New Orleans: Immunotherapy—The Game Is Still in Town

21 Nov 2003

At the 33rd Annual Meeting of the Society for Neuroscience held last week in New Orleans, at least two dozen presentations dealt with current efforts to develop a vaccine for Alzheimer’s disease. This bold approach suffered a setback last year when meningoencephalitis in 6 percent of patients halted Elan/Wyeth’s (AN-1792 phase 2a trial), but it has since bounced back with a variety of approaches. Asked privately, a majority of scientists said they believe that vaccination will eventually work. These are among the most actively pursued questions: How best to avoid a harmful T cell response with clever design of Aβ construct and adjuvant choice; how well does amyloid clearance correlate with cognitive improvement; and is passive or active immunization the better way to go. Reflecting the ongoing pathophysiological debate about whether soluble oligomers/small aggregates or mature plaques do the most damage, some researchers place their bets more on targeting the former while others bank on clearing the latter. In truth, most researchers say that both sides overlap, and that perhaps a two-pronged approach could be found. Below are selected highlights.

Roger Nitsch at the University of Zurich recapped his group’s ongoing analysis of the still-blinded Zurich cohort of the AN-1792 trial (133.8). His data indicates that patients who formed antibodies declined less rapidly than those without a measurable immune response (see ARF related news story). Only some of the neuropsychological instruments used revealed a benefit; ADAS-Cog did not. The Zurich cohort had no plasma or CSF changes of Aβ. When grouped by the amount of antibody patients produced (high, medium, low), cognitive benefit correlated such that people with the highest titers tended to do best. This high-responder group included two patients who developed meningoencephalitis, Nitsch said. Overall, 18 people came down with the side effect, of whom 12 recovered within weeks but six remain with cognitive or neurological deficits, Nitsch added (see also Orgogozo et al., 2003). The side effect did not correlate with antibody titers, suggesting a T cell response.

MW Pride at Wyeth Research in Pearl River, New Jersey, with colleagues there and at Elan Biopharmaceuticals, reported on the cellular immune response by patients in the AN-1792 trial. The scientists isolated peripheral blood mononuclear cells (PBMCs) from patients in the phase 1 trial and phase 2a trials, and stimulated them with Aβ. Differences in the cytokine profile of the responding cells suggested that specific T cell responses elicited from phase 1 PBMCs were TH-2 biased, while those from the phase 2a were biased toward a proinflammatory TH1 response, the scientists write in their abstract. Both responses were directed against the C-terminus of Aβ, which is generally cut away in second-generation immunogens.

The reason for this differential response is still not entirely clear, but the adjuvant used, QS-21, is potent and known to elicit TH1-biased T cell responses, which can lead to autoimmune reactions.

David Cribbs of University of California, Irvine, with colleagues elsewhere, tried to redirect this TH1-response toward a presumably safer TH2-like response by using the adjuvant mannan. To enhance antigen presentation, Cribbs conjugated multiple copies of this group’s Aβ1-28 peptide to mannan in the form of “antigen trees.” Injecting small amounts of this into mice elicited responses directed at the first 15 amino acids of Aβ. The antibody types (mostly IgG) and cytokine profile indicated the response was biased toward TH2, Cribbs told the audience. To curtail the TH1 risk further, the team substituted the endogenous T cell epitope of Aβ with the synthetic T cell epitope PADRE (this is because a T cell activation is desirable to boost antibody titers, but need not be directed against Aβ/APP). This chimeric immunogen elicited high anti-Aβ antibody titers, yet T cells from mice immunized with it fail to respond to Aβ or APP, Cribbs said (133.6).

Hideo Hara of the National Institute of Longevity Sciences in Obu City, Japan, presented initial data on an oral vaccine that decreased amyloid deposits in transgenic mice without any adjuvant, nor with an apparent T cell response. This approach packages Aβ into an AAV vector and stimulates a mild humoral response via Aβ presentation to the mucosal immune system in the intestine, Hara reported (201.2).

One major question in the field concerns the role of microglia (see related New Orleans news story). Are they key to the removal of amyloid? Prior work by scientists at Elan and elsewhere had suggested that the Fc tails of Aβ antibodies bind Fc receptors on microglia, which activates them to ingest amyloid deposits. This is widely considered a major mechanism of amyloid clearance, and Donna Wilcock, with David Morgan and colleagues at University of South Florida, Tampa, developed an ingenious way to test it (133.1). She injected either full-length Aβ antibodies or F(ab)2 fragments lacking an Fc domain into frontal cortex or hippocampus of transgenic mice, and then suppressed microglial activation pharmacologically with dexamethasone, a new NSAID, or the antibiotic minocycline. The more these drugs dampened microglial activation, the less compact amyloid was removed near the injection site. F(ab)2 fragments were unable to activate microglia and were similarly inept at getting compact amyloid removed. Intriguingly, however, the fragments did clear diffuse deposits quite efficiently, suggesting that different mechanisms could be harnessed for the removal of mature plaques and the soluble pool. The work also suggests, though, that NSAIDs that inhibit microglial activation might undercut the effect of a vaccine and should perhaps not be administered simultaneously, the researchers said. The study is in press at Neurobiology of Disease.

A simple yet beautiful demonstration of how neurons perk up once plaques are gone came from Julianne Lombardo, working with Brian Bacskai and Brad Hyman at Massachusetts General Hospital in Charlestown. Neuronal dendrites that grow through and around plaques are known to look abnormal: Rather than growing straight through the neuronal parenchyma, these neurites are curvy and strangely swollen. Could clearing plaques restore their morphology, or is such a brain beyond repair? To address this question, the researchers injected a single dose of antibody into the cortex of PDAPP mice, sacrificed them, and examined their brains with immunohistochemistry. After four days, and lasting for about a month, amyloid plaques disappeared and, amazingly, the neurites simply straightened themselves out once the obstacles were gone. This experiment did not address corresponding behavioral benefits, and whether the aging human brain would right itself similarly is an open question. Still, the result suggests that the brain may be able to correct itself after a passive immunotherapy, and it extends a growing understanding of the plasticity of the adult brain. This work is in press at J. Neuroscience.

These images show SMI32 (anti-neurofilament) staining in the brain of a PDAPP mouse. The left side shows neurites in an untreated area of the cortex, away from where the antibody was applied. There is a large amyloid plaque within this field (not shown), and numerous distorted, curvy neurites. The right side shows a field of neurites within the 10d5 anti-A& antibody treatment area, showing neurites with normal, straight trajectories. This surprising "restoration" of distorted neurites occurs within four days after antibody treatment, and lasts at least up to 1 month.

A variety of different vaccination approaches clear amyloid and improve performance of various models. Elan and Wyeth scientists led by Peter (133.3) compared full-length Aβ1-42 with the amino-terminal fragment Aβ1-7, and passive immunization with monoclonals either against plaques (the 3D6) or against soluble Aβ (the m266 antibody). The Elan group still considers vaccines that trigger microglial phagocytosis most efficient, as both passive administration of 3D6 and active treatment with the small N-terminal Aβ fragment cleared amyloid pathology. A second presentation by Elan scientists (201.13) showed that AD patients in the AN-1792 trial had produced antibodies mainly against the first nine amino-terminal residues of Aβ, again suggesting the peptide’s C-terminus is dispensable for a strong humoral response.

As expected, the m266 antibody did not clear plaque deposits; this implies that rapid improvements in a learning task seen with this antibody occur because it interferes with acute effects of soluble Aβ, perhaps on synapses (see ARF related news story). Other scientists speculated that soluble Aβ might explain the day-to-day variations in cognition frequently seen in AD patients, rather than the gradual decline over time. Several groups presented work on antibodies which appear to be oligomer-specific and could remove soluble pools of Aβ. While such novel antibodies could yield rapid effects on cognition, the studies have not yet clearly demonstrated that these antibodies are specific to Aβ. Further research must show that they don’t cause problems by interacting with other physiological proteins that can oligomerize, such as insulin.

The m266 antibody has gained fame also because it fueled the peripheral sink hypothesis of AD vaccination, which holds out hope that passive immunization could “draw” out brain Aβ (see ARF related news story). This study raised a question about the staying power of this effect; in other words, can plasma Aβ increases after an antibody injection really predict future decreases in brain Aβ? Addressing this issue was a longitudinal study by the research collaboration between Steven Paul, Ron DeMattos at Eli Lilly and Company in Indianapolis, Dave Holtzman at Washington University, St. Louis, and colleagues. The scientists injected m266 into APPV717F mice at four and eight months of age, measured their plasma Aβ levels the next day, let them age, and at 12 months quantified their amyloid burden immunohistochemically. Results differed somewhat between Aβ42 and 40, but overall, the scientists reported that relative increases in plasma Aβ soon after immunization indeed correlated with, i.e., predicted, amyloid burden in the hippocampus and cortex at an older age (842.19).

Pritam Das and VG Howard, working with Todd Golde at the Mayo Clinic in Jacksonville, Florida, reported that passive immunization with an unusual antibody that recognizes the C-terminal end of soluble Aβ (most antibodies bind the other end) effectively reduced brain levels of Aβ40 and 42, while increasing plasma levels 25-fold. An ongoing longitudinal study will address cognitive effects, if any, pathology, and possible mechanisms (133.13).

Cindy Lemere’s group at Brigham and Women’s Hospital, Boston, presented the latest data on their immunization of the Caribbean vervet monkeys, following her presentation at last year’s CSH meeting (scroll to Lemere in ARF related news story). There, Lemere had reported high antibody titers and reductions in CSF Aβ after immunizing five aged monkeys with Aβ42. In New Orleans, Lemere reported that brain levels of insoluble Aβ42 were reduced by two-thirds in the immunized monkeys, while soluble Aβ40 levels were unchanged. Brains of immunized monkeys contained no Aβ42-immunoreactive plaques in six regions checked, but controls did (133.5). These results recapitulate results in mice and make this non-human primate an alternative model for vaccine development and other AD studies. The monkeys contained no tangles but did have hyperphosphorylated tau in neurites near plaques, Lemere said. The next immunization study will assess cognition in the monkeys before and after treatment. Lemere’s lab also presented characterizations of the mouse immune response following intranasal vaccination, and reported that genetic background and choice of adjuvant can greatly influence the nature of the immune response to immunization with Aβ and its peptide fragments (201.1, 201.11), suggesting that choice of adjuvant may be key in developing the human vaccine, too (see also Furlan et al., 2003).

Einar Sigurdsson and colleagues at New York University School of Medicine reported that a modified version of their K6Aβ1-30 vaccine (see ARF related news story), in which a hydrophobic region around amino acid 18 and 19 was altered, improved the performance of Tg2576 mice in the radial arm maze, even though antibody titers were low and the amyloid burden shrank only modestly, affecting only small plaques. The work suggests that massive amyloid clearance and high titers may not be necessary for a therapeutic effect (133.10).

Though approaches vary, it appears that the strongest benefit to date lies in preventing future deposition and mild cognitive improvement in older animals with severe deposition; neuritic plaques generally stay in place. No new trials were officially announced, though speculation was rampant. This news summary can’t be comprehensive. My apologies to all whose work went unmentioned; as always, I encourage additions and corrections. You can view abstracts mentioned in this story at the SfN/ScholarOne website.—Gabrielle Strobel.

References

News Citations

Paper Citations

Orgogozo JM, Gilman S, Dartigues JF, Laurent B, Puel M, Kirby LC, Jouanny P, Dubois B, Eisner L, Flitman S, Michel BF, Boada M, Frank A, Hock C. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology. 2003 Jul 8;61(1):46-54. PubMed.
Furlan R, Brambilla E, Sanvito F, Roccatagliata L, Olivieri S, Bergami A, Pluchino S, Uccelli A, Comi G, Martino G. Vaccination with amyloid-beta peptide induces autoimmune encephalomyelitis in C57/BL6 mice. Brain. 2003 Feb;126(Pt 2):285-91. PubMed.

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New Orleans: New Approaches to Lift Microglia Mysteries

21 Nov 2003

The role of inflammation in Alzheimer’s disease—beyond serious dispute, yet still enigmatic—gained much attention at the 33rd Annual Meeting of the Society for Neuroscience, held last week in New Orleans. While immunotherapy labs are retooling their approaches (see ARF related news story), other studies approached the functions of a key inflammatory operative—the microglial cell—by means of gene expression analysis. For example, Anna Parachikova, working with Carl Cotman at University of California, Irvine, presented a poster (629.26) suggesting that MHC class 2 antigens are upregulated in hippocampus early on in AD. She extracted mRNA from postmortem frontal cortex and hippocampus of seven people with mild dementia/MCI and 14 controls, and compared expression of 12,000 gene sequences. For further analysis, Parachikova used the genes in which expression changes came up as significant in two separate statistical analyses. Overall, those genes in which expression changed in the cortex tended to be upregulated (many of those were inflammatory markers), whereas gene changes in the hippocampus tended to be downregulations, Parachikova explained. A noticeable exception was MHC class 2 genes, in which expression in the hippocampus increased, as did their protein levels in Western blots. It is unclear whether this increased expression of genes involved in antigen presentation in the hippocampus occurs only on microglia or perhaps also on neurons, Parachikova said (see ARF related news story; see also Comparative Genomics section in ARF related news story). She suggests her study has picked up a region-specific increase in MHC 2 expression at the threshold to AD, which indicates that early in the disease the hippocampus contains microglia capable of devouring cells that present antigens together with MHC 2, and reinforces the view that inflammation is not a late event in the AD cascade.

That phagocytosing microglia would be prowling the AD hippocampus is neither new nor surprising, given that they are the resident macrophages in the brain. After all, most brain pathologies stimulate these cells in some way. Still, their role in neuroprotection versus destruction are ill-defined. Many scientists think that microglial activation is initially “adaptive” but gets out of hand in chronic neurodegeneration, in AD possibly egged on by the persistent presence of amyloid deposits. When does a protective activation tip over into a damaging one? Equally unclear are the functions of a microglial cell compared to those of infiltrating macrophages. To get a molecular handle on such questions, Monica Carson of the Scripps Research Institute in La Jolla, California, compared gene expression in resting versus activated microglia to identify genes that microglia express in response to various inflammatory stimuli (527.5). She then used in-situ hybridization to characterize expression of these genes in normal mice and models of multiple sclerosis and AD.

Among the genes upregulated in response to AD-related cytokines, she found the microglial receptor TREM-2 and its adaptor protein DAP-12. The function of these genes is poorly understood, but Carson said they rev up the microglia’s ability to present antigens and activate T cells. TREM-2 and DAP-12 expression in normal brain is highest in entorhinal cortex and hippocampus, and the APP23 transgenic mice from Novartis Pharmaceuticals showed robust expression of these genes in microglia near amyloid plaques. Overall, the profiles of gene expression among microglial populations were highly heterogeneous, varying by brain region and surrounding pathology. In addition to T cell reactions (Monsonego et al., 2003), individual variations in these microglial responses might help explain why a subset of patients developed inflammation in Elan’s halted vaccine trial, Carson suggested.

Among dozens of presentations on microglial biology and activation, several corroborated this notion of microglial heterogeneity. To name but one, Michael Dailey and colleagues at the University of Iowa in Iowa City imaged slices of mouse and rat hippocampus with two-channel fluorescence and time-lapse confocal microscopy (743.13). These authors noted marked differences in the expression of early activation markers such as NFkB or leukocyte function antigen (LFA-1), as well as in the microglia’s motility in response to injury. Together, these studies reinforce the complexity of the task at hand, namely, of turning to therapeutic advantage, or at least disarming, these still-enigmatic cells.

Does brain pathology lure outside macrophages? Another lingering question about microglia appears close to an answer, however. It is this: Are the activated microglia found near amyloid deposits prior residents of the brain, or have at least some of them recently immigrated from across the blood-brain barrier? Microglia derive from blood-derived monocytes made throughout life in the bone marrow. Tarja Lappetelainen, working with Jari Koistinaho and others at the University of Kuopio, Finland, brought classic immunology to bear on this issue (945.4). Using irradiation, the scientists first wiped out the immune system of 21-month-old APP/PS1 transgenic mice and non-transgenic controls, and then transplanted back the bone marrow from mice overexpressing green fluorescent protein. Four weeks later, they measured by flow cytometry whether the graft had taken hold and was producing blood cells; it was. Fourteen weeks later, they checked for green fluorescent cells in various brain areas. Both mice strains had equal numbers of GFP cells, indicating that bone marrow-derived cells continuously filter into the brain. While some of these cells appeared to cluster around amyloid plaques, on the whole this study detected no difference between the wild-type and APP/PS1-trangenic mice in the amount of infiltrated GFP-positive cells. Does that mean there is no specific response by the peripheral immune system to CNS amyloid pathology? Probably not. “The mice used in this study were very old, with fully established Aβ plaque pathology and related gliosis already at the start of the experiment. Therefore, it is not surprising that the extent of infiltration is not greater in the transgenic mice. Clearly, more studies are needed to investigate the effects of blood-derived microglia on the development and progression of Aβ pathology,” comments coauthor Milla Koistinaho. You can view abstracts mentioned in this story at the SfN/ScholarOne website.—Gabrielle Strobel.

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New Orleans: Symposium Probes Why Synapses Are Suffering

25 Nov 2003

Are you sometimes tempted to view APP and presenilin merely as raw material and machine for Aβ production, respectively? If so, think again, because that narrow spotlight is opening up to a broader understanding of how these two proteins function and might contribute to Alzheimer’s in their own right, as it were. At the annual meeting of the Society for Neuroscience held earlier this month in New Orleans, Jie Shen of Brigham and Women’s Hospital in Boston led a symposium on synaptic changes in Alzheimer’s disease, a topic that overlaps in large part with new areas of investigation for APP and PS. The event drew a packed auditorium, in part, perhaps, because the original Richard Morris of water maze fame was on hand, as well. (Developed in the 1980s for the study of hippocampal memory, this mouse swimming pool has become de rigueur for any Alzheimer’s mouse lab trying to prove their favorite intervention is meaningful in vivo.) New functions proposed for APP and PS included axonal transport, formation of the synapse, and synaptic transmission and its related signal transduction pathways. One provocative upshot of this new data—briefly debated at the symposium—was that presenilin mutations may cause memory loss and neurodegeneration not just through a toxic gain of function—the widely accepted notion—but in a more complex manner that involves also the loss of separate physiological functions of PS in learning and memory. Here’s a summary of the symposium presentations, plus another talk that tied in tau with axonal transport.

Presenilin: A Memory Molecule?
Shen first reminded the audience that synaptic loss remains the best early correlate of cognitive impairment in AD, and is accompanied by a “dying back” of dendrites and axons, and by aberrant sprouting (for a recent review, see Hashimoto et al., 2003). Of the known familial AD mutations, only 16 are in APP, and they are rare (see APP mutations directory; see also ARF related news story). By contrast, the literature contains about 143 mutations in presenilins 1 and 2 (see PS mutations directory; for the latest additions, see Tedde et al., 2003), and they are far more common. Do they cause AD through a gain of function or a loss of function? Speaking for the former are their dominant inheritance pattern and evidence that they increase Aβ42 levels. At the same time, however, PS mutations are scattered throughout the coding sequence of the two genes, and this is consistent with a partial loss of function, Shen said.

Shen took a genetic approach to understand the normal function of presenilin in the postnatal brain. Since PS 1 and 2 are important for embryonic development, knockout mice die too early for the study of these proteins' roles later in life. Shen and colleagues, therefore, generated a line of neuron-specific conditional PS1 knockout mice, which are normal anatomically, have lower Aβ production, and show a surprisingly subtle spatial memory deficit (see ARF related news story). Next, Shen’s group crossed these conditional PS1 knockouts with APPswe+717e transgenic mice made in Lennart Mucke’s lab and asked whether inactivating PS1 could prevent the amyloid pathology in these mice without side effects. (Besides exploring the normal role of PS, this experiment mimics genetically a γ-secretase inhibitor treatment for AD; see also ARF related New Orleans story.) As expected, these double mutants have neither amyloid pathology nor the related gliosis and inflammation. However, collaborative studies with Alfredo Kirkwood’s group at Johns Hopkins University showed that eliminating PS1 did not rescue the LTP impairment. It did not completely rescue the behavioral deficits of the APP transgenics, either, Shen's and Morris' groups found (see also 295.13). The double mutants temporarily performed better on an associative memory task, but by the age of six months, had fallen behind again, plus they performed poorly in spatial reference tests. “This mixed phenotype highlights the importance of comprehensive testing of AD models using multiple behavioral paradigms,” said Shen.

Up to this point, PS2 could compensate for PS1, potentially muddying the results. To shut this escape hatch, Shen’s lab crossed their conditional PS1 knockout to a PS2 knockout strain from Karen Duff’s group, creating mice that express PS1 until a month after birth and then continue life without any presenilin in their excitatory forebrain neurons. These mice, too, seem fine at two months of age, but go downhill from there, Shen told the audience. At this age, prior to any pathology, the mice show mild deficits in associative and spatial memory, as well as in short-term plasticity and LTP. The latter prompted the researchers to investigate NMDA receptor-mediated responses and found reductions there, as well. What’s more, NMDA receptor and CaMKinase II levels in synaptoneurosome preparations were reduced. Postdoc Carlos Saura presented these data in a slide talk (239.2). By six months, the mice were severely impaired in spatial learning and memory, failing to learn conditioned fear or the Morris water maze. The mice also had shrunken white matter, and by nine and 16 months (few mice survive to this age), they suffer progressive neurodegeneration, with reductions in synaptophysin levels, dendritic number and complexity, number of cortical neurons, and cortical volume. Gliosis accompanies this process, Shen reported.

What molecular webs might underlie this profound phenotype? Shen’s suspicion fell on the CREB/CREM pathway, in part because prior work has established it as a mediator of synaptic plasticity, hippocampus-dependent learning, and in part because mice mutant in these factors show impaired LTP and neurodegeneration (see Pittenger et al., 2002; Chen et al., 2003). Indeed, at two months, these mice had downregulated a list of genes whose expression depends on CREB, including BDNF, c-fos, and others. Total levels of CREB itself or its phosphorylated version were unchanged, but levels of CREB-binding protein (CBP) were down, suggesting that presenilin is somehow necessary to maintain normal levels of CBP. Tau was hyperphosphorylated and p25 upregulated.

Shen invited the audience to ponder a working model for memory impairment in which problems with presenilin function would, as one of the earliest steps, lead to impaired synaptic plasticity through a downregulation of the CREB pathway. While at first blush these data seem to flatly contradict the amyloid hypothesis, Shen explained that the story is more complex than that. "There is truth to both sides as some PS mutations clearly increase Aβb42 generation. I think that, depending on the downstream targets, PS mutations can lead to the disease through both loss and gain of function," she concluded.

Richard Morris, of the University of Edinburgh, Scotland, noted that finding a suitable behavioral model for AD is a formidable task because the clinical diagnosis of this heterogeneous disease requires memory impairment plus at least one other cognitive disturbance, for example aphasia or deficits in decision-making. Even just the memory component features differences in working, episodic, remote, and semantic memory, Morris explained, with striking deficits in episodic memory early in the disease. How can one model that? By now, numerous mouse models show behavioral deficits (for example, the Tg2576, App23, CRND8, and SAMP8 strains), most of which are assessed using the water maze and the radial arm maze introduced by Gary Arendash and Dave Morgan at the University of South Florida in Tampa.

In earlier studies of the PDAPP mouse, with Guiquan Chen at the University of Edinburgh and Karen Chen and others at Elan Biopharmaceuticals in south San Francisco, Morris tried to distinguish between the ability to encode, store, and retrieve memories (Chen et al., 2000). To that end, he devised a serial spatial-learning task that involves longitudinal testing of an animal and allows for the teasing apart of the different processes of memory. A key result of this work was that these animals seem to forget what they had learned faster because of a specific defect in memory retrieval. Morris noted that a decline in excitatory transmission might underly this effect.

APP: A Synapse Builder?
Hui Zheng of Baylor College of Medicine in Houston presented data implying a physiological role for APP and some of its family members in synapse formation. She proposed that impairments in this function could perhaps also contribute to the synaptic deficits in AD. Zheng pointed out that Aβ is but one of many APP processing products, all of which are affected by FAD mutations. She said it remains unclear whether the synaptic damage in vivo occurs via Aβ, other APP cleavage products, APP-related signaling pathways, or a combination of these. Zheng added that other APP family members such as APLP1 or 2 are also suspects, given that they are abundantly expressed in neurons and found in dendrites.

Zheng and colleagues have further examined their APP-knockout mouse (Zheng et al., 1995). In addition to gliosis, an LTP defect, and behavioral deficits in the water maze, this mouse also moves around less and has a weak grip. The mice have normal numbers of neurons, but their dendrites were abnormally short. Then, Zheng crossed APLP2-knockout mice, which appear normal, with the APP knockouts and found that the offspring died soon after birth, much like a similar set of mice created in Ulrike Muller’s lab (Heber et al., 2000). A growing number of double knockouts of either APP and APLP1 or 2 now exist, Zheng said, offering a chance to sort out the more subtle synaptic functions of these proteins.

The locomotor defect and weak grip of the mice inspired Zheng to study their neuromuscular junctions as a peripheral model of what might be going wrong with synaptic development in the late embryonic stage. The scientists found that the double knockouts had fewer synaptic vesicles on the presynaptic side, as well as a defect in the proper distribution and positioning of presynaptic vesicles opposite acetylcholine receptors on the postsynaptic membrane. Slightly later, these faulty synapses sprouted neurites excessively, Zheng reported. It’s not clear yet whether the vesicle defect has to do with transport or some other aspect of vesicle release and recycling, but it is clear that the neuromuscular junctions were defective in electrophysiological measures of presynaptic function. This synapse-forming role of APP/APLP2 is the first in-vivo demonstration of a physiological function of the APP family of proteins, Zheng said. Whether something similar happens in central synapses, and whether this could explain the behavioral and LTP deficits in APP-knockout mice, are open questions at this point, she added.

Starving Synapses: Axonal Transport a Common Theme?
If the APP family and the presenilins play important physiological roles at synapses, then surely trouble looms if their delivery dries up. In this sense, blockages of axonal transport could provide another mechanism of AD pathogenesis, and indeed, a number of groups have described axonal transport defects in different neurodegenerative diseases in recent years (see ARF related news story; ARF news; ARF news). At the symposium, Larry Goldstein of University of California, San Diego, set the stage by describing just how enormous a transport burden neurons face. Their “railway” system of tracks (the microtubules) and engines (the kinesin and dynein families of motor proteins) shuttle most of the neuron’s biosynthesis products down the axon and other materials (such as NGF) back up to the nucleus. Axons are narrow and easily jammed by stalled vesicles; distances are immense. Goldstein briefly recapped his lab’s prior work on huntingtin (see ARF related news story) and on Alzheimer’s, including Drosophila studies suggesting that APP serves as an anchor for kinesin (see ARF related news story), which mediates transport of vesicles containing APP, BACE, and γ-secretase to the synaptic terminals (see ARF related news story). This work led to a working model in which APP interacts with kinesin, perhaps with JIP-1b as a scaffold (see Inomata et al., 2003), to transport a cargo vesicle that contains the APP processing machinery. Transport defects could be physical, i.e., plugging of the axon by protein aggregates, and/or could occur in a positive feedback loop whereby stalling of vesicles would increase local processing of APP in the axon. Increased APP or impaired kinesin function could also set off this speculative cycle, as could endocytosis of Aβ, Goldstein said.

Goldstein then presented new experiments in mice that aimed to test the prediction that APP overexpression should poison axonal transport by titrating APP away from kinesin binding. Postdoc Gorazd (Goghy) Stokin first studied the morphology of cholinergic axons in basal forebrain of APPSwe mice made by Richard Bochelt, and found numerous axonal swellings, some as large as 10 microns (see also 445.6). In the electron microscope, these swellings resemble those seen in axons of APP-overexpressing fruit flies and contain numerous vesicles and organelles, including mitochondria. Some look as though they are degenerating. Initial data from an ongoing analysis of human AD tissue hint that it, too, contains similar axonal swellings, Goldstein added. Could these axonal swellings be precursors of the dystrophic neurites that are a hallmark of the AD brain?

Next, Stokin crossed the APP transgenic mice to strains deficient in a kinesin light chain gene to ask whether reducing the amount of kinesin would disrupt axonal transport in mice as it did in flies. When he looked for axonal swellings in cortex, he found none in wild-type mice, some in the APP transgenics, and abundant swellings in the double-transgenics. To ask whether reduced transport increases APP processing, the scientists measured amyloid pathology in the double-transgenic mice and found that reducing kinesin accelerated the age-related increase in Aβ levels and plaque deposition. In preliminary experiments, the scientists also saw the distribution of plaque deposition shift in such a way that the fraction in the entorhinal cortex (which projects through the perforant pathway to the dentate gyrus of the hippocampus and is affected early in AD) appeared to increase relative to deposition in the dentate gyrus (see ARF related news story). While these issues need further study, Goldstein argued that APP processing likely is regulated by kinesin in some way.

And, in turn, kinesin may be regulated by tau. In a separate session in New Orleans, Eva-Maria Mandelkow talked about a new relationship between tau and the movement of APP vesicles down the axon (336.6). She proposed that the kinase cascade MARKK-MARK controls this process by phosphorylating tau and causing it to come off the microtubules. Her presentation revolved around tau’s role in limiting vesicle transport through its competition with kinesin for microtubule binding. Overexpression of tau leads to a redistribution of organelles back to the cell body and thus starves the synapse, Mandelkow explained. Her lab has created conditional tau transgenic mice to study subtle changes early in the disease process, long before hyperphosphorylated tau detaches entirely from microtubuli, causing them to disintegrate. While these mice are still too young to use in studying brain aging, cortical neurons cultured from the mice yielded some clues. These neurons lose their dendrites and axons, and quickly die. In normal neurons, anterograde transport of APP-containing vesicles predominates, but in the tau-overexpressing neurons, anterograde transport slows down and retrograde transport predominates. Interestingly, activating the MARKK-MARK pathway in these cells restored anterograde transport, suggesting that the increased MARK activity observed in degenerating neurons may be a protective response by neurons trying to contain tau. Mandelkow believes that even a small increase in tau may be sufficient to disrupt the flow of traffic to the synapse (Timm et al., 2003).

Stepping back to take a broader view, Goldstein even wonders if different neurodegenerative diseases could reflect divergent outcomes of a common dysfunction such as axonal transport, much like different types of cancer are all outcomes of common problems with cell cycle control. A lot of future work is needed to test this leap, some of it being a search for kinesin SNPs that cause subtle differences in kinesin activity and could, over time, impair axonal transport. You can view abstracts mentioned in this story at the SfN/ScholarOne website.—Gabrielle Strobel.

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Inomata H, Nakamura Y, Hayakawa A, Takata H, Suzuki T, Miyazawa K, Kitamura N. A scaffold protein JIP-1b enhances amyloid precursor protein phosphorylation by JNK and its association with kinesin light chain 1. J Biol Chem. 2003 Jun 20;278(25):22946-55. PubMed.
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New Orleans: Out Go Classic γ-Secretase Inhibitors, In Come More Dexterous NSAIDs?

26 Nov 2003

Many academic scientists find news about industry research hard to find. It travels by word of mouth, formal publications appear late, if ever. This is true of γ-secretase inhibitors, which for a while were prime candidates for the next generation of AD drugs. Then, additional targets for this complex enzyme were discovered and rumors about side effects cast doubt over this therapeutic strategy. So it was noteworthy that Eric Parker from the Schering-Plough Research Institute in Kenilworth, New Jersey, reported at the Society for Neuroscience meeting in New Orleans what many may have heard already: At least some γ-secretase inhibitors cause mechanism-based side effects so severe as to disqualify them from clinical testing.

Parker reported on experiments with a potent γ-secretase inhibitor that also inhibits Notch cleavage (LY-411,575), and a less potent stereoisomer to serve as a control (133.11). The work was conducted in large part by Gwen Wong, who is now at ALS-TDF. The researchers fed LY-411,575 for 15 days to wild-type mice and CRND8 APP transgenics, then they measured plasma and brain Aβ levels, studied effects on the immune system by flow cytometry, and examined tissues histologically.

As expected, LY-411,575 decreased plasma and brain Aβ levels. However, at the concentrations where it did so, it also increased the size of certain immature thymocyte and B lymphocyte populations while reducing the number of mature T cells in the thymus and mature B cells in spleen and blood. Specifically, LY-411,575 blocked the physiological transition of certain double-negative (CD4-CD8-) populations of immature lymphocytes (e.g., the Cd44+/25+ set) to their single-positive, differentiated state (e.g., the CD44+/25- set). What’s more, the normal cellular architecture of the mice’s intestinal villi looked abnormal, and the mucosa showed goblet cell hyperplasia. At the higher inhibitor doses tested, the mice lost weight and died, probably due to the intestinal side effects, Parker said. The histology of the brain, liver, kidney, lung, heart, adrenal gland, bone marrow and stomach appeared normal. The T cell effects predictably resulted from inhibition of Notch cleavage, as Notch is known to function in thymocyte development, but the B cell and intestinal effects were unexpected, Parker added.

These results still leave open the possibility of inhibiting γ-secretase with other inhibitors that distinguish between APP and Notch. Alas, recent reports suggest that’s easier said than done, at least with classic strategies of fitting competitive inhibitors into the enzyme’s catalytic pocket. Researchers at Merck, Sharpe and Dohm in Harlow, United Kingdom, reported this summer that a range of such inhibitors drawn from six different compound classes were all unable to distinguish clearly between APP and Notch in vitro (Lewis et al., 2003; see also Shearman section in ARF related news story). Others have reported that γ-secretase inhibitors cause developmental defects consistent with Notch inhibition in fruit flies (Micchelli et al., 2002) and zebra fish (Geling et al., 2002). For a review, see Tsai et al., 2002).

At the same time, however, a flurry of meeting presentations on NSAIDs is pointing to a new way of achieving this goal. These studies explore in more detail the mechanism by which certain NSAIDs inhibit γ-secretase in non-competitive ways. At this point, results vary depending on the assay, dose, and compounds used, and some data contradict each other. Overall, however, the new hope is that an existing NSAID can be found—or, more likely perhaps, a new one designed—that tweaks γ-secretase allosterically, i.e., outside of the active site. It would have to do so in such a way that APP cleavage shifts away from generating Aβ42 and toward Aβ38 (which by all accounts so far is safe to have in increased amounts), while leaving alone proteolysis of Notch and other targets, such as ErbB-4. For more detail, view abstracts 295.2, 295.7, 295.21, 295.22, 336.8, 336.9, 523.3, 549.4, 729.1, and 876.14 at the SfN/ScholarOne website.—Gabrielle Strobel

References

News Citations

Paper Citations

Lewis HD, Pérez Revuelta BI, Nadin A, Neduvelil JG, Harrison T, Pollack SJ, Shearman MS. Catalytic site-directed gamma-secretase complex inhibitors do not discriminate pharmacologically between Notch S3 and beta-APP cleavages. Biochemistry. 2003 Jun 24;42(24):7580-6. PubMed.
Micchelli CA, Esler WP, Kimberly WT, Jack C, Berezovska O, Kornilova A, Hyman BT, Perrimon N, Wolfe MS. Gamma-secretase/presenilin inhibitors for Alzheimer's disease phenocopy Notch mutations in Drosophila. FASEB J. 2003 Jan;17(1):79-81. PubMed.
Geling A, Steiner H, Willem M, Bally-Cuif L, Haass C. A gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep. 2002 Jul;3(7):688-94. PubMed.
Tsai JY, Wolfe MS, Xia W. The search for gamma-secretase and development of inhibitors. Curr Med Chem. 2002 Jun;9(11):1087-106. PubMed.

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New Orleans: Plant Chemical a Protein Aggregation Buster?

03 Dec 2003

By Erene Mina. While modern medicine races into the future, it tends to overlook the possibility that remedies might lie in the past. At the 33rd Annual Meeting of the Society for Neuroscience, Anthony Fink and M. Zhu of the University of California, Santa Cruz, reported that baicalein, a flavanoid used in traditional Chinese medicine, binds tightly to α-synuclein and even disaggregates fibrils (SfN abstract 132.12).

Fink’s group monitored fibrillization of α-synuclein with thioflavin-T in the presence of various concentrations of baicalein. They found fibrillization was inhibited after 45-hour incubation of 70µM α-synuclein with 20μM baicalein. Furthermore, baicalein had no effect when it was added to fibril seeds of α-synuclein, indicating that baicalein works prior to the nucleation step of fibrillization.

Baicalein was also added at different time points in the fibrillization process and monitored via thioflavin-T fluorescence and light scattering. In the preliminary stages of fibril assembly, baicalein was able to inhibit fibril formation completely. When baicalein was introduced at later time points, it blocked fibril assembly and even disaggregated the α-synuclein fibrils.

After treating fibrils for four hours with baicalein, Fink and Zhu found that the fibrils had disaggregated into oligomers. Atomic force microscopy revealed that these were globular as well as donut-shaped oligomers, about 32-45nm in diameter. This is intriguing because some scientists think that donut- or pore-shaped oligomers precede fibrillization (for a review, see Volles and Lansbury, 2003). Moreover, baicalein has been shown to inhibit lipid peroxidation; α-synuclein is also known to bind lipids.

Fink’s group also reported that this phenomenon was seen when Aβ fibrils are treated with baicalein. However, from what is currently understood about the toxicity of Aβ and amyloid oligomers generally, it does not seem as though disaggregating fibrils into oligomers is preferable. Amyloid fibrils have long been thought to contribute to neurotoxicity, but this idea is slowly being replaced by the notion that amyloid intermediates exert much of the toxicity. Fink and colleagues have yet to test the toxicity of the oligomers produced by this process (See also Lebeau et al., 2001). You can view abstracts mentioned in this story at the SfN/ScholarOne website.—Erene Mina is a Ph.D. student at University of California, Irvine.

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References

Paper Citations

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.
Lebeau A, Esclaire F, Rostène W, Pélaprat D. Baicalein protects cortical neurons from beta-amyloid (25-35) induced toxicity. Neuroreport. 2001 Jul 20;12(10):2199-202. PubMed.

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New Orleans: Kelly Dineley Reports from Satellite Social

03 Dec 2003

This meeting report is anchored by Kelly Dineley, with individual presentations written by the respective investigators.

At the 33rd Annual Meeting of the Society for Neuroscience in New Orleans, approximately 100 scientists gathered to discuss informally what roles the interaction between Aβ and the nicotinic acetylcholine receptor (nAChR) play in normal cognitive function and Alzheimer's disease. This meeting was prompted by the increasing number of reports of high-affinity interactions between Aβ peptides and nAChRs. The type of interaction (agonist vs. antagonist; competitive vs. non-competitive) varies, as does the subtype (α7 vs. α4/β2) and location (presynaptic vs. postsynaptic vs. somatic) of the nAChR involved.

After a brief introduction, David Sweatt, Baylor College of Medicine, Houston, Texas, posed the following six questions which were addressed through open discussion. I have answered them based upon that dialogue:

1. Does Aβ interact with nAChRs? Yes. There are several examples in the literature, as well as approximately 12 abstracts submitted to the present SfN meeting. Which receptors? There are reports for α4/β2 and α7-containing neuronal nAChRs, as well as the muscle nAChR.

2. Does Aβ activate or inhibit nAChRs? There is evidence in the literature for both. On the surface these reports appear contradictory; however, the biological preparations and methods of detection vary among laboratories. These differences indicate that the cell population, subcellular location, accessory proteins, and posttranslational modifications may influence the net effect of Aβ on nAChRs.

3. Is this interaction good or bad? Possibly both, depending on the concentration and aggregation state of the peptide. Sally Frautschy, University of California, Los Angeles, reported that infusion of Aβ peptide into cerebral ventricles of rodents initially improves cognitive performance, followed later by impairment (as Aβ accumulates, in theory; see ARF related news story). Clearly, further study is needed.

4. What is the normal physiological role of Aβ-nAChR interaction? It is noteworthy that non-demented brain contains picomolar concentration of Aβ. a concentration sufficient for interaction with nAChRs. This suggests that Aβ-nAChR interaction has a biological function under normal physiologic conditions. Published studies indicate that Aβ-nAChR interaction leads to second messenger system activation, intracellular Ca2+ increases, and increased neurotransmitter release. Other studies report that Aβ interaction with nAChRs leads to receptor antagonism, indicating that the precise location and conditions under which receptor-peptide interaction takes place can lead to quite varied outcomes. Thus far, it appears to be a complicated relationship, and we will gain clarity only through further research.

In addition, a role in Aβ metabolism was cited, since the literature has documented nAChR-mediated endocytosis. The role of astrocytes and a possible link to inflammation have precedence in the literature, as well. Finally, since it appears that muscle nAChRs and Aβ interact, there are implications yet to be explored for muscle function and peripheral amyloid disease.

5. What are the therapeutic opportunities based on Aβ-nAChR interaction? Discussion of this question mainly led to more questions: Should we block the interaction? What if nAChRs serve to remove Aβ from other deleterious interactions? Should we provide more nAChR-like binding sites as a decoy for Aβ-nAChR interaction? Is there a basal level of good Aβ-nAChR interaction? Our understanding of Aβ-nAChR interaction is too inchoate to decide these issues.

6. What important future directions might this research take? One suggestion was to define clearly where Aβ and nAChRs interact. Based on known nAChR and Aβ biology, participants urged investigation of potential intracellular interactions between the two proteins, in addition to the well-documented extracellular contact.

An additional point was to encourage a more accurate structural description of the β-amyloid used for study. Thus far, most investigators report that they prepare freshly solubilized, nonfibrillar Aβ for experimentation. However, few report direct investigations into the structure of their Aβ mixtures. Below are summaries of some individual presentations.

Kelly Dineley, University of Texas Medical Branch, Galveston, presented preliminary data obtained from cognitive testing of a new mouse model for altered nAChR function and β-amyloid overproduction. The model is a genetic cross between mice null for α7 nAChRs and the Tg2576 strain. Tg2576 mice exhibit an associative learning deficit at five months of age as measured with the rodent fear-conditioning paradigm. Coincident with this is an upregulation of α7 nAChRs and dysregulation of ERK MAPK signaling in the hippocampus of these animals (see also SfN abstract 945.13). Proper ERK MAPK signaling is necessary for rodent fear learning. Five-month-old Tg2576 are impaired in the contextual test for fear learning 24 hours following two pairs of conditioned stimulus (CS:cue) and unconditioned stimulus (US:footshock). This impairment is overcome with a more rigorous training schedule of five pairs of CS-US. Five-month-old animals lacking α7 nAChRs which also produce excessive β-amyloid are further impaired in the contextual test for fear learning in that five pairs of CS-US fail to impart memory of the context in which CS-US were delivered during training 24 hours prior. These results indicate that the relationship between β-amyloid and α7 nAChRs is more complex than a simple ligand-receptor interaction. If that were the case, one would expect that knocking out α7 nAChRs would alleviate the contextual learning deficit induced by excessive β-amyloid production. Since the deficit worsens, it implies that α7 nAChRs may be important for buffering the toxic effects of elevated β-amyloid. These animals are currently being evaluated for plaque load, Aβ oligomer load, and total Aβ burden, as well as for inflammatory status.

Jerryl Yakel, NIEHS, Research Triangle Park, North Carolina

We found that Aβ1-42 inhibits whole-cell and single-channel nAChR currents from rat CA1 stratum radiatum interneurons in hippocampal slices at concentrations as low as 100 nM. This inhibition appears specific for neuronal nAChRs, because Aβ1-42 had no effect on glutamate or serotonin 5-HT3 receptors, and may be a direct effect on the channel rather than an indirect effect via a signal transduction cascade. In addition, the magnitude of Aβ1-42 inhibition was dependent on the subtype of nAChR. When investigating the block of the single-channel currents by Aβ1-42, the non-α7/62 pS channel was significantly more inhibited (i.e., 54 percent) than was the α7-containing 38 pS channel. In addition to the full-length Aβ1-42 peptide, we previously showed that the fragment of Aβ1-42 including amino acid residues 12-28, Aβ12-28, also inhibited these channels. Recently, we showed that propionyl-amyloid β-protein (31-34) amide (Pr-Aβ31-34), a small fragment of Aβ1-42 which previously was reported to block the neurotoxic effects associated with Aβ1-42 on cholinergic neurons of the rat magnocellular nucleus basalis in vivo, and Aβ31-35, also blocked nAChRs, in a rapid and dose-dependent manner with a similar potency to Pr-Aβ31-34.

Darwin Berg, University of California, San Diego

We've found that Aβ specifically and preferentially blocks the α7-nAChR response both in chick ciliary ganglion neurons and in rat hippocampal neurons (Liu et al., 2001). The blockade is reversible, voltage-independent, and non-competitive. The peptide does not block α3-containing nAChRs under our conditions or non-cholinergic ionotropic receptors. So there does appear to be specificity; we did not test it on α4/β2-nAChRs. Interestingly, the blockade can be compensated by using specific albumins to potentiate the remaining response (Conroy et al., 2003). The potentiation appears to depend on specific sequences within the albumin rather than on absorbed components, and is likely to involve the extracellular N-terminal domain of the α7-nAChR. Synthetic compounds devised to mimic the albumin effect may have significant therapeutic value. We have not seen Aβ-induced currents attributable to α7-nAChR activation, though we tested the peptide under a variety of conditions and concentrations with and without potentiators present. It is quite possible that Aβ interacts with α7-nAChRs in a variety of ways, depending on the cell type and the history of the receptor, vis-a-vis posttranslational modification status. Recently, we have identified postsynaptic molecular scaffolds associated with α7-nAChRs in neurons. The scaffolds involve PDZ-containing proteins and mediate calcium-dependent downstream signaling as exemplified by nicotinic regulation of gene expression. The scaffolds introduce a new complexity to possible consequences of Aβ/α7-nAChR interactions, since the composition of the scaffold varies with receptor subtype and host cell. Further, the receptor is subject to rapid nicotine-induced SNARE-dependent trafficking, presenting the possibility of receptor-mediated Aβ internalization. These findings suggest numerous mechanisms by which Aβ may impact nicotinic signaling in the nervous system.

Robert Nichols, Drexel University College of Medicine, Philadelphia

We assessed the effect of Aβ peptides on nicotine-evoked changes in presynaptic Ca2+ level via confocal imaging of isolated presynaptic nerve endings from rat hippocampus and neocortex. Picomolar Aβ1-42 was found to directly evoke sustained increases in presynaptic Ca2+ via nAChRs. The direct effect of Aβ was found to be sensitive to α-bungarotoxin, mecamylamine, and dihydro-β-erythroidine, indicating involvement of both α7-containing nAChRs and non-α7-containing nAChRs. Prior depolarization strongly attenuated subsequent Aβ-evoked responses in a manner dependent on amplitude of the initial presynaptic function. Together, these results suggest that the sustained increases in presynaptic Ca2+ evoked by Aβ may underlie disruptions in neuronal signaling via nAChRs.

Jie Wu, Barrow Neurological Institute, Phoenix, Arizona

We employed patch-clamp techniques to elucidate acute effects of Aβ1-42 on human α4/β2 nAChRs that are heterologously expressed in SH-EP1 cells. One nM Aβ1-42 reduced both peak and steady-state components of nicotine-induced currents, accelerated acute desensitization, and slowed channel relaxation. Aβ1-42 modulates α4/β2 nAChR-mediated currents in a concentration-dependent manner, and needs appropriate Aβ pretreatment. Comparison of effects of Aβ1-42 on α4/β2 and α7 nAChR-mediated currents indicates that at pathological concentration (1nM), Aβ1-42 significantly modulates α4/β2 nAChRs function without affecting α7 nAChRs. At pharmacological concentrations (>100 nM), Aβ1-42 modulates both α4/β2 nAChRs and α7 nAChRs, with more dramatic effects on α7 nAChRs.

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References

News Citations

Paper Citations

Liu Q, Kawai H, Berg DK. beta -Amyloid peptide blocks the response of alpha 7-containing nicotinic receptors on hippocampal neurons. Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4734-9. PubMed.
Conroy WG, Liu QS, Nai Q, Margiotta JF, Berg DK. Potentiation of alpha7-containing nicotinic acetylcholine receptors by select albumins. Mol Pharmacol. 2003 Feb;63(2):419-28. PubMed.

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New Orleans: Aβ Oligomers and Memory: Now They Are Good…

03 Dec 2003

By Erene Mina. As evidence mounts against oligomers as culprits of Aβ neurotoxicity (see ARF related New Orleans story), their only defense hinges on the possibility that they may have some normal physiological function—even a slightly neurotrophic effect—on cognition which has yet to be characterized. Sally Frautschy’s group at the VA Medical Center in Sepulveda, California, presented data at the 33rd Annual Meeting of the Society for Neuroscience suggesting that Aβ oligomers can be acutely beneficial for spatial memory performance in rats, but that chronic exposure eventually leads to cognitive decline that parallels Alzheimer’s pathogenesis (SfN abstract 240.11).

Presenting author Marni Harris-White and colleagues infused rats with synthetic, SDS-stable low-molecular weight Aβ oligomers (monomers, dimers, trimers) using a mini osmotic pump that continually delivered the Aβ into a cerebral ventricle. Throughout the 3-month infusion, the researchers tested spatial memory at 6, 9, and 12 weeks in the Morris water maze. The rats spent 3 days training to swim to a visible platform, and then the researchers tested acquisition using a hidden platform. To test for memory retention, the rats attempted the maze again following a 24-hour delay. The researchers reported a steady improvement in memory acquisition and retention starting at 6 weeks and carrying through until 9 weeks post-infusion. However, by 12 weeks post-infusion, the animals began to show a mild decline in acquisition coupled with more pronounced deficits in spatial memory retention, characterized by the rats spending less time in the target quadrant. This apparent worsening of memory retention was correlated with a reduction in crucial post-synaptic proteins, such as PSD-95, in the cortex and hippocampus.

In summary, soluble, low-molecular-weight β-amyloid oligomers seem to support memory acquisition and retention early on. However, their contribution to excitatory neurotransmission is short-lived, as this initially beneficial synaptic stimulation eventually sentences the brain to cognitive deterioration. What remains to be elucidated is how an arguably toxic species of Aβ can be even remotely beneficial or have trophic activity in neurons, proving that this is no open-and-shut case. You can view abstracts mentioned in this story at the SfN/ScholarOne website.—Erene Mina is a graduate student at the University of California, Irvine.

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New Orleans: Aβ Oligomers and Memory: …Now They Are Bad

03 Dec 2003

Notwithstanding some redeeming features (ARF related New Orleans story) soluble Aβ oligomers have an overwhelmingly bad reputation as suspected mediators of synaptic dysfunction in Alzheimer’s. A number of accusing fingers have pointed to the havoc they wreak in vitro and in vivo (see, for example, ARF related news story and ARF related story), and at the 33rd Annual Meeting of the Society for Neuroscience, several groups incriminated these oligomers more deeply. Here are selected highlights:

Sylvain Lesne, working with Karen Hsiao Ashe and colleagues at the University of Minnesota in Minneapolis, further analyzed Aβ oligomers in an attempt to find out which are the precise species that impair memory in young Tg2576 mice, even before these mice develop plaques (Kotilinek et al., 2002). Using a detergent, the researchers extracted soluble proteins from these APP-overproducing mice throughout life, from embryonic stages to the elderly age of around 20 months, and then identified Aβ oligomers using immunoblots (SfN abstract 772.1).

The scientists were surprised to find trimers present throughout all ages tested. Their amounts did not change with advancing age, suggesting they are not behind the memory problems. Multimers of those trimers, namely hexamers and dodecamers, appeared with a time course paralleling the gradual loss of memory. By contrast, tetramers began appearing at 13 months, when the memory loss was already in full swing, and their emergence did not worsen it. This leads the Ashe group to suspect that trimers are the fundamental form of Aβ oligomers in their mice, and that multimeric forms of them are at the root of the mice’s cognitive problems.

Dennis Selkoe of Brigham and Women’s Hospital Boston, described how his group has further characterized the soluble Aβ oligomers that are made intracellularly and then secreted by Chinese hamster ovary (CHO) cells overexpressing human APP (667.7). This group considers these secreted Aβ oligomers more “natural” than synthetic oligomers, and it appears that they may be structurally different and more potent, too. Selkoe presented new data indicating that of several proteases known to degrade Aβ monomers (neprilysin, IDE, and plasmin were tested), only plasmin readily breaks down oligomers, as well. Selkoe also showed data on two synthetic small molecules made by pharmaceutical companies that can inhibit the formation of Aβ oligomers in medium and in cells.

He then cited prior work by Dominic Walsh, Igor Klyubin, and others showing that injecting low nanomolar concentrations of these oligomers from conditioned medium inhibits hippocampal LTP in live rats (see ARF related news story). In that paper, the scientists had shown that γ-secretase inhibitors could prevent this oligomer-induced LTP block. In New Orleans, Selkoe presented new data indicating that anti-Aβ antibodies, as well, can neutralize this LTP block, suggesting that this might be a third way (besides microglial plaque clearance and passive, peripheral sink effects) by which AD immunotherapy might eventually become useful (see ARF related New Orleans story).

Recently, this group has developed a size exclusion chromatography protocol to isolate Aβ oligomers from this conditioned medium and shown that four different Aβ antibodies recognize oligomers collected in this procedure. This allowed them to test if these oligomers had biological activity affecting learned behavior. To do that, Selkoe and Walsh collaborated with James Cleary, Ashe, and colleagues in Minneapolis. The researchers chose a rat behavior test called the Alternating Lever Cyclic Ratio (ALCR) lever-pressing procedure, in which rats are trained to keep switching between two levers, pressing them until they receive a food pellet. Rats can make perseveration errors, i.e., they don’t alternate levers properly any more, and switching errors, i.e., they switch to the wrong lever. The scientists consider this test more sensitive than previous instruments, and other researchers including Cleary have used it before to test Aβ-induced learning deficits in rats and the ability of NSAIDs to protect against them (see O’Hare et al., 1999; Richardson et al., 2002). Cleary microinjected into rat lateral brain ventricles the conditioned medium, Aβ oligomers, or Aβ monomers isolated by chromatography. Conditioned medium and oligomers, but not monomers, caused the rats to commit both kinds of errors (see also 772.11). The doses used were physiologically comparable to those seen in AD brain.

Taken together, this leads Selkoe to conclude that a biochemically isolated, defined species of Aβ oligomer—in the absence of monomer and fibrils—can disrupt hippocampal LTP and a learned behavior. Many prior studies had wrestled with the technical difficulty of using mixtures of Aβ species, leaving doubt over which component may have had which effect. Despite this new data, many questions remain about the synaptotoxic mechanisms of Aβ oligomers, and how they could be targeted therapeutically, Selkoe added.

Qinwen Wang, Mike Rowan, and Roger Anwyl of Trinity College in Dublin, Ireland, in collaboration with Selkoe and Walsh (who is based both at University College, Dublin, and Brigham and Women’s Hospital Boston), looked closely at how Aβ oligomers affect LTP (904.10). They performed electrophysiology on rat brain slices harboring the perforant pathway from the entorhinal cortex to dentate granule cell synapses in the hippocampus, a nerve projection that shows damage early on in AD. Both synthetic Aβ oligomers and oligomers secreted by APP-overexpressing CHO cells indeed inhibited the induction of LTP that normally follows high-frequency stimulation, but the natural oligomers were much more potent, Wang et al. found.

Since phosphorylation of excitatory neurotransmitter receptors might play into the mechanism underlying this phenomenon, the scientists next asked which kinases might affect it. They applied the Aβ oligomers in the presence of various kinase inhibitors, and reported that two different inhibitors of c-Jun N-terminal kinase (JNK) counteract the LTP inhibition caused by Aβ oligomers. The Cdk5 inhibitors butyrolactone and roscovitine did, as well, as did a p38MAP kinase inhibitor, but a p42/44 MAP kinase inhibitor did not. All of these kinases have been previously implicated in Alzheimer’s in various ways, but their precise role, if any, on learning and memory remains poorly understood (see, for example, Liu et al., 2001; Fisher et al., 2002, Savage et al., 2002; Sun et al., 2003.

Wang et al. presented one more experiment addressing the question of which transmitter receptor might be at play here. They suggest that the metabotropic glutamate receptor (mGluR) might be one, as two antagonists against it prevent the Aβ oligomer effect. By contrast, an angatonist of the α7 nicotinic acetylcholine receptor (α7nAChR) did not (but see alsoARF related New Orleans story). Together, this suggests that Aβ oligomers inhibit LTP through the mGluR5 receptor and that the kinases JNK, Cdk5, and p38 MAP kinase might be involved. (Cdk5 is also suspected of phosphorylating the NR2A subunit of the NMDA receptor complex, see ARF related news story). Again, this update on oligomer news can’t be comprehensive; other noteworthy presentations on Aβ oligomers included 20.13, 133.7, 841.2, 525.22, 772.4, 772.5, 772.9, 841.21, and 876.5. As always, additions and corrections are welcome. You can view abstracts mentioned in this story at the SfN/ScholarOne website.—Gabrielle Strobel.

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Kotilinek LA, Bacskai B, Westerman M, Kawarabayashi T, Younkin L, Hyman BT, Younkin S, Ashe KH. Reversible memory loss in a mouse transgenic model of Alzheimer's disease. J Neurosci. 2002 Aug 1;22(15):6331-5. PubMed.
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Richardson RL, Kim EM, Shephard RA, Gardiner T, Cleary J, O'Hare E. Behavioural and histopathological analyses of ibuprofen treatment on the effect of aggregated Abeta(1-42) injections in the rat. Brain Res. 2002 Nov 1;954(1):1-10. PubMed.
Liu F, Ma XH, Ule J, Bibb JA, Nishi A, DeMaggio AJ, Yan Z, Nairn AC, Greengard P. Regulation of cyclin-dependent kinase 5 and casein kinase 1 by metabotropic glutamate receptors. Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11062-8. PubMed.
Fischer A, Sananbenesi F, Schrick C, Spiess J, Radulovic J. Cyclin-dependent kinase 5 is required for associative learning. J Neurosci. 2002 May 1;22(9):3700-7. PubMed.
Savage MJ, Lin YG, Ciallella JR, Flood DG, Scott RW. Activation of c-Jun N-terminal kinase and p38 in an Alzheimer's disease model is associated with amyloid deposition. J Neurosci. 2002 May 1;22(9):3376-85. PubMed.
Sun A, Liu M, Nguyen XV, Bing G. P38 MAP kinase is activated at early stages in Alzheimer's disease brain. Exp Neurol. 2003 Oct;183(2):394-405. PubMed.

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New Orleans: Michael O'Neill Reports on Nicotinic Receptors in Parkinson's

05 Dec 2003

A satellite meeting to the 33rd Annual Meeting of the Society for Neuroscience, held last month in New Orleans, addressed nicotine and nicotinic receptors, and their potential for Parkinson's disease therapy. Organized by Maryka Quik, Parkinson's Institute, Sunnyvale, California, and Al Collins, University of Colorado, Boulder, the event drew around 300 people. The goal of this symposium was to bring together researchers with expertise in the pathogenesis of Parkinson's disease and scientists investigating basic and clinical aspects relating to neuronal nicotinic receptors. Advisors to the organizers included Paul Clarke, McGill University, Montreal, Sue Wonnacott, University of Bath, United Kingdom, and Dino Di Monte, also of the Parkinson's Institute.

Fourteen short talks covered three main areas: the biology of nicotinic receptors and their role in dopaminergic function; the potential of nicotinic drugs in the symptomatic treatment of Parkinson's disease; and smoking, nicotine and protection against nigrostriatal damage.

Clearly, a lot of progress had been made in the basic biology in the last decade. In the first three talks—by Collins, Wonnacott, John Dani, Baylor College of Medicine, Houston—we learned that nicotinic receptors are very complex. At least 10 α and four β subunits exist, which can combine to form functional heteromeric and homomeric receptors. Additional complexity arises from the fact that many of these receptor complexes desensitize rapidly, and until recently, pharmacological tools to study the various subtypes did not exist. However, the use of knockout mice and the combined use of molecular biology, neurochemistry, and electrophysiology has provided evidence that at least the α4, β2, α6 subunits are important in the regulation of dopamine release.

The use of nicotinic receptor drugs for symptomatic treatment is also complicated. As mentioned above, multiple subtypes of receptor exist, and targeting dopaminergic function in substantia nigra and striatum may be difficult without also modulating reward pathways in the ventral tegmental area and nucleus accumbens. That said, however, it is clear that activation of nicotinic receptors can produce behavioral and biochemical changes in the rodent brain. Moreover, a large and accumulating body of evidence suggests that nicotine can improve cognition and alter plasticity processes; this may be of particular interest with regard to Alzheimer's disease and other, nonmotor symptoms of PD.

G. Ross, University of Hawaii, provided a nice overview that clearly illustrated that smokers have a decreased incidence of PD. This effect has been reported in several independent studies and is probably one of the major drivers for researchers to try and work out how this effect is being produced. A series of talks (R. Tyndale, Lorise Gahring, University of Utah, Salt Lake City; Michael O'Neill, Eli Lilly and Co., Windlesham, UK) presented preclinical data showing that nicotine has protective properties in various in-vitro culture systems and in some rodent models of PD. The potential neuroprotective mechanisms discussed included altering neurochemistry, receptor expression and signaling pathways; antioxidant properties; induction of growth factors; and induction of metabolic enzymes. However, it was clear that the mechanism(s) are not yet elucidated and that data are mixed (i.e., some papers reporting protection; others, no effect; or indeed, increase of injury). In addition to clarifying the data with nicotine itself, further work is needed to test more selective ligands (nicotine has effects of multiple receptor subtypes), and to understand which receptor subtypes are responsible.

The final talks illustrated some of the newer models of PD that may be used to study further the protective actions of smoking/nicotine. These include the use of paraquat in mice (DiMonte); rotenone in rats (Timothy Greenamyre, Emory University, Atlanta); and MPTP in primates (Quik). The first two models are slow and progressive; they differ from other models in that ubiquitin- and α-synuclein-positive inclusion bodies are present. These inclusion bodies at least in part mimic Lewy bodies, a hallmark of PD in the human brain.

Overall, this was an enjoyable meeting with lots of interaction among cellular, neurochemical, and behavioral scientists. The availability of more selective ligands, knockout mice, and newer models of PD will hopefully allow this area to progress rapidly in the next three to five years. Unfortunately, the conference coincided with the Parkinson's Foundation Symposium, which was in progress a few blocks away at Hotel Monaco. It would be nice to see a follow-up conference, perhaps with an even greater presence of PD specialists, in the future.—Michael O'Neill.

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New Orleans: Jason Shepherd Reports on New Mouse Models

05 Dec 2003

Two new approaches for modeling neurodegeneration in mice were unveiled at the 33rd Annual meeting of the Society for Neuroscience in New Orleans. Frank LaFerla’s group at University of California, Irvine, generated mice in which selective subpopulations of neurons can be ablated in a time-dependent manner (Abstract 773.13; view abstracts at the SfN/ScholarOne website). Most methods to lesion-specific neuronal subfields rely on excitotoxicity; they act on relatively nonspecific populations of neurons and are hard to control temporally. LaFerla’s group combined inducible transgenic expression of the diphtheria toxin A chain (DT-A)—a potent cytotoxin in eukaryotic cells—with the binary tetracycline (tet-off) promoter system to selectively eliminate Ca2+-calmodulin kinase II (CaMKII)-expressing neurons in transgenic mice in a time-dependent manner. Induction of DT-A transgene expression resulted in a selective and focal loss of neurons in specific brain regions, with CA1 hippocampal neurons emerging as the most vulnerable population, followed by the neocortex, dentate gyrus, striatum, and basal forebrain. The CA3 region remained intact and was affected only after extreme periods of DT-A expression. The DT-A expression was clean, with no leaking during development when the transgene is repressed; in this way, the transgenic mice develop normally and lesions on adult brains can be assessed directly. This system can now be used to explore a number of questions on the consequences of focal cell loss. Since DT-A expression can be turned off at will, one can look at recovery after the lesion and use various manipulations or putative therapeutics to increase recovery. Jason Shepherd is a coauthor of this study.

Wouldn’t it be nice to have a live biosensor, a way to image neurodegeneration and cellular injury without having to kill the mice? Tony Wyss-Coray’s group at Stanford University developed a new transgenic model to do just that (Abstract 773.14). The cytokine TGF-β1 is known to be increased in AD and is also rapidly induced in response to injury, for example, as triggered by the toxin kainate. TGF-β1 signals via Smad, which translocates to the nucleus, binds to Smad binding elements (SBEs), and in this way activates transcription of various genes. Taking advantage of this, Wyss-Coray’s group engineered mice that overexpress an SBE-luciferase reporter gene. The luciferase protein bioluminesces in the presence of its substrate luciferin (which can be injected), and an ultrasensitive, cooled CCD camera can capture this bioluminescence inside the brain of mice. TGF-1 activity, therefore, turned on luciferase, making the mice “glow.” The scientists found that TGF-β1 activity was highest in the brain, specifically in the hippocampus and cortex. After intraperitoneal injection of kainate, luciferase activity showed a biphasic induction at one hour and 24 hours after injection. The researchers also showed that the reporter gene could be imaged in live, anaesthetized animals in real time. These mice can be used to assess the progression and outcome of neurodegenerative disorders. The consequences of chronic degeneration in these animals need to be further assessed, Wyss-Coray added. These mice could be crossed to mouse models of neurodegeneration, and could help in assessing the role of inflammation in various disease paradigms.—Jason Shepherd is a graduate student at Johns Hopkins University School of Medicine, Baltimore, Maryland.

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New Orleans: Probing Alzheimer’s Gene Background

09 Dec 2003

If the leery mother-in-law and the cautious would-be employer scrutinize the background of their prospect at hand, AD scientists would be well-advised to do the same. That, at least, is the view of Bruce Lamb at Case Western Reserve University in Cleveland, Ohio, and he has taken his own advice. At the 33rd Annual Meeting of the Society of Neuroscience held last month in New Orleans, Lamb described the latest analysis of his strains of congenic mice that differ by background only—a novel approach to study AD genetics that has not, as yet, been widely applied in the AD field.

In brief, the reasoning goes like this: Alzheimer’s is such a heterogeneous syndrome that even members of a single family carrying the same FAD mutation vary wildly in when they get the disease, how long and severe a course it takes, and in their symptoms and pathology. Assuming that the consequences of APP processing underlie AD pathogenesis, this striking variability means that other genes lurking in these patients’ backgrounds influence how the mutant APP is processed, how its cleavage products interact with other factors, and how the Aβ peptide is metabolized or deposited, Lamb told the audience. He also noted that the known AD genes represent only about 30 percent of AD risk; several human genetics labs have been trying for a decade to identify the rest.

The mouse situation mirrors the human in the sense that numerous strains of APP- and APP/PS-transgenic mice exist, but they vary widely in their phenotype and are difficult to compare directly (see Alzforum Research Model directory). Lamb and colleagues started a long-term project to identify modifying genes in AD without having to make prior mechanistic assumptions. They first packaged the entire human gene from various FAD mutations into yeast artificial chromosomes (YACs are basically large cloning vectors) and made transgenic mouse lines with them (Lamb et al., 1997, Lamb et al., 1999). At the meeting, Lamb focused on the APPSwe mutation, showing how extensive backcrossing of a YAC APPSwe line with three inbred mouse lines led to the creation of congenic strains that carry the same APPSwe gene on different genetic backgrounds (abstract 336.12, peruse abstracts at the SfN/ScholarOne website).

If the mutant APP was the only factor driving the phenotype, these strains should all be alike. Alas, they are not. While the congenic strains do show the same levels of APP holoprotein, at young ages they already differ in their production of C-terminal fragments (CTF) and in their brain and plasma Aβ levels. At the elderly age of 20 months, mice of one strain, called B6, have plaques littering their cortex, while those of another, D2, remain plaque-free despite their APPSwe gene. This suggests that background genes override the effects of the mutant APP gene in D2, Lamb said.

Lamb added that congenic mice also offer insight into another knotty problem—the interaction between genes and environmental confounders. Taking, for example, the link between cholesterol and amyloid deposition, Lamb asked whether background genes could account for some of the differences observed in the cholesterol response. Again, mice carrying the APPSwe gene on the B6 background developed increases in Aβ when fed a high-cholesterol diet, but the D2 background did not, suggesting that background genes influence cholesterol’s effect on Aβ metabolism. These are preliminary data.

In general, AD transgenic mice are on hybrid backgrounds. Many researchers using such strains have made anecdotal observations about marked background effects on the given phenotype they are studying, but most have viewed this as an experimental nuisance rather than an opportunity. Few, if any, have made a formal, systematic effort to capitalize on these background effects to identify novel modifying genes. Lamb’s group, with first author Emily Lehman, just published some of their data (Lehman et al., 2003), but for now, the identity of those modifying genes remains a mystery.—Gabrielle Strobel

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References

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Lamb BT, Call LM, Slunt HH, Bardel KA, Lawler AM, Eckman CB, Younkin SG, Holtz G, Wagner SL, Price DL, Sisodia SS, Gearhart JD. Altered metabolism of familial Alzheimer's disease-linked amyloid precursor protein variants in yeast artificial chromosome transgenic mice. Hum Mol Genet. 1997 Sep;6(9):1535-41. PubMed.
Lamb BT, Bardel KA, Kulnane LS, Anderson JJ, Holtz G, Wagner SL, Sisodia SS, Hoeger EJ. Amyloid production and deposition in mutant amyloid precursor protein and presenilin-1 yeast artificial chromosome transgenic mice. Nat Neurosci. 1999 Aug;2(8):695-7. PubMed.
Lehman EJ, Kulnane LS, Gao Y, Petriello MC, Pimpis KM, Younkin L, Dolios G, Wang R, Younkin SG, Lamb BT. Genetic background regulates beta-amyloid precursor protein processing and beta-amyloid deposition in the mouse. Hum Mol Genet. 2003 Nov 15;12(22):2949-56. Epub 2003 Sep 23 PubMed.

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Can’t Close Spigot? Try Opening Drain: New Tack on Amyloid Degradation

24 Dec 2003

Scientists prospecting the amyloid hypothesis for new treatment strategies have set their sights on the idea of boosting the enzymatic destruction of the Aβ peptide. A flurry of recent papers and presentations at the 33rd Annual Meeting of the Society for Neuroscience, held last month in New Orleans, Louisiana, indicate that a growing number of research labs are investigating the proteases that can chew up Aβ. They are trying to gain an understanding of which ones are relevant in vivo, whether tweaking them affects amyloid deposition and relevant disease markers, and whether this could be done safely. While Aβ degradation remains poorly understood, results from several different approaches are beginning to converge.

Traditionally, Aβ production has received a much larger share of attention than has Aβ degradation (and indeed, on the production side, the current hot topic of investigation has shifted toward other APP cleavage products and away from Aβ itself). What’s more, while therapeutic attempts to curtail production—chiefly γ- and β-secretase inhibitors and vaccination protocols—are wending their way through the (mostly pre-) clinical pipeline, it has become as plain as day that these approaches are challenging and risky. So, at least theoretically, enzymatic breakdown of monomeric Aβ shines as the next-best hope of cutting off the cascade of detrimental consequences that occur when this peptide begins to accumulate, aggregate, and deposit. Below is a news account of recent developments.

Aβ-Degrading Enzymes: Lifesaver for Mice?
The most recent paper on this topic appeared in the December 18 Neuron. In it, Malcolm Leissring, Wesley Farris, and Dennis Selkoe of Brigham and Women’s Hospital, and colleagues, report that chronic elevation of the two most extensively studied Aβ proteases in neurons did not overtly harm mice. Instead, it reduced the mice’s brain Aβ levels and pathology, and even prevented premature death. The scientists created two strains of transgenic mice, one that overproduced insulin-degrading enzyme (IDE—see ARF related news story, ARF news story), and one that overproduced neprilysin, a neural endopeptidase studied intensely by Takaomi Saido’s lab (see below). The former mouse strain expressed twice the normal level of IDE and its enzymatic capacity was doubled; the latter strain expressed eight times the normal neprilysin level and also had a corresponding increase in activity. Leissring and colleagues then bred each of these two strains with APPSwInd mice (Mucke et al., 2000; also see APP Research Models).

Strikingly, this appeared to rescue a premature death phenotype that the authors observed when raising APPSwInd mice. Mouse models of AD amyloidosis generally have neither massive neuronal death nor full-blown AD. Even so, some lines of APP transgenic mice have been reported to die prematurely, prior to overt plaque deposition Hsiao et al., 1995), and Leissring and colleagues observed a similar effect in the APPSwInd mice, with one in five perishing by six months of age. This rate dropped to about one in 20 in the IDE+APP double-transgenics, and to nil in the neprilysin+APP cross. When quantifying brain Aβ levels in these crosses, the scientists found them cut in half in the IDE+APP transgenics, and down by 90 percent in the neprilysin+APP mice. Further experiments suggest that the proteases degraded chiefly Aβ monomers, not its aggregated forms. The extent of this reduction is surprising, the authors write, given that this particular model produces a 10,000-fold increase in Aβ over endogenous mouse brain levels, and it leads them to suggest that a even a small enhancement of Aβ proteolysis might over time have a therapeutic benefit. In addition, the plaque load of the IDE+APP mice was halved and the neprilysin+APP mice had barely any plaques at all. Antibody markers for astrocytosis, microgliosis, and dystrophic neurites were also down in the crosses.

This October, a different in-vivo study yielded broadly similar findings. Scientists led by Thomas Beach at Sun Health Research Institute in Sun City, Arizona, infused the neprilysin inhibitor thiorphan into the cerebral ventricles of rabbits for five days, and then found increases in cerebral and CSF Aβ levels Newell et al., 2003).

Both IDE and neprilysin are known to cleave many other substrates besides Aβ, for example, insulin. Could a drug raising their activity ever be safe? This question has no answer yet, but Leissring et al. note that the IDE or neprilysin single-transgenic mice are healthy, fertile, and without neuropathological abnormalities. This is but a rough first-pass check for possible mechanism-based side effects. For one, the transgenic mice had IDE and neprilysin upregulated chronically only in the brain, as the transgenes were driven by the neural CamKII promoter. (For this reason, the Neuron study also does not address links between insulin metabolism and AD; see ARF Live Discussion). For another, unexpected side effects in humans sometimes crop up which were not seen in extensive mouse studies. To pick a recent example from AD research, encephalