/3 with pRb acts to regulate migration, demonstrating a unique mechanism by which Rb proteins regulate this process.

Azad Bonni, Harvard Medical School, provided a detailed update on the role of the Cdh1-anaphase promoting complex (APC) in the cell-intrinsic control of axonal growth and patterning. Cdh1-APC is a ubiquitin ligase that acts to ensure the correct progression of the cell cycle. The integration of developing neurons involves an ordered series of events, including polarization, growth, differentiation, and eventually death. Neuronal connectivity is predominantly regulated by extrinsic cues; however, recent evidence suggests that cell-intrinsic mechanisms also exist and can control distinct aspects of axonal, dendritic, and synaptic development. The effects of Cdh1-APC range from regulation of axon growth and patterning to synapse development and neuronal survival. This led Bonni to ask two questions: what are the downstream substrates of Cdh1-APC, and how is this process regulated? SnoN, a transcriptional regulator, is a substrate of neuronal Cdh1-APC, and studies in knockdown models demonstrate that SnoN promotes both axonal growth and serves a key role in TGF-β signaling in proliferating cells. TGF-β is well known to AD researchers as being an important growth factor that is increased in AD brain (Flanders et al., 1995). Additionally, the overproduction of TGF-β by astrocytes is neuroprotective, and TGF-β1 knockouts exhibit increased neuronal death (see ARF related news story). Bonni and colleagues took this a step further, demonstrating that TGF-β inhibits axonal growth by regulating the APC pathway to promote the ubiquitination and degradation of SnoN.

Tying everything together, Lloyd Greene, Columbia University, New York, discussed how cell cycle molecules regulate survival and death of postmitotic neurons in development and disease. Upon apoptotic stimulation, cyclin-dependent kinases become activated in neurons. This event triggers the sequential phosphorylation of Rb family members, and de-repression of transcription factors, such as Myb, which acts through the cooperative activation of the JNK/cJun and FOXO pathways, and the proapoptotic molecule BIM. Notably, BIM has been found to be elevated in the AD brain, in addition to pRb and other cell cycle molecules.

The cell cycle is activated in various disorders involving neuron death, and many researchers believe it may ultimately evolve into an avenue for therapeutic development. Though related on many levels, these disease states (e.g., Parkinson disease, ALS, stroke, spinal cord injury, etc.) are also very diverse. It is still an open question whether the pathways discussed in this symposium specifically tie into AD, or are of a more general nature. Interestingly, in a series of talks on Monday afternoon (slide sessions 444.4 to 6), Robert Vassar’s lab, Northwestern University, Chicago, outlined a link between Cdk5 and the upregulation of BACE1 in AD. Since increased BACE1 may be a major factor in the development of sporadic AD, there may be a relationship between both BACE1 and tau hyperphosphorylation at different levels in the pathogenic process. In his closing remarks, Karl Herrup reminded the attendees that, “as neuroscientists, we need to move beyond the description of the cell cycle that has been propagated by those in the cancer field, because the regulation of the cell cycle in the neuron is much more nuanced than in a cell simply growing in a dish.” Indeed, this symposium emphasized the atypical role of the cell cycle in neurons, revealing further evidence that all neurons may not leave mitosis for good. It is the examination of these subtleties within neurodegenerative pathways that will allow research to determine if what goes around actually comes around.—Rachel R. Ahmed.

Rachel Ahmed is a Ph.D. student at the University of Kentucky.

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References

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Paper Citations

Höglinger GU, Breunig JJ, Depboylu C, Rouaux C, Michel PP, Alvarez-Fischer D, Boutillier AL, Degregori J, Oertel WH, Rakic P, Hirsch EC, Hunot S. The pRb/E2F cell-cycle pathway mediates cell death in Parkinson's disease. Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3585-90. PubMed.
McClellan KA, Slack RS. Novel functions for cell cycle genes in nervous system development. Cell Cycle. 2006 Jul;5(14):1506-13. PubMed.
Flanders KC, Lippa CF, Smith TW, Pollen DA, Sporn MB. Altered expression of transforming growth factor-beta in Alzheimer's disease. Neurology. 1995 Aug;45(8):1561-9. PubMed.

San Diego: Oligomers Live Up to Bad Reputation, Part 2

19 Nov 2007

Oligomer-only Alzheimer Disease?

Of all other presentations on Aβ oligomers, a talk on Wednesday morning by Takami Tomiyama, who works with Hiroshi Mori at Osaka City University, Japan, created the greatest stir in the audience. Tomiyama told of a new variant of AD that appears to be due predominantly to oligomers. As such, it would offer the first direct evidence in humans that Aβ oligomers are sufficient to bring on AD symptoms in the absence of fibrils. Different aggregation forms of Aβ coexist in the human AD brain and until now, scientists have been unable to tease apart their relative contributions, except as reported above. That’s because methods to identify one over the other in live people, for example, oligomer-specific brain imaging ligands, do not exist as yet. Tomiyama presented a possible genetic separation of clinical AD from plaque formation. He told the audience that their clinic had examined a 59-year-old Japanese woman with a pedigree of early onset familial AD. She had a classic clinical presentation. The scientists found a novel mutation within the Aβ sequence of her APP gene. The patient and her symptomatic sister were homozygous for this mutation, while other, unaffected siblings were heterozygous for it, raising the possibility that the mode of inheritance might be recessive. This would be highly unusual, as pathogenic APP mutations are typically autosomal-dominant. The scientists next screened a sample of more than 5,300 Japanese people gathered by the Japanese Genetic Study Consortium for Alzheimer's Disease for the new mutation, and they found three additional families carrying it.

When Tomiyama and colleagues next characterized the mutation in terms of Aβ production and aggregation, they came in for a surprise. Not only did the mutation reduce total Aβ secretion without affecting the Aβ42/40 ratio, but the mutant peptide also failed to aggregate into fibrils. Thioflavin T fluorescence and electron microscopy suggested as much, and on subsequent Western blotting, too, the mutant Aβ peptide displayed a unique aggregation behavior. It formed oligomers more abundantly than do wild-type peptides but did not form fibrils; instead, it seemed to get firmly stuck in the oligomer conformation. Finally, the scientists assessed how the mutant Aβ would affect synaptic plasticity. They injected it into rat lateral ventricles and soon after stimulated the Shaffer collateral pathway. When recording EPSPs from the hippocampus, the scientists noted that wild-type Aβ slightly reduced LTP in this experiment, as others have found, but the new Aβ mutation did so more potently. In total, these families offer a human genetic validation for the hypothesis that synaptic and cognitive impairment in AD may be more closely tied to oligomers than fibrils and plaques, Tomiyama said.

Several scientists in this session told this reporter that this was the most exciting talk they had heard at the conference so far, and that they were eager to see the paper published. They noted that it will be interesting to follow the heterozygous relatives, one of whom reportedly has MCI, to see if they, too, in time develop AD. Independent investigators in the field said that this work may have profound implications for the underlying biology of AD. One question the data reported at the conference raised in their minds is what kind of pathology this mutation might cause in the brains of affected patients. No tissue is available for autopsy to date, but Tomiyama said in the question-and-answer period following his talk that a PIB PET scan in the proband was negative (PIB binds primarily fibrillar plaques). Other investigators emphasized that it will be important to perform careful imaging studies on these families to ascertain that their disease truly is AD. In a conversation after Tomiyama’s talk, Mori agreed with this point. He noted that the patients were diagnosed using DSM-IIIR and NINCDS-ADRDA criteria, and said that though they were clinically typical for AD, they indeed may not have AD as traditionally defined. “To have AD, do you have to have senile plaques? This is exactly my question. Even once we have autopsy material and do not find plaques, we cannot answer that question,” he said. Mori emphasized, however, that the disease represents a rare variant of AD. To his mind, it presents an opportunity to test the hypothesis that AD is a disease of synaptic failure.

In the same session, Anna Lord, in Lars Lannfelt’s group at Uppsala University in Stockholm, Sweden, presented new data on that group’s exploration of the Arctic mutation in APP, which, like the still-undisclosed new Japanese mutation, falls into the Aβ sequence of APP. Discovered by the Swedish group in 2001, the Arctic mutation has done much to separate effects of oligomeric Aβ from those of fibrillar forms, and in San Diego Lord continued the story. Previously, Lord and colleagues had characterized a transgenic mouse line made to express this mutation plus the Swedish APP mutation. They had found that pathology in these mice began with early intraneuronal Aβ accumulation and then continued with plaque pathology later (Lord et al., 2005).

At the time, the scientists had no means to measure Aβ oligomers in vivo. Now they do. With Hillevi Englund, Dag Sehlin, and Frida Ekholm Pettersson in the same group, the Swedish scientists used their monoclonal IgG mAb158 to develop a sandwich ELISA. This new assay is specific for Aβ oligomers/protofibrils, and it detects large oligomers down to a concentration of 1 picomolar, about 5,000-fold better than it detects low-molecular-weight Aβ (monomers to tetramers). In a poster presented at the AD/PD conference last March in Salzburg, Austria, Pettersson showed that unlike 6E10, a widely used anti-Aβ antibody in the field, Ab158 does not bind APP in its native state. In the hands of this group, 6E10 also did not distinguish between low-n oligomers or monomer and larger protofibrils, see also Englund et al., 2007. (First, a word about nomenclature: these Swedish investigators refer to Aβ oligomers as “protofibrils,” partly because they follow an early naming by David Teplow, and partly because they focus on species larger than dimers or trimers. The protofibrils in their hands are above 60 kDa in molecular weight but fall apart upon SDS treatment, as did the ones found by Shankar and colleagues. Theoretically, the correlates to the Swedes’ “protofibrils” in the human AD extracts would have eluted first, in the solvent of the size exclusion column used by the Boston/Dublin team.)

In San Diego, Lord reported that the mAb158 sandwich ELISA detects synthetic protofibrils, as well as protofibrils in biological samples from a variety of mouse models. The scientists detected and quantified protofibril levels in their Arctic mouse, in the APP+PS1 model, and in the Tg2576 model. In all of these mouse strains, protofibrils predated the appearance of plaques. The Arctic strain contained by far the highest levels, and protofibril levels correlated with Aβ burden but not with amyloid. This finding led some scientists attending the session to speculate that the Japanese Aβ mutation eventually upon autopsy might yield a similar picture of diffuse deposits but no neuritic plaques.

Lord also presented initial behavioral data on the Arctic mice, reporting that the mice did poorly in finding the platform in the Morris water maze at 4 to 8 months of age, prior to plaque deposition. She also reported that levels of the protofibrils rise in the mice between 8 and 14 months of age but then plateau, whereas formic-acid extracted insoluble Aβ (the stuff in neuritic plaques) keeps increasing steeply with age as the mice continue to deposit more plaques. (This last finding jibes with findings from Karen Ashe’s group, who found the Aβ*56 species by looking for forms of Aβ that stay stable over certain periods of age.) In summary, Lord said that oligomers/protofibrils represent an early, neurotoxic stage of amyloid pathology and could serve as a marker of cognitive impairment. (Lord disclosed an ownership interest in BioArctic Neuroscience, a company co-founded by Lannfelt that attempts to develop immunotherapies for AD.)

Adding to the field’s toolbox, another group also reported their generation of new sandwich ELISAs selective for Aβ oligomers. This reporter missed this presentation, but according to the abstract (search SfN abstracts for No. 444.13), these assays use the oligomer-selective Nab61 monoclonal antibody (Lee et al., 2006) , as well as others. M. Kim, Virginia Lee, and colleagues at the University of Pennsylvania in Philadelphia write in the abstract that it can measure oligomers in human brain extracts and other biological fluids, as well as the response to anti-amyloid treatment in a mouse model of amyloidosis. Along the same line of anti-amyloid treatment, Igor Klyubin at Trinity College, Dublin, and collaborators there and at four other institutions, presented a poster (Abstract 485.5) also suggesting that systemic passive immunization might work. These investigators intravenously injected anti-Aβ antibodies into rats. They found that this rescued not only the inhibition of LTP that these authors had previously reported with cell-secreted Aβ oligomers (Walsh et al., 2002), but also a similar LTP inhibition that was caused by Aβ oligomers obtained from CSF of patients with AD.—Gabrielle Strobel.

This is Part 2 of a three-part summary of presentations about Aβ oligomers at the Society for Neuroscience conference held 3-7 November 2007. See Parts 1 and 3.

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References

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Paper Citations

Lord A, Kalimo H, Eckman C, Zhang XQ, Lannfelt L, Nilsson LN. The Arctic Alzheimer mutation facilitates early intraneuronal Abeta aggregation and senile plaque formation in transgenic mice. Neurobiol Aging. 2006 Jan;27(1):67-77. 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.
Lee EB, Leng LZ, Zhang B, Kwong L, Trojanowski JQ, Abel T, Lee VM. Targeting amyloid-beta peptide (Abeta) oligomers by passive immunization with a conformation-selective monoclonal antibody improves learning and memory in Abeta precursor protein (APP) transgenic mice. J Biol Chem. 2006 Feb 17;281(7):4292-9. PubMed.
Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan MJ, Selkoe DJ. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 2002 Apr 4;416(6880):535-9. PubMed.

External Citations

San Diego: Oligomers Live Up to Bad Reputation, Part 3

20 Nov 2007

A Direct Comparison

20 November 2007. A major point of confusion in this field has been that different research groups are working with different kinds of Aβ oligomer. The sources, the methods, and the tools used to characterize them differ enough to leave people scratching their heads about which kind of Aβ does what, and what best to work with themselves as they enter the field. Now, labs are beginning to report a side-by-side comparison of some of those species, and one such presentation is summarized below (see reference to a second, published one, further below). Miranda Reed, a postdoctoral fellow in Karen Ashe’s group at University of Minnesota Medical School in Minneapolis, presented data from a collaborative study with Jim Cleary’s lab at the Minneapolis VA Medical Center, and with MaryJo Ladu at the University of Illinois at Chicago. Reed started by noting that oligomers made from synthetic Aβ as used in ADDLs and other synthetic forms, naturally occurring Aβ derived from the transfected 7PA2 cell line, and transgenic mouse-derived Aβ*56, as used by Lesne et al. had largely driven the public literature on oligomeric Aβ species and raised two simple questions: 1) Does source matter? and 2) Does size matter?

To address these questions, the scientists compared the three types of Aβ in the same experiment. It involves training healthy young rats on an alternating lever-pressing task, performing surgery to infuse the respective Aβ preparation into their brains, and then testing the rats to assess how the injection has affected their performance (Cleary et al., 2005). Some scientists consider this a more sensitive measure of learning and memory than tests of transgenic mice in mazes (partly because rats are smarter than mice). Reed measured two types of error: approach errors and perseveration errors.

Reed reported that a dimer-enriched size exclusion chromatography fraction from conditioned medium of the 7PA2 cells caused the highest rate of both types of errors from that source, while a fraction enriched in trimers showed a non-significant trend toward increased errors. Their potency was in the nanomolar range. Among different fractions isolated from Tg2576 mouse brain, a purified trimer fraction increased approach errors, but the effect was weak and variable. Aβ*56 showed the largest increase in both types of error and was the only species to show a concentration-response relationship. The overall effect was less potent than fractions from 7PA2 medium, as both species were tested at micromolar concentrations. Synthetic Aβ was tested as an undefined mixture of oligomers that ran as monomers, trimers, and tetramers but not dimers on SDS-PAGE, which breaks down higher-order synthetic Aβ oligomers. It, too, produced both types of error when applied in the micromolar range. In toto, all preparations elicited errors except for monomers; the main difference lay in their potency and reliability.

The laboratories of Bill Klein at Northwestern University, Chicago, and Jorge Busciglio at University of California, Irvine, have shown that Aβ oligomers localize to synaptic sites, and in San Diego they reported on their continuing research on which processes might end up being disrupted there (see ARF related news story). Working with his colleagues Atul Deshpande, Erene Mina, and Charlie Glabe, all at UC Irvine, Busciglio last year published a side-by-side comparison of the effects that micromolar and nanomolar concentrations of ADDLs, and other high-n oligomers generated in Glabe’s lab, exerted on human fetal cortical neurons in culture. These oligomers, as well as ADDLs, are a heterogenous mixture and range widely in molecular weight. ADDLs appear to partially overlap in several species with the oligomers made in Glabe’s lab, including the 56 kDa dodecamers, Busciglio clarified by e-mail. The 90 kDa species also are a significant component, and those appear more similar in size to the protofibrils Lannfelt’s group is studying. At the higher concentrations, both these forms of Aβ oligomer were toxic at multiple levels, ranging from speeding up calcium influx to activating a mitochondrial death pathway. They were toxic not only at the synapses but also at other cellular membranes. The main difference between the high-n oligomers and the ADDLs was that the former tended to kill the neurons within a day, whereas the latter took up to a week to do so. Nanomolar concentrations of both preparations caused a similar but milder toxicity (Deshpande et al., 2006).

Why Do Oligomers Bind Synapses?
In San Diego, Busciglio addressed the related question of what draws soluble Aβ oligomers to synapses preferentially. He hypothesized that synaptic transmission might be at play, and described experiments in rat hippocampal slices and human cortical neurons that suggest that more Aβ oligomers cluster on the synapses when synaptic activity is stimulated pharmacologically. In contrast, blocking synaptic firing with the poison TTX reversed this effect. NMDA receptors appear to mediate this localized accumulation, as the oligomers co-localized with NR2B subunits after stimulation. Busciglio’s data also implicate nicotinic acetylcholine receptors in this synaptic receptor targeting. Furthermore, Busciglio reported that when these scientists added monomers and stimulated the preparations, they actually observed oligomeric formation at synapses, implying that overactivity at excitatory synapses may induce oligomer formation from monomers secreted at synaptic sites.

Metal ions such as zinc (Zn2+) and copper (Cu2+) may attract and further aggregate Aβ to the synaptic cleft since they are released at glutamatergic synapses during neurotransmission, Busciglio said. In these experiments, adding the chelator drug clioquinol reduced Aβ’s synaptic accumulation. (See Ritchie et al., 2003 for clinical trial results on clioquinol, and Caragounis et al., 2007 and Lau et al., 2007 for data on clioquinol’s mode of action, published this fall.)

Among the antibodies the scientists used is the polyclonal “officer” (OC) from Glabe’s lab, which labels fibrillar, donut-shaped oligomers (Kayed et al., 2007; see ARF Eibsee report). The neuron does not “eat” the “donuts”— that is, they remain on the cell surface and are not endocytosed or otherwise internalized during this experimental period of neurotransmission, Busciglio said. Rather, they appear to exert a toxic effect from the outside. As seen with immunohistochemistry on postmortem brain tissue of people who had had AD, these oligomers co-localized with the presynaptic marker synaptophysin, as well as with postsynaptic markers PSD 95 and NR2B, Busciglio showed.

Synapses Let Go, Then Shrivel
A welcome new development at the Neuroscience conference was a broadening of the research effort in that a growing number of laboratories were joining the fray. For example, Barbara Calabrese of the Scripps Research Institute in La Jolla, California, showed a series of elegant experiments visualizing what Aβ oligomers do to both sides of the synapse. Calabrese works with Shelley Halpain at Scripps, who is known for her basic research on spine dynamics and the role of the cytoskeleton in synaptic biology. In a collaboration with Eddie Koo at the University of California, San Diego, these investigators started testing the effects of Aβ oligomers secreted into the culture medium of 7PA2 cells (Calabrese et al., 2007). “This cell line allowed us to use picomolar concentrations of Aβ oligomer and to look at early effects on synapses separately from neurotoxicity. The changes we see do not kill the neurons,” Calabrese told ARF.

In her talk, Calabrese showed how she applied 40 to 80 picomolar concentrations of Aβ oligomers to primary hippocampal neurons from embryonic rats co-cultured with glia. These mixed cultures form synapses and stay alive for weeks. Calabrese showed that markers of presynaptic vesicles became fewer and smaller in glutamatergic synapses, but not in GABAergic terminals. The latter were impervious to Aβ oligomers. This finding might help explain the old observation that GABAergic, i.e., inhibitory, neurons are selectively spared in AD, and it suggests that neural circuits might shift to a more quiescent state in the presence of Aβ oligomers, Calabrese said. (Morphologically speaking, glutamatergic synapses tend to sit on the spine head, whereas GABAergic terminals generally synapse onto the dendrite’s shaft in between the spine protrusions.)

These changes happened quickly, within a couple of hours of application. This puts them in the right time frame for representing a potential cellular substrate for impaired synaptic plasticity, Calabrese added.

Calabrese noted not only a drop in number and size of glutamatergic nerve terminals and spines, but also that the remaining spines changed shape. Time-lapse imaging brought to light a new form of structural synaptic plasticity: in these movies, some previously normal-looking spines developed long, thin necks but stayed attached to their presynaptic sides, whereas other spines plain separated and collapsed. Yet other spines first elongated, then collapsed. These data address one debate in the field, namely, the question of whether Aβ oligomers affect the pre- or post-synaptic side first. Calabrese first saw the two sides uncouple, then the post-synapse collapse. This phenomenon must be understood in the biological context of “morphing,” she added, a term that describes that the synaptic connection constantly and normally undergoes some level of morphological change, even in the absence of Aβ oligomers.

Interestingly, these experiments also revealed that the spines are dynamic in more ways than that. Not only do they reappear a day after washout, they also somehow become resistant to the oligomers if those oligomers stay in the culture medium for 2 days. It’s not that the oligomers degrade in that time—transferring the same medium to a fresh batch of neurons will make the spines on those naïve neurons cave in. Rather, the spines somehow compensate for the continued presence of the oligomers. But they only do so for a time. When Calabrese washes out the oligomers, then re-applies them, the synapses shrink once again, and when she continuously applies them for 10 days, the spines do not recover spontaneously anymore. Even under this sustained assault, however, the neurons do not die. The effect is restricted to their synapses, which rapidly lose strength in electrophysiological recordings, but by itself it is not toxic to the neuron as a whole, Calabrese reported.

How this works is unclear at present. Calabrese and colleagues noticed that the spine changes disappeared when they added either a drug that blocks all types of nicotinic acetylcholine receptors, or the NMDA receptor antagonist memantine (aka Namenda). On this red-hot question, many groups are honing in on potential receptor targets of oligomeric forms of Aβ, as well as on receptor-independent mechanisms involving membrane integrity.—Gabrielle Strobel.

This concludes our report of presentations about Aβ oligomers at the Society for Neuroscience conference held 3-7 November. See also Parts 1 and 2.

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References

News Citations

Paper Citations

Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ, Ashe KH. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci. 2005 Jan;8(1):79-84. PubMed.
Deshpande A, Mina E, Glabe C, Busciglio J. Different conformations of amyloid beta induce neurotoxicity by distinct mechanisms in human cortical neurons. J Neurosci. 2006 May 31;26(22):6011-8. PubMed.
Ritchie CW, Bush AI, Mackinnon A, Macfarlane S, Mastwyk M, MacGregor L, Kiers L, Cherny R, Li QX, Tammer A, Carrington D, Mavros C, Volitakis I, Xilinas M, Ames D, Davis S, Beyreuther K, Tanzi RE, Masters CL. Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting Abeta amyloid deposition and toxicity in Alzheimer disease: a pilot phase 2 clinical trial. Arch Neurol. 2003 Dec;60(12):1685-91. PubMed.
Caragounis A, Du T, Filiz G, Laughton KM, Volitakis I, Sharples RA, Cherny RA, Masters CL, Drew SC, Hill AF, Li QX, Crouch PJ, Barnham KJ, White AR. Differential modulation of Alzheimer's disease amyloid beta-peptide accumulation by diverse classes of metal ligands. Biochem J. 2007 Nov 1;407(3):435-50. PubMed.
Lau TL, Gehman JD, Wade JD, Masters CL, Barnham KJ, Separovic F. Cholesterol and Clioquinol modulation of A beta(1-42) interaction with phospholipid bilayers and metals. Biochim Biophys Acta. 2007 Dec;1768(12):3135-44. PubMed.
Kayed R, Head E, Sarsoza F, Saing T, Cotman CW, Necula M, Margol L, Wu J, Breydo L, Thompson JL, Rasool S, Gurlo T, Butler P, Glabe CG. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol Neurodegener. 2007;2:18. PubMed.
Calabrese B, Shaked GM, Tabarean IV, Braga J, Koo EH, Halpain S. Rapid, concurrent alterations in pre- and postsynaptic structure induced by naturally-secreted amyloid-beta protein. Mol Cell Neurosci. 2007 Jun;35(2):183-93. PubMed.

San Diego: “Calcinists” See Years of Compensation Prior to Alzheimer’s

21 Nov 2007

If our summary on Aβ oligomers from the recent conference of the Society for Neuroscience delivered an overdose of news on synaptotoxic Aβ, take this story as an antidote. Held 3-7 November in San Diego, California, the conference featured a wealth of presentations to reflect a broadening knowledge base about this disease, and the role of presenilin and calcium in AD pathogenesis is one of the exciting examples. Perturbations to the neuronal calcium balance in both aging and AD have been described for some years and have led to a calcium hypothesis of brain aging and dementia. What is new is an evolving concept tying intraneuronal calcium increases in important functional ways to known players in AD risk and pathogenesis, that is, to presenilin (PS) mutations, APP mutations, ApoE4, synaptic dysfunction, apoptosis. In particular, new research is trying to assess these links in model systems at early stages, long before visible pathologies have formed and could confound results. In essence, the new calcium research is reaching for a grasp of how subtle disease-promoting changes play against compensatory mechanisms that the neuron deploys to maintain homeostasis. “Calcinists” view the emergence of amyloid and tau pathology as the result of a breakdown of this compensation. As a conference offering, below is a summary of one such presentation by Grace (aka Beth) Stutzmann at Rosalind Franklin University of Medicine and Science, North Chicago, Illinois. For a broader view, read her eloquent review, published last month (Stutzmann, 2007).

In her talk, Stutzmann focused on the role of presenilin (PS) mutations and the ryanodine receptor. Unlike presenilin, this receptor is not a household term among “Alzheimerologists.” In a nutshell, Stutzmann proposed that, in mice expressing mutant human presenilin 1, the ryanodine receptor is hyperexcitable. Prone to dumping large amounts of Ca2+ from ER stores preferentially into distal dendrites and synaptic spines, it tends to end up dampening the activity of those synapses.

For background, Stutzmann first reminded the audience that neurons maintain a deep valley of nanomolar Ca2+ in the cytosol vis-à-vis millimolar Ca2+ outside the cell membrane on one side, and high micromolar Ca2+ inside the endoplasmic reticulum (ER) on the other side. Multiple different calcium channels and pumps in the cell membrane and the ER membrane are necessary to keep Ca2+ distributed across these gradients. In previous work, Stutzmann had set up a system that combines whole-cell patch clamp recordings with 2-photon imaging of calcium flows. This is done in thick, 300-micron cortical slabs from various mouse models. She routinely isolates three types of calcium response:

One through the plasma membrane (where action potentials trigger calcium flow through voltage-gated Ca2+ channels)
One through the IP3 receptor in the ER membrane (triggered by photolysis of caged IP3)
One through the lesser-known ryanodine receptor (using its agonist caffeine and its blocker dantrolene)

Together, this experimental setup serves as a “holistic” system of assessing neurophysiologic consequences of specific calcium changes, Stutzmann said.

Stutzmann had shown before that neurons from triple transgenic mice created in Frank LaFerla’s lab, where she had been a postdoctoral fellow, readily show much stronger calcium outflows from the ER than do those of non-transgenic mice. By comparing this strain with a different model overexpressing only mutant PS1, and with an APP/tau double transgenic, she attributed this defect in calcium management to presenilin. The present focus on the ryanodine receptor arose with the twin observations that PS1-transgenic mice have elevated levels of ryanodine receptor protein, and that blocking this receptor normalized excessive calcium outflow from the ER. In the presenilin transgenic mice, the ryanodine receptor mediated the majority of this particular calcium flow, up from 20 percent in normal mice to 70 percent in the mutant mice (Stutzmann et al., 2006).

At the conference, Stutzmann showed newer data suggesting that the ER within the distal dendrites and even spines releases the greatest relative increases in ryanodine-triggered calcium in the PS1-mutant mice; it is not the ER around the nucleus and cell body where most textbook diagrams depict it to be. (The ER is known to extend into dendrites and even into spine heads; see picture below.) Those distal ER tips show little ryanodine receptor-mediated Ca2+ release in non-transgenic mice, but in PS1-mutant they increase this flow dramatically by 10-fold. By contrast, the ER in the cell body about doubled its ryanodine receptor-mediated outflow. This suggests that the synaptic areas of the ER are functionally separate from what is going on in the cell body, Stutzmann said.

Three-dimensional Reconstruction of the Smooth ER (Purple) in a Rat Hippocampal CA1 Dendritic Segment
The left side shows that the ER in the dendrite is contiguous with the ER entering the thin neck of a dendritic spine (grey). The smooth ER in the head of the spine (right) is thought to provide synapse-specific regulation of calcium release, and modulate incoming synaptic signals (Reproduced from SynapseWeb; Spacek and Harris, 1997).

Does this mean anything to cortical neurons? These cells integrate multiple signals, and then generate a summed reaction. Stutzmann tried to model this behavior by assessing calcium release from the ER in response to either synaptic stimulation or ryanodine receptor activation alone, or both in combination. In the latter experiment, she saw a supra-additive depletion of ER calcium stores in the PS1-transgenic mice. Further experiments showed that this leads to greater membrane hyperpolarization. And this, in turn, hampered the synapses’ ability to generate trains of action potentials in response to electrical stimulation, essentially making the neuron less excitable (Stutzmann et al., 2007). “A main point here is that, depending on where it is dumped from the ER, this extra calcium will have very different functional consequences for the neuron,” Stutzmann said. (Links between ryanodine receptor function and mitochondria and apoptosis, for example, have been reported, as well.)

The existing literature indicates no change in synaptic transmission or synaptic plasticity in the triple transgenic mice at the young ages of 4 to 6 weeks, and Stutzmann’s group confirmed these findings. Yet she felt she had not looked hard enough. Maybe there was a compensatory effort hidden behind the curtain of this normal-looking output? “Calcium plays such a big role in synaptic transmission and plasticity, I don’t see how you can mess with it on a major scale and not affect those functions,” Stutzmann added. To peek behind the curtain, she blocked the ryanodine receptor with the drug dantrolene. In non-transgenic mice, this did away with about half the LTP output, but in PS1-transgenic mice it abolished LTP completely.

To Stutzmann’s mind, this suggests that the ryanodine receptor calcium stores in the dendritic tips of the ER contribute to synaptic function quite differently when there is a PS1 mutation. That the neurons at this age still manage to generate normal-looking LTP means that they are compensating, she said. This happens before any AD-like histopathology can be detected. Speculating about PS1-mutant FAD, this could imply that people are born with these different calcium dynamics and compensate well, until years of effort to maintain homeostasis exhaust the neuron and it switches from compensation to pathology. One future experiment would be to slightly dial down the ryanodine receptor over longer periods of time with drugs such as dantrolene, and ask whether that can keep AD-like pathology and behavioral deficits at bay in those models.

This summary covers but one area of active research on calcium flows in AD models. Effects of presenilin on ER calcium were established by a number of groups (Tu et al., 2006; Nelson et al., 2007; LaFerla, 2002), and the link between the ryanodine receptor, calcium, and AD is even older (Querfurth et al., 1998). Current questions focus on the relative contributions of the ryanodine versus the inositol trisphosphate IP3 receptor, as well as on how the two interact. This writer invites comments on these issues to broaden the discussion.—Gabrielle Strobel.

References

Paper Citations

Stutzmann GE. The pathogenesis of Alzheimers disease is it a lifelong "calciumopathy"?. Neuroscientist. 2007 Oct;13(5):546-59. PubMed.
Stutzmann GE, Smith I, Caccamo A, Oddo S, Laferla FM, Parker I. Enhanced ryanodine receptor recruitment contributes to Ca2+ disruptions in young, adult, and aged Alzheimer's disease mice. J Neurosci. 2006 May 10;26(19):5180-9. PubMed.
Spacek J, Harris KM. Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendritic spines of the immature and mature rat. J Neurosci. 1997 Jan 1;17(1):190-203. PubMed.
Stutzmann GE, Smith I, Caccamo A, Oddo S, Parker I, Laferla F. Enhanced ryanodine-mediated calcium release in mutant PS1-expressing Alzheimer's mouse models. Ann N Y Acad Sci. 2007 Feb;1097:265-77. PubMed.
Tu H, Nelson O, Bezprozvanny A, Wang Z, Lee SF, Hao YH, Serneels L, De Strooper B, Yu G, Bezprozvanny I. Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations. Cell. 2006 Sep 8;126(5):981-93. PubMed.
Nelson O, Tu H, Lei T, Bentahir M, de Strooper B, Bezprozvanny I. Familial Alzheimer disease-linked mutations specifically disrupt Ca2+ leak function of presenilin 1. J Clin Invest. 2007 May;117(5):1230-9. Epub 2007 Apr 12 PubMed.
Laferla FM. Calcium dyshomeostasis and intracellular signalling in Alzheimer's disease. Nat Rev Neurosci. 2002 Nov;3(11):862-72. PubMed.
Querfurth HW, Haughey NJ, Greenway SC, Yacono PW, Golan DE, Geiger JD. Expression of ryanodine receptors in human embryonic kidney (HEK293) cells. Biochem J. 1998 Aug 15;334 ( Pt 1):79-86. PubMed.

External Citations

San Diego: MHC Class I and Complement—Holding Down Second Jobs in the Synapse

21 Nov 2007

Adapted from a story that originally appeared on the Schizophrenia Research Forum.

It may be a surprising idea, but it is becoming clearer from work done in several labs that complement factors and MHC Class I proteins are regulators of synapse formation during fetal development, synapse plasticity, and synapse repair. This function occurs without apparent T cell involvement or any presentation of self or non-self peptides. It also now appears that these proteins may be relevant to neural disorders such as autism and schizophrenia, as discussed at the 2007 meeting of the Society for Neuroscience, held 3-7 November in San Diego, California. Lisa Boulanger of the University of California, SD, led a symposium titled "The New Neuroimmunology: Immune Proteins in Synapse Formation, Plasticity, and Repair." Parts of the research presented appeared on ARF before, see Bar Harbor Report 2006, ARF related news story, and ARF news story). Below is an update of this emerging story.

Carla Shatz of Harvard University first demonstrated that MHC Class I proteins were functionally required for development and plasticity of the CNS in 2000 (Huh et al., 2000). Today Shatz presented work from her laboratory—which primarily focuses on the visual cortex ocular dominance models of plasticity—showing that Class I proteins co-localize with PSD-95, considered by many a “master organizer of synapses” (see Goddard et al., 2007). Shatz demonstrated that Class I proteins are expressed at high levels in neurons of both somatosensory cortex as well as hippocampus. Using a double knockout mouse that lacks both β2-microglobulin (β2M) and the transporter associated with antigen processing 1 (TAP1), which dramatically reduces the surface expression of all MHC Class I proteins, Shatz and her colleagues showed that Arc (activity-regulated cytoskeletal-associated protein) induction in the visual cortex is abnormally widened after visual stimulation of the double knockout mice. These data suggest that MHC Class I proteins regulate the process of synaptic plasticity in the ocular dominance model used. Shatz proposed that the gene PirB (paired immunoglobulin-like receptor B) encodes the receptor for MHC Class I in neuronal synapses, which was shown previously to be expressed in neurons in the brain, and functions to limit experience-dependent plasticity in the visual cortex (Syken et al., 2006).

Staffan Cullheim of the Karolinska Institute in Stockholm, Sweden, used the same double β2M/TAP1 knockout mice to examine the role that Class I proteins play in the elimination of synapses following nerve injury (Thams et al., 2007; Cullheim and Thams, 2007). The data presented focused on the role of activated microglia and MHC Class I protein specificity in the synapse removal process after axotomy, and suggested that there may be a differential effect of these proteins in excitatory (NMDA) versus inhibitory (glycine or GABA) synapses, with more elimination of inhibitory synapses.

Ben Barres of Stanford University, Palo Alto, California, shifted the focus from MHC Class I proteins to the role of components of the complement cascade C1q and C3 to act as “punishment signals” and cause axon atrophy and withdrawal. Using a new imaging method called array tomography (Micheva and Smith, 2007), Barres showed that C1q co-localizes to developing CNS synapses using immunofluorescent staining of 70 nm sections of developing mouse brain. Barres further showed that in C1q or C3 knockout mice, synapse refinement that normally occurs in early postnatal development (P5 through P30) was defective, resulting in more synapses, not more neurons. Barres presented a model in which immature astrocytes clustering near the developing synapses release C1q and C3 into the synapse to prune and eliminate synapses. An important implication of Barres’s presentation is the tantalizing idea that inhibitors of the complement cascade may have the potential to block neurodegeneration.

The final talk of the symposium was by Lisa Boulanger. Boulanger has continued on with the work she had done as a postdoctoral fellow in Shatz’s laboratory, and has pursued studies of MHC Class I proteins in synaptic pruning and plasticity to explore the potential implications for both autism and schizophrenia. Boulanger is no stranger to the autism field, having published a thoughtful article on abnormal development of brain connectivity in autism in 2004 (Belmonte et al., 2004). In her talk, Boulanger examined the electrophysiological responses of β2M/TAP1 double knockout mice in paired pulse inhibition (an experimental model of sensory gating deficits in schizophrenia that is widely used, if not widely accepted), AMPA receptor fEPSP, and NMDA-induced chemical LTD, which is similar to low frequency stimulation induced LTD.

Surprisingly, the last test, which results in a stable LTD in wild-type mice, instead induced a robust LTP in the β2M/TAP1 double KO mice. Pursuing these studies further, Boulanger demonstrated that NMDA treatment caused a dramatic increase in surface AMPA receptor expression. Boulanger proposed that the increase in AMPA receptors was a result of increased internalization of AMPA, accompanied by a dramatic increase in recycling AMPA receptors back to the synaptic surface to cause a homeostatic shift of net increase.

Boulanger proposed that MHC proteins are tied to neural diseases, speculating that maternal immune challenge increases the risk of the unborn fetus to such diseases (see Patterson, 2007 and Schizophrenia Research Forum news story). In the model that Boulanger proposed, induction of a maternal immune response in mice increases maternal cytokines that are capable of entering the fetal blood circulation. These cytokines, if exposed to the developing nervous system of the fetus, may regulate MHC Class I levels in neurons. The question remains, Do changes in neuronal MHC Class I expression mediate changes in the development of the fetal brain?—Gwendolyn T Wong.

References

News Citations

Paper Citations

Huh GS, Boulanger LM, Du H, Riquelme PA, Brotz TM, Shatz CJ. Functional requirement for class I MHC in CNS development and plasticity. Science. 2000 Dec 15;290(5499):2155-9. PubMed.
Goddard CA, Butts DA, Shatz CJ. Regulation of CNS synapses by neuronal MHC class I. Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6828-33. PubMed.
Syken J, GrandPre T, Kanold PO, Shatz CJ. PirB restricts ocular-dominance plasticity in visual cortex. Science. 2006 Sep 22;313(5794):1795-800. PubMed.
Thams S, Oliveira A, Cullheim S. MHC class I expression and synaptic plasticity after nerve lesion. Brain Res Rev. 2008 Jan;57(1):265-9. PubMed.
Cullheim S, Thams S. The microglial networks of the brain and their role in neuronal network plasticity after lesion. Brain Res Rev. 2007 Aug;55(1):89-96. PubMed.
Micheva KD, Smith SJ. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron. 2007 Jul 5;55(1):25-36. PubMed.
Belmonte MK, Allen G, Beckel-Mitchener A, Boulanger LM, Carper RA, Webb SJ. Autism and abnormal development of brain connectivity. J Neurosci. 2004 Oct 20;24(42):9228-31. PubMed.
Patterson PH. Neuroscience. Maternal effects on schizophrenia risk. Science. 2007 Oct 26;318(5850):576-7. PubMed.

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External Citations

San Diego: Microglia Enter Enrichment Stage, Human Brain Imaging of Neurogenesis

26 Nov 2007

At the 37th conference of the Society for Neuroscience, held earlier this month in San Diego, Sangram Sisodia’s group at the University of Chicago, Illinois, presented new data that exemplify how the expanding field of environmental enrichment and adult neurogenesis has inspired AD research. Sisodia presented a special twist on this emerging story with experiments supporting the idea that the presenilin mutations that cause familial AD lead to defects in hippocampal neurogenesis (Wen et al., 2002; Wen et al., 2004). A growing number of laboratories are probing different aspects of how environmental enrichment, or other forms of physical and mental exercise, stimulate the growth of neural precursor cells in mice, dogs, and even humans. The goal is to both understand underlying mechanisms and their relevance to aging and AD, and to build a body of knowledge upon which to base therapeutic interventions. And helping to narrow the leap from beast to human, a paper in Science offered news on a method to image neural progenitor cells in the brains of people.

That environmental enrichment boosts learning and memory goes back to an original observation by none other than Donald Olding Hebb (Hebb, 1947), who is better known for his theory of synaptic plasticity. That hippocampal neurogenesis continues into adulthood in rodents and mammals is more recent knowledge, but established, as well (e.g. Lie et al., 2004). Sisodia’s lab began this line of research when Orly Lazarov, then in his lab, discovered that keeping mice in cages filled with toys and climbing structures reduced amyloid-β levels and deposition in APP/PS1-transgenic mice (Lazarov et al., 2005).

In San Diego, Sisodia and postdoc Karthik Veeraraghavalu presented data on the potential role of presenilin in mediating effects of environmental enrichment. Sisodia began by establishing that mice living in the “fun” cages had more neurogenesis in their hippocampi. The researchers obtained absolute numbers by cutting the hippocampus into series of slices and counting every BrdU-labeled cell. The most avid users of the running wheel seemed to lead the pack. By contrast, adult mice expressing either the δE9 or the M146L FAD mutations in presenilin 1 derived no benefit from environmental enrichment. They did have some neurogenesis in their hippocampus, but their BrdU counts stayed flat no matter how eagerly they scampered over their colorful playthings, suggesting that presenilin somehow influences the enrichment-induced proliferation of neural progenitor cells (NPCs).

The scientists asked if this difference arose from within the neural precursors or a different cell type. To do that, they developed neurosphere cultures of hippocampal NPCs and primary microglia. Intriguingly, they found that the effect appeared to come from the glia. NPCs from human wild-type PS1 transgenic mice proliferated when cultured with microglia from wild-type PS1 mice, but failed to do so when cultured with microglia from mice expressing FAD-linked PS1, Sisodia reported. Moreover, the differentiation of wild-type PS1 NPCs toward neurogenic lineages was markedly inhibited when they were co-cultured with microglia from mutant PS1 mice.

The conditioned medium from PS1 microglia fully recapitulated the wild-type NPC responses in co-culture assays. This suggests that the glia secrete some mystery factors that are dependent on expression of presenilin 1. Analysis of the cytokine and chemokine profile of wild-type versus mutant microglia generated a list of polypeptides whose mRNA and protein levels changed, including CXCL16, MCP-1, eotaxin, and others, Sisodia reported. While still evolving, this work suggests that environmental enrichment activates normal microglia to release proteins that support the proliferation and neurogenesis of neural precursors in the hippocampus. Microglia expressing these pathogenic presenilin mutations do not respond to enrichment in this way, suggesting that they need proper presenilin function to translate the environmental stimulus into a changed chemokine output.

But like everything in the brain, one cell type does not tell the whole story. NPCs expressing either of these two FAD presenilin mutations show cell-autonomous deficits, as well. On a poster, Veeraraghavalu showed that self-renewal and differentiation of these NPCs were impaired in neurosphere cultures taken from the subventricular zone of these mice. In those cells, the problem may have to do with Notch, a well-studied substrate of presenilin. The NPCs do express the Notch signaling machinery, and priming this pathway by expressing downstream target genes revived their flagging proliferation, the Chicago scientists reported. Further experiments also pointed to the interpretation that a partial loss of Notch processing may account for the defects of these mutant NPCs.

When cell-based and animal research develop mechanistic plot lines, the question of how pertinent this is to human aging and disease invariably arises. On this question, a collaboration of scientists at the State University of New York (SUNY) at Stony Brook and Cold Spring Harbor Laboratories on Long Island, NY, just handed the field a cool new tool. Led jointly by Mirjana Maletic-Savatic and Grigori Enikolopov, first author Louis Manganas and others reported in the November 9 issue of Science their development of the first biomarker to identify NPCs non-invasively in the brain. Using proton nuclear magnetic resonance spectroscopy (1H-NMR), the scientists characterized in vitro a lipid metabolite specific to NPCs. Then they moved to a complementary method that is suitable for live tissue imaging, that is, 1H-MRS done in an MRI scanner. This enabled them to identify NPCs in the hippocampus, first of rats, then of people. Manganas and colleagues detected the NPC biomarker in the hippocampus of adults and also looked at changes with age by imaging preadolescents, adolescents, and adults (yes, you guessed right: levels dropped off quite steeply by one’s thirties). The data gathered so far with this method confirm prior findings that demonstrated a small but continuing trickle of neurogenesis in adult life in humans.

If confirmed, this biomarker could help analyze NPCs and evaluate the efficacy of therapeutic interventions in a range of neurological and psychiatric disorders, the authors write. A press release issued by Cold Spring Harbor Laboratories quotes Walther Koroshetz, deputy director of the Neurological Disorders and Stroke, as saying: “The ability to track these cells in living people would be a major breakthrough in understanding brain development in children and continued maturation of the adult brain. It could also be a very useful tool for research aimed at influencing NPCs to restore or maintain brain health.”—Gabrielle Strobel

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References

Paper Citations

Wen PH, Shao X, Shao Z, Hof PR, Wisniewski T, Kelley K, Friedrich VL, Ho L, Pasinetti GM, Shioi J, Robakis NK, Elder GA. Overexpression of wild type but not an FAD mutant presenilin-1 promotes neurogenesis in the hippocampus of adult mice. Neurobiol Dis. 2002 Jun;10(1):8-19. PubMed.
Wen PH, Hof PR, Chen X, Gluck K, Austin G, Younkin SG, Younkin LH, DeGasperi R, Gama Sosa MA, Robakis NK, Haroutunian V, Elder GA. The presenilin-1 familial Alzheimer disease mutant P117L impairs neurogenesis in the hippocampus of adult mice. Exp Neurol. 2004 Aug;188(2):224-37. PubMed.
Lie DC, Song H, Colamarino SA, Ming GL, Gage FH. Neurogenesis in the adult brain: new strategies for central nervous system diseases. Annu Rev Pharmacol Toxicol. 2004;44:399-421. PubMed.
Lazarov O, Robinson J, Tang YP, Hairston IS, Korade-Mirnics Z, Lee VM, Hersh LB, Sapolsky RM, Mirnics K, Sisodia SS. Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice. Cell. 2005 Mar 11;120(5):701-13. PubMed.

San Diego: Klotho Spins Threads Linking Aging and AD Research

04 Dec 2007

In Greek mythology, Klotho is a goddess of fate. She spins the thread of life, while her sister Lachesis determines the length of the thread and sister Atropos cuts it when the time has come. Japanese scientists 10 years ago reached for this divine trio in naming a gene they accidentally discovered to influence lifespan in mammals, and now new research, presented at last month’s Society for Neuroscience convention, reinforces how apt this imagery truly is. In San Diego, California, Carmela Abraham of Boston University reported first results of a molecular analysis of klotho expression in the brain in aging and AD models. She showed that klotho levels not only wane with age but also drop off steeply in AD transgenic mice. Her group’s further research on klotho cleavage by ADAM proteases describes a protein at the crossroads of aging, oxidative stress, insulin signaling, and APP cleavage—and the research might just open up a fresh angle for therapeutic intervention in age-related neurodegenerative disease. In essence, the researchers find, the faster we lose Klotho, the sooner Lachesis and Atropos can move in.

Abraham’s lab initially stumbled across klotho during a study of brain aging in rhesus monkeys for whom cognitive decline had been established. That study focused on white matter, and a microarray experiment comparing gene expression in young versus old rhesus monkey brain pointed to a 74 percent drop in klotho mRNA in aging white matter (see Duce et al., 2008). Previously, scientists had reported premature aging with cognitive deficits in klotho knockout mice, whereas mice overexpressing klotho lived especially long and expressed copious amounts of antioxidant genes (Kuro-O et al., 1997; Kurosu et al., 2005). A human klotho polymorphism has been associated with longevity and robust health. In short, klotho is a mammalian lifespan gene. It falls into a group with other genes that impair insulin receptor signaling and dramatically increase longevity in worms and fruit flies.

Previous research by others had also shown that klotho gets secreted and acts much like a hormone. In peripheral organs, particularly the kidney, klotho promotes calcium resorption and vitamin D signaling, protecting bones against osteoporosis, for example.

“The question for us is, How does it work in the brain?” Abraham told ARF in San Diego. To that end, her group studied klotho expression and cleavage. On a poster, Abraham showed how biochemical experiments confirmed the microarray data at the protein level, indicating that klotho levels decrease with age in monkey, rat, and mouse brain. Klotho protein was most abundant in the choroid plexus. This capillary/ependymal organ dangling from the brain’s ventricles releases klotho into the CSF. Klotho concentration falls off in samples from AD brain. In the brain itself, klotho expression was highest in the hippocampus, followed by striatum, thalamus, medulla, and spinal cord, Abraham and first author Sonia Podvin reported. What’s more, 12-month-old APP/PS1-transgenic mice with plaques expressed less klotho than did littermates without amyloidosis, the scientist found.

Next, the Boston researchers analyzed klotho cleavage. The gene sequence predicts klotho to be a type 1 transmembrane protein (the type of protein that is cleaved by α-, β-, and γ-secretases), and its cleaved ectodomain appears to be that piece that functions like a hormone. Klotho cleavage generates a long 130 kDa and a short 65 kDa fragment. On a separate poster, as well as in a paper in the December 3 Proceedings of the National Academy of Sciences, first author Ci-Di Chen and colleagues identified the sheddases, aka α-secretases, that cleave klotho. In COS-7 cells and also in rat kidney slices, those enzymes were none other than ADAM 10 and ADAM 17, the metalloproteases thought to cleave APP as well as other AD-relevant proteins such as TNFα. (The human genome contains additional ADAM enzymes, and some of those could cleave klotho, as well, the authors note.)

Thickening the plot, insulin turned out to stimulate this cleavage and subsequent release of the klotho ectodomain, Chen and colleagues reported. Further experiments indicated that insulin promotes klotho cleavage by affecting the proteolytic activity of ADAM 10 and/or 17 rather than change the proteases’ expression (Chen et al., 2007). As insulin receptor signaling stimulates cleavage of klotho, its released ectodomains in turn activate expression of antioxidant enzymes. So do the sirtuin longevity genes. Importantly, elevated klotho might then feed back to inhibit insulin receptor signaling, Abraham proposes. The transgenic klotho overexpressors are insulin-resistant in a way that is clearly beneficial for their health. Klotho’s relationship, if any, to sirtuins and the FoxO transcription factors need to be explored, Abraham said.

Clearly, klotho per se is not specific to AD. It counteracts age-related processes in multiple ways. In San Diego, for example, Dwight German, Kevin Rosenblatt, Makoto Kuro-O, and colleagues at the University of Texas Southwestern Medical Center in Dallas reported data suggesting that klotho protects nigrostriatal dopamine neurons from MPTP toxicity in vivo in mice. Even so, areas of molecular overlap with AD mechanisms deserve study, Abraham said. First, the same sheddases that cleave klotho also cleave APP to generate the protective sAPPα fragment and may preclude Aβ generation. One question to explore here is whether insulin receptor signaling boosts sAPPα release by the same mechanism, i.e., via ADAM 10 and/or 17. Second, one proposed mechanism of Aβ oligomer toxicity focuses on insulin receptor signaling, (see ARF related news story), and indeed, AD is sometimes called “type 3 diabetes.” Abraham’s group is studying whether there are links between klotho and Aβ. The investigators also focus on characterizing the klotho promoter, which they suspect may sustain oxidative damage with age. Theoretically, then, small-molecule drugs that keep klotho expression high could keep Lachesis and Atropos at bay.—Gabrielle Strobel.

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References

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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.
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.

San Diego: Exploring the Function of APP, Part 1

05 Dec 2007

Despite a 20-year courtship, researchers have gotten to know rather little about amyloid-β precursor protein (APP). Yes, it undergoes sequential cleavage via amyloidogenic and non-amyloidogenic pathways, and it is a key player in the pathology of Alzheimer disease, but the normal function of the precursor is still up for grabs. Is it a signaling receptor, a transcription factor, a transporter of intracellular cargo? What role does it play in embryogenesis, neuronal development, apoptosis, tumorigenesis? Does it have pathological roles outside of being the source of Aβ? Progress is being made, albeit slowly. At this year’s Society for Neuroscience annual meeting, held last month in San Diego, California, an ancillary symposium explored the normal and pathological roles of APP. Titled “Function of APP Gene Family Members and Clues to AD Pathogenesis: Studies from Worms to Mammals,” and organized by Sanjay Pimplikar, Case Western Reserve University, Cleveland, Ohio, and Suzanne Guenette, Massachusetts General Hospital, Boston, the symposium was intended to provide an informal platform where researchers would be free to discuss, debate, and speculate on the normal and pathological roles of APP.

The first few talks dealt with the role of APP in lower organisms. Chris Li, City College of the City University of New York, kicked things off by outlining her work on the role of APL-1, a homolog of APP in the worm Caenorhabditis elegans. APL-1 is essential for survival in these worms. Without it, larvae cannot molt properly and never transition to their second stage. This point of death provides a platform to study individual domains of APL-1, said Li. Her group has made a series of APL-1 constructs and tested their ability to rescue lethality in APL-1 knockouts. With this approach they found that neither the transmembrane domain nor the cytoplasmic end of APL-1, which harbors a putative G protein binding domain, is essential for rescue. Rather, the extracellular part of the protein seems to be the business end. Expressing either the E1 or E2 extracellular domains was sufficient to rescue the APL-1 knockouts (see Hornsten et al., 2007). This finding is starkly reminiscent of recent work from Ulrike Muller and colleagues at Germany’s University of Heidelberg. These investigators showed that sAPPα, the α-secretase cleaved extracellular domain of APP is sufficient to rescue APP knockout phenotypes in mice (see below).

What is the function of the E1 and E2 APL-1 domains? This question is currently under investigation, said Li. What does appear certain is that the worms die because neuronal APP function fails, not for other reasons. That’s because only pan-neuronal expression of constructs led to rescue of APL-1 knockouts.

Sanjay Pimplikar described his studies on APP function in Danio rerio, better known to non-ichthyologists as the zebrafish. These tropical minnows have two APP homologs, APPa and APPb, both highly similar to human APP, especially at the C-terminal end. The human and fish AICD is 100 percent conserved, said Pimplikar, and in the complete cytoplasmic end of the protein there are only three conserved substitutions in human versus fish APP.

Pimplikar has used antisense oligonucleotides to knock down APP expression in fish embryos. He reported that silencing APPb alone was sufficient to induce severe morphological changes and to disrupt embryogenesis as early as 9-12 hours post-fertilization. The reason for these developmental problems is not known, but Pimplikar reported that human APP (hAPP) was capable of partially rescuing the APPb phenotype. Expression of hAPP reduced the number of deformed embryos and increased the number of normal embryos, though not to levels seen in wild-type zebrafish. Interestingly, hAPP with the Swedish mutation failed to rescue, which suggests that although the cytoplasmic end of hAPP is most similar to APPb, the extracellular domain is important for function, too. In this regard, Pimplikar suggested that APP processing and function may be intimately linked and inseparable.

The main advantages of working with zebrafish and worm models are the ease and speed of addressing specific questions. “The beauty of C. elegans is that we know exactly where all the neurons are and genetics experiments can be done so readily,” Guenette told ARF. But she also stressed that because of the greater biological complexity of mammals, it is important to replicate findings in mammalian models.

Guenette outlined some of her group’s work deciphering the role of APPs and their binding partners Fe65 and Fe65L1 in mouse brain development. Last year, Guenette reported that knocking out both Fe65 proteins gave a phenotype that resembled triple APP/APLP1/APLP2 knockouts (see Guenette et al., 2006). In the Fe65 double knockout (KO) mice, neurons appeared in the wrong places, particularly in the cortex. These so-called marginal zone heterotopias also appear in Muller’s APP triple KOs. Guenette found that in the Fe65 double KOs, levels and pattern of expression of APP were normal, as were levels of the intracellular domain AICD and APP C-terminal fragments. The data suggested that if Fe65 ensures proper brain development, then it does not do it by modulating APP levels or processing. But could Fe65 have some other effect on APP?

In San Diego, Guenette reported a subtle alteration in APP biology in Fe65 double KOs. She reported that treating wild-type cells with the glutamate analog NMDA elicits production of a high-molecular-weight form of APP that is substantially reduced in Fe65-negative cells. Guenette showed that this APP is not due to alternative splicing, but to post-translational modification by glycosylation. It is not yet clear if this high-molecular-weight form of APP is related to the developmental effects seen in Fe65 knockout mice. “Characterization of modified forms of APP can be pretty tricky,” said Guenette, but she noted that glycosylation can affect binding of Notch and its ligands. “The question for us is, Does glycosylation interfere with any signal that is essential for development?” she said. Guenette suggested that reduced glycosylation could be restricted to cells in a specific region of the brain, as that would explain why she does not see a difference in glycosylated APP in total brain lysates of the Fe65 knockout mice.

Tracy Young, from Dennis Selkoe’s lab at Harvard Medical School, also addressed the role of APP in development. Some of Young’s data was presented at a subsequent slide session. Young uses in utero electroporation of short hairpin RNAs to knock down APP to create mouse mosaics, with some cells expressing APP while others do not. This may be a crucially important point, because some of the phenotypes observed are not seen in APP knockout mice.

Young traced the APP-deficient cells by virtue of a green fluorescent protein (GFP) construct that is coexpressed with the shRNA. She showed how precursor cells that lack APP fail to migrate past the cortical plate. This does not seem to be a problem of motility, since the cells appear to migrate laterally. In fact, when Young focused on embryonic day-16 brains, 3 days after electroporation, she was able to see that GFP cells migrate out of the intermediate zone, before getting trapped at the cortical plate (ARF related conference story. Interestingly, the trapped cells begin to express the neuronal marker MAP2, suggesting that they begin to differentiate into neurons despite having their journey cut short.

By what mechanism does APP contribute to neuronal migration? Young has started to perform rescue experiments with various APP constructs to tease out factors that retard precursors at the cortical plate. She found that the migration defect does not appear when she electroporates in human APP constructs that are not recognized by the shRNA and are therefore expressed. Both the 751 and the 695 amino acid isoforms of APP rescue migration, as do APLP1, APLP2, and even APP with the Swedish mutation, Young reported. However, she found that the complete holoprotein is required; the extracellular or intracellular domains alone failed to rescue. Young also reported that rescue with full-length APP depended on an intact NPTY motif, found in the cytoplasmic tail. This motif binds a variety of proteins, including phosphotyrosine-binding proteins such as Dab1. In fact, Young reported that electroporation of Dab1 shRNAs lead to a similar phenotype as APP shRNA, with cells getting trapped at the cortical plate. Interestingly, Dab1 expression could partially rescue APP knockdown but not the other way around. Young suggested that this means that Dab1 works downstream of APP. It is worth noting that Dab1 has been shown to increase cell surface expression and processing of APP (see Parisiadou and Efthimiopoulos, 2006).

Both Hongmei Li from Tom Sudhof’s lab at the University of Texas Southwest Medical Center, Dallas, and Muller also addressed the role of various parts of the APP molecule. Li noted that although plenty of molecules have been discovered to bind to the cytoplasmic tail of APP, 80 percent of APP is in the extracellular space. Which is more important, she asked, the head or the tail of APP? To address this, Li attempted to rescue APP/APLP2 double KO mice.

Despite having what appears to be normal brain morphology and normal expression of synaptic protein, about 80 percent of these animals die early. Li tried to keep them alive with a flag-tagged construct that expresses only sAPPβ. Knocked in to the APP KO mice, this construct did not extend survival past postnatal day 21. It did bind to the molecular chaperone GRP78, otherwise known as BiP, however. Li said that presently it is unclear whether this binding is physiologically relevant or is merely related to misfolding of the flagged-APP protein. It is also unclear if the sAPPβ gets retained in the endoplasmic reticulum or is released from the cells.

For her part, Muller has had better success with rescue experiments using the extracytoplasmic end of APP. In San Diego, she summarized findings published last summer that sAPPα is sufficient to rescue APP KO mice (see Ring et al., 2007 and commentary).

Muller introduced a stop codon into the APP gene such that knock-in mice only expressed secreted sAPPα, the extracellular part of APP that is normally cleaved off by α-secretase. Muller found that this knock-in was sufficient to rescue the prominent phenotypes of APP-deficient mice, including their small body weight, weak grip, learning deficit in the Morris water maze, and loss of hippocampal synaptic plasticity. When crossed with APP/APLP2 double knockouts, the knock-in also survived longer. Muller noted that sAPPα levels are lowered in Alzheimer disease, which may partially explain learning and memory deficits in patients.

Overall, one important theme that emerged from the symposium was the role of the various parts of the APP molecule, said Guenette. In both C. elegans and in mice, the extracellular piece of APP, E1 or E2 in the case of worms or sAPPα in rodents, can rescue APP deficiency. In worms this seems particularly noteworthy, since these animals only have one APP homolog, APL-1, while in mice, APLP1 and APLP2 may partially compensate for APP loss. The ability of sAPPα to rescue at least some of the lethality seen in APP/APLP2 knockouts indicates the importance of this part of the molecule in mammals, as well. But the fact that the full-length molecule seems necessary to rescue developmental defects in mice suggests that different parts of the APP molecule may have different targets.—Tom Fagan.

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

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References

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Hornsten A, Lieberthal J, Fadia S, Malins R, Ha L, Xu X, Daigle I, Markowitz M, O'Connor G, Plasterk R, Li C. APL-1, a Caenorhabditis elegans protein related to the human beta-amyloid precursor protein, is essential for viability. Proc Natl Acad Sci U S A. 2007 Feb 6;104(6):1971-6. PubMed.
Guénette S, Chang Y, Hiesberger T, Richardson JA, Eckman CB, Eckman EA, Hammer RE, Herz J. Essential roles for the FE65 amyloid precursor protein-interacting proteins in brain development. EMBO J. 2006 Jan 25;25(2):420-31. PubMed.
Parisiadou L, Efthimiopoulos S. Expression of mDab1 promotes the stability and processing of amyloid precursor protein and this effect is counteracted by X11alpha. Neurobiol Aging. 2007 Mar;28(3):377-88. PubMed.
Ring S, Weyer SW, Kilian SB, Waldron E, Pietrzik CU, Filippov MA, Herms J, Buchholz C, Eckman CB, Korte M, Wolfer DP, Müller UC. The secreted beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J Neurosci. 2007 Jul 18;27(29):7817-26. PubMed.

San Diego: Exploring the Role of APP in Transit

06 Dec 2007

At this year’s Society for Neuroscience annual meeting, held 3-8 November in San Diego, California, a special ancillary symposium called “Function of APP Gene Family Members and Clues to AD Pathogenesis: Studies from Worms to Mammals” explored the normal and pathological roles of APP. An emerging theme was that different parts of the APP molecule might play different roles in the development of the nervous system (see Part 1 of this news story). Curiously, different parts of the APP molecule also seem to have non-developmental roles, both within and outside of neurons.

One APP domain that may play a developmental role is the NPTY motif that occurs in the C-terminal of the protein. An APP construct lacking this motif rescues APP knockout deficits but also reduces turnover of holo APP, increasing cell surface expression and reducing production of Aβ (see Part 1 of this story). This NPTY motif may have pleiotropic effects, since it binds Dab1 (see Part 1 of this story) and other key players in APP biology, including Fe65 (see Part 1). One of those effects could be to regulate intracellular transport. Stefan Kins and colleagues at the University of Heidelberg, Germany, recently showed that the NPTY motif is crucial for transport of synaptic markers in Drosophila (see Rusu et al., 2007). However, Kins’s data suggest it may not be important for the trafficking of APP itself, and with that addressed a debated question in the field (see ARF related conference story and crosslinks). In San Diego, Kins reported that a variety of different APP constructs, including those lacking the C-terminus, are transported to axons just fine in cultured neurons. Even the speed of transport is unaffected by removing the C-terminus.

Kins also reported that APP transport may be linked with that of other synaptic proteins and also with activity of the small GTPase Rab3. Using APP antibodies to immunoisolate proteins from the low-density membrane fraction of wild-type mouse brain, he showed that synaptic proteins snap25, syntaxin1b, and synapsin, Rab3 GTPase, its activating protein Gap (both p130 and p150 subunits), and Rab3 partners RIM and Munc13-1 are all pulled down. However, in a testament to the specificity of the antibody, none of these proteins could be isolated from APP knockout tissue using the same approach. The experiments suggest that all these proteins coexist with APP in cellular vesicles. So could transport of APP and Rab3, which is abundant in synaptic vesicles, be intertwined? Kins asked. He demonstrated that APP anterograde transport was diminished in genetically modified cells expressing Rab3 locked in the GTP bound state. Further, he found that Kinesin-1, normally present in APP-immunoisolations, was lost in APP-membrane isolations from mouse brains that predominantly contain Rab3 locked in a GTP bound state. All together, these findings link transport of synaptic proteins to APP and interconnect for the first time APP targeting and transport mechanisms. Interestingly, other Rab family members have been linked to APP processing (see, for example, Laifenfeld et al., 2007) and, via reduced isoprenylation, the ability of statins to lower Aβ production (see ARF related news story).

Angels Almenar from Larry Goldstein’s lab at University of California, San Diego, also addressed the role of APP in axonal transport, particularly the idea that it may couple cargo to kinesin. Almenar has taken a direct approach to this question, purifying synaptic vesicles and examining their contents. Using mass spectroscopy, Almenar has identified 280 potential candidates that might co-transport with APP and are currently being analyzed.

“The analyses of APP transport is still an important issue for AD research as it allows insights in the complex regulation of APP processing and the putative function of APP,” Kins told ARF after the symposium. “Interestingly, although Angels Almenar and I characterized very different APP membrane compartments, both analyses established a connection between APP with synaptic vesicles. This argues that APP dysfunction in AD may directly contribute to altered synaptic transmission and neurodegeneration,” he said.

The transport theme was echoed in a slightly different way by Hui Zheng from Baylor College of Medicine in Houston, Texas. Zheng suggested that one of APP’s primary roles is to ensure that a different kind of transporter, the high-affinity choline transporter (CHT), is appropriately expressed in presynaptic terminals. Because CHT recycles choline back to presynaptic neurons for re-synthesis into acetylcholine, loss of CHT could explain cholinergic deficits that occur in Alzheimer disease (see ARF related news story).

To study the relationship between APP and CHT, Zheng’s group has focused on the neuromuscular junction as a model system. In this peripheral, cholinergic synapse, neuronal synaptophysin normally is perfectly juxtaposed to muscle-bound αBungarotoxin, which has high affinity for the acetylcholine receptor. In APP and APLP2 double knockout mice, however, synaptophysin and the toxin only partially co-localize, as synaptophysin is diffusely distributed along axons. Zheng demonstrated that the same pattern emerges in CHT knockout animals, suggesting that APP and CHT may somehow cross paths.

In support of this idea, Zheng’s group demonstrated that CHT is absent from presynaptic terminals in APP/APLP2 double knockout mice, suggesting that APP may somehow help target CHT to its normal location (see Wang et al., 2007). To test this, her lab has made both pre- and post-synaptic APP knockouts where the protein is absent from either neurons or the neuromuscular junction, respectively. Zheng reported that the phenotype of the post-synaptic knockout is more severe. How can this be? Zheng speculated that an intercellular interaction between APP and CHT localizes the latter to the neuromuscular junction.

A symposium on APP can hardly get by without mention of Aβ. On that note, Orly Lazarov, who has established her own laboratory at the University of Illinois at Chicago, outlined some of her work linking APP processing with environmental enrichment (Lazarov et al., 2005). In that work, she demonstrated that mice exposed to enriched environments have fewer plaques and less Aβ40 and 42 than do animals kept in normal, boring lab cages. The reduction in Aβ species may be thanks to enhanced clearance, because Lazarov and colleagues found that environmental enrichment increased the levels of the Aβ-degrading enzyme neprilysin. In San Diego, Lazarov reported that she is currently conducting microarray analysis on environmentally enriched mice to better understand crosstalk between APP processing, synaptic activity, and transcriptional activation. In related news, Sam Sisodia presented data on the involvement of presenilin mutations and microglia in the response of mice to living in a more stimulating environment (see ARF related news story).

And last, but by no means least, Huaxi Xu from the Burnham Institute of Medical Research, La Jolla, California, took the audience into uncharted waters by studying the role of APP in non-neuronal tissue. Though APP is widely expressed in mammalian tissues, relatively scant attention is being paid to its role outside the nervous system. It may be prudent to take off the blinkers and look to extraneuronal roles of APP, given that loss of presenilin activity has been linked to skin lesions and tumorigenesis. Could this be related to APP processing? Earlier this year, Xu and colleagues reported that expression of the epidermal growth factor receptor (EGFR) goes up in fibroblasts lacking presenilin or nicastrin (see Zhang et al., 2007). Interestingly, normal EGFR levels are restored by adding back the relevant proteins, i.e., presenilin or nicastrin, but also by adding AICD, the APP intracellular domain (but not NICD, the Notch intracellular domain) to APP/APLP2 double knockouts which also have an increase in EGFR. These results suggest that production of AICD may attenuate EGFR expression. Indeed, Xu reported pulse-chase experiments showing that it was synthesis of the receptor and not its degradation that was altered by the various knockout and rescue experiments.

How does AICD regulate EFGR expression, Xu asked? It appears to work, in concert with Fe65, on the receptor’s promoter. Xu demonstrated chromatin immunoprecipitation experiments showing AICD bound to the EGFR promoter, while AICD together with Fe65 could attenuate elevated receptor levels in APP/APLP2 double knockout fibroblasts.

Taken together, these experiments suggest that APP processing may play a role in preventing tumorigenesis, since many cancers are driven by EGFR-mediated responses.

On a similar note, Xu introduced a new AICD-related protein called 168, which induces apoptosis. The protein decreased the membrane potential in mitochondria, which is an indication of mitochondrial dysfunction, and it increases activity of the apoptotic caspase-3. These effects seem to be related to AICD because APP/APLP2 double knockout cells exhibit less apoptosis than wild-type cells when transfected with 168 constructs, and 168-mediated caspase-3 cleavage was enhanced on addition of AICD. The results, if confirmed, indicate that in certain tissues AICD may protect against tumorigenesis in more ways than one, decreasing EGFR expression and enhancing programmed cell death.

Overall, the symposium demonstrated that while a lot has been learned in recent years about the non-amyloidogenic roles of APP, broad consensus is still elusive. Progress in mammalian research has been complicated by the presence of different isoforms, but with double and even triple knockouts and rescue experiments at hand, researchers are finally getting a grip on the various roles the different cytosolic and extracellular domains of APP are playing. Perhaps the major gaping hole in APP knowledge is how the extracellular domain exerts its effects.—Tom Fagan.

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

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References

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Rusu P, Jansen A, Soba P, Kirsch J, Löwer A, Merdes G, Kuan YH, Jung A, Beyreuther K, Kjaerulff O, Kins S. Axonal accumulation of synaptic markers in APP transgenic Drosophila depends on the NPTY motif and is paralleled by defects in synaptic plasticity. Eur J Neurosci. 2007 Feb;25(4):1079-86. PubMed.
Laifenfeld D, Patzek LJ, McPhie DL, Chen Y, Levites Y, Cataldo AM, Neve RL. Rab5 mediates an amyloid precursor protein signaling pathway that leads to apoptosis. J Neurosci. 2007 Jul 4;27(27):7141-53. PubMed.
Wang B, Yang L, Wang Z, Zheng H. Amyolid precursor protein mediates presynaptic localization and activity of the high-affinity choline transporter. Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):14140-5. PubMed.
Lazarov O, Robinson J, Tang YP, Hairston IS, Korade-Mirnics Z, Lee VM, Hersh LB, Sapolsky RM, Mirnics K, Sisodia SS. Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice. Cell. 2005 Mar 11;120(5):701-13. PubMed.
Zhang YW, Wang R, Liu Q, Zhang H, Liao FF, Xu H. Presenilin/gamma-secretase-dependent processing of beta-amyloid precursor protein regulates EGF receptor expression. Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10613-8. PubMed.

San Diego: Merck Reports BACE Inhibition in Primates

11 Dec 2007

The 37th annual conference of the Society for Neuroscience offered morsels of news on β-secretase-1 (BACE1), the enzyme that carries hope for a next generation of little white pills to treat Alzheimer disease. The effort to exploit Robert Vassar and Martin Citron’s discovery 8 years ago (Vassar et al., 1999) of this APP scissor for drug development has hit technical snags over the years. Even so, the general sense is that by now, a handful of pharmaceutical and biotech companies have compounds that are currently being optimized and at least a couple—one by CoMentis and possibly one by Takeda—have entered initial human tests. Whatever clinical data exist are unpublished, but in the meantime, read our brief summary of two preclinical developments reported last month in San Diego.

Adam Simon of Merck in West Point, Pennsylvania, presented a poster on his company’s study of a new, still-unnamed aminopyridine-oxadiazole inhibitor. First author Sethu Sankaranarayanan and colleagues tested its ability to lower Aβ40 and 42 in body fluids by infusing it intravenously into a so-called “cisterna magna-ported” rhesus monkey model (previously described in an ARF related news story). In this model, an in-dwelling catheter in the monkey’s cisterna magna at the base of the neck enables scientists to take repeated small samples of cerebrospinal fluid (CSF) to assess in real time how a drug affects a given set of readouts. Using six monkeys, the scientists infused two different doses of the BACE inhibitor for 4 hours, and for each experiment took samples before, during, and after the infusion. They measured a dose- and time-dependent reduction of up to 40 percent of Aβ40 and 42 in CSF, as well as a 60 percent reduction of Aβ40 in plasma. (When asked about plasma Aβ42, Simon said that it has been seen to change before, but the assay data do not meet the group’s technical standards so they don’t show them.) This is significant because one difficulty with developing BACE inhibitors had been that the relationship between inactivating the enzyme and the resulting drop of Aβ is not linear; for example, genetic halving of BACE in heterozygous knockout mice achieved only a modest reduction of Aβ concentration.

In the Merck study, CSF levels of the BACE1 cleavage product sAPPβ decreased by 10 percent. Theoretically, a reduction in BACE1 activity would leave APP available to sheddases and predict a corresponding increase in α cleavage. In practice, CSF sAPPα levels merely trended upwards in a non-dose-dependent fashion, indicating that there are layers of biological complexity that the scientists don’t understand yet, Simon said.

The Merck team began addressing concerns that BACE1 inhibition might cause demyelinating side effects, which had been implicit in the discovery last year that neuregulin-1 is a physiological BACE1 substrate during myelination early in life (Willem et al., 2006; Hu et al., 2006). Sankaranarayanan and colleagues first confirmed these findings using BACE1 knockout mice developed in Philip Wong’s laboratory. Then they infused Merck-3, a different, potent experimental BACE1 inhibitor (which is not suitable for human drug development because it does not enter the brain well; see Stachel et al., 2004) for a week into the brain ventricle of adult mice expressing wild-type human APP off a yeast artificial chromosome (see also ARF related news story). The inhibitor blocked BACE1, but levels of neuregulin-1, and various markers of myelination, did not change. This leads the Merck team to conclude, for now, that neuregulin-1 cleavage by BACE is important primarily during development and may not pose a problem in aged people. Careful studies looking for interactions of BACE inhibition with white matter changes in people or old mice have not been conducted.

The inhibitor presented here is better than previous ones in that it enters the brain and does not get extruded again by the efflux pump P-glycoprotein, as had earlier compounds by Merck, Wyeth, and other companies. However, in the process of “hiding” a BACE inhibitor from this pump, potency can diminish (see Moore et al., 2007). The inhibitor on the SfN poster is a different one from the ones discussed in this paper. Yet other scientists noted that the doses given to the monkeys were fairly high, and that its structure did not appear to suggest ideal drug properties. Large pharmaceutical companies frequently present data on projects that are behind their internal cutting edge, or on compounds that are not the real McCoy they are developing clinically. When asked about inhibitors in trials, Simon intoned the standard “Sorry, can’t confirm or deny.”

One thing this new Merck inhibitor cannot do is to be orally available, i.e., effective when swallowed. (Pharma companies much prefer pills to infusions. Merck collaborates with Sunesis to develop orally available BACE1 inhibitors.) On that front, researchers at Wyeth in Princeton, New Jersey, presented new data. Their BACE1 inhibitor WAY-258131 is a small molecule that, when fed to Tg2576 mice either once or repeatedly, decreased plasma and brain Aβ. When added to food for 3 months to 6-month-old PSAPP transgenic mice, WAY-258131 reduced their brain plaque load, first author David Riddell and colleagues reported on a poster. They also showed that the compound was able to reverse memory deficits in young, plaque-free transgenic mice, but not in old mice, invoking again the question of whether a secretase inhibitor can be enough once AD is established (see ARF related SfN 2005 story) For an open-access, up-to-date, and well-written review of all things BACE, see Cole and Vassar, 2007.—Gabrielle Strobel.

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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.
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.
Hu X, Hicks CW, He W, Wong P, Macklin WB, Trapp BD, Yan R. Bace1 modulates myelination in the central and peripheral nervous system. Nat Neurosci. 2006 Dec;9(12):1520-5. Epub 2006 Nov 12 PubMed.
Stachel SJ, Coburn CA, Steele TG, Jones KG, Loutzenhiser EF, Gregro AR, Rajapakse HA, Lai MT, Crouthamel MC, Xu M, Tugusheva K, Lineberger JE, Pietrak BL, Espeseth AS, Shi XP, Chen-Dodson E, Holloway MK, Munshi S, Simon AJ, Kuo L, Vacca JP. Structure-based design of potent and selective cell-permeable inhibitors of human beta-secretase (BACE-1). J Med Chem. 2004 Dec 16;47(26):6447-50. PubMed.
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Cole SL, Vassar R. The Alzheimer's disease beta-secretase enzyme, BACE1. Mol Neurodegener. 2007;2:22. PubMed.

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San Diego: α-Synuclein Protofibrils Create Sense of Déjà Vu in PD, DLB

18 Dec 2007

In some ways, Alzheimer disease research is about a decade ahead of research on Parkinson’s. As PD research expands and catches up, observers of both fields can’t help but notice how the latter appears to follow rather surprisingly in the tracks of the former. For example, one idea that is gaining currency in PD research holds that it is a conspiracy of oligomeric/protofibrillar species of the presynaptic protein α-synuclein that harms neurons, more so than do mature fibrils or the pathologic hallmark, i.e., the Lewy bodies. At the 37th annual conference of the Society for Neuroscience last month in San Diego, California, two presentations illustrated how this line of research is evolving in the wings of similar work on AD.

The hypothesis that monomeric α-synuclein passes through a series of aggregation states starting with a native monomer and ending with insoluble fibrils, and that the intermediate states may well be the worst, goes back to Peter Lansbury at Boston’s Brigham and Women’s Hospital (Goldberg and Lansbury, 2000; Volles and Lansbury, 2003). The number of labs working to test this hypothesis is steadily growing. In San Diego, Martin Ingelsson of Uppsala University, Sweden, presented early data of a new line of investigation that aims to characterize the fibrillization propensity of the α-synuclein mutations known to be at the root of early onset Parkinson’s. Previously, the larger Uppsala group led by Lars Lannfelt has been studying protofibril formation by the arctic Aβ mutation in familial early onset AD. As in AD, the heat is on in PD research to find the most toxic α-synuclein species. Unlike in AD, however, the work is still in vitro and no candidates have as yet been isolated from either genetic or sporadic PD brain (for comparison, see ARF related news story).

In PD, the A30P and A53T mutations in α-synuclein are known to cause early onset disease. More recently, a third mutation, E46K, proved to cause early onset dementia with Lewy bodies. DLB is an understudied double whammy of a bad disease, which robs people of their cognition and movement. In addition, rare α-synuclein duplications cause early onset PD, whereas triplications cause early onset DLB. With regard to the aggregation propensity of these mutant proteins, earlier work by others has shown that the A30P mutation slows aggregation down, whereas A53T speeds it up. In San Diego, Ingelsson reported how he, Joakim Bergström, and their colleagues compared side by side the speed with which the three mutant forms aggregate. This experiment confirmed the previous finding, and discovered further that the E46K mutation causing DLB was the most aggressive of all.

Next, the Swedish scientists combined size exclusion chromatography with high-performance liquid chromatography (SEC-HPLC) to characterize the intermediate species that form over time when wild-type α-synuclein is incubated in vitro and left to associate with itself. At the beginning, Ingelsson reported, the researchers saw only monomer. After about a day, smaller species than the monomer began appearing. These initial species could represent truncated monomer, invoking the speculation that they might act as seeds for aggregation, Ingelsson said. (Some scientists also suspect truncated species of tau of seeding aggregation.) At a later time point, aggregation proceeded to oligomeric forms larger than 600 kDa. Putting a precise molecular weight on these oligomers is difficult because their apparent weight on the chromatogram may not reflect their true size, Ingelsson said. But the Swedes are confident that one major species they are seeing are large protofibrils comprising about 50-100 monomers each. They do not see low-N oligomers in this system. When added to HEK cells in initial tests of toxicity, only the protofibrils managed to kill significant numbers of the cells; neither the monomer nor the fully formed fibrils did. (Tests in primary neurons are underway, Ingelsson said.)

Clearly, aggregation in vivo is more complicated because α-synuclein interacts with a multitude of factors, some of which are likely attempts to try to prevent damage to the cell. AD research has shown one such line of protection to come from the chaperone HSP70 and its partner CHIP, which mark the Aβ peptide for degradation if it cannot be properly folded (Kumar et al., 2007). In this regard, too, α-synuclein is following in the footsteps of Aβ in that its misfolding and aggregation appear to be subject to rescue attempts and disposal by CHIP, at least in vitro. Researchers at Massachusetts General Hospital had reported that CHIP can degrade α-synuclein (Shin et al., 2005). In San Diego, Julie Tetzlaff, a postdoctoral fellow in Brad Hyman and Pam McLean’s laboratory there, followed up by asking whether this happened to monomers or oligomers. To do that, Tetzlaff used a new cellular imaging trick, where she split green fluorescent protein and fused each non-fluorescent half to one α-synuclein molecule. When the α-synuclein monomers interact, dimers fluoresce. Using this bimolecular fluorescence complementation (BiFC) assay in transfected cells, Tetzlaff observed that CHIP reduced the fluorescence. A series of further experiments suggested that CHIP recognizes toxic oligomeric forms of α-synuclein and mediates their degradation. The present data are from human neuroglioma cells; work with dopaminergic cell lines is ongoing, Tetzlaff said. Beyond these two research groups, others as well have picked up on parallels in the aggregation of these two proteins. The focus lies on exploiting the similarities not only for mechanistic studies but also toward biomarker development in Parkinson disease.—Gabrielle Strobel.

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References

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Paper Citations

Goldberg MS, Lansbury PT. Is there a cause-and-effect relationship between alpha-synuclein fibrillization and Parkinson's disease?. Nat Cell Biol. 2000 Jul;2(7):E115-9. PubMed.
Volles MJ, Lansbury PT. Zeroing in on the pathogenic form of alpha-synuclein and its mechanism of neurotoxicity in Parkinson's disease. Biochemistry. 2003 Jul 8;42(26):7871-8. PubMed.
Kumar P, Ambasta RK, Veereshwarayya V, Rosen KM, Kosik KS, Band H, Mestril R, Patterson C, Querfurth HW. CHIP and HSPs interact with beta-APP in a proteasome-dependent manner and influence Abeta metabolism. Hum Mol Genet. 2007 Apr 1;16(7):848-64. PubMed.
Shin Y, Klucken J, Patterson C, Hyman BT, McLean PJ. The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways. J Biol Chem. 2005 Jun 24;280(25):23727-34. PubMed.

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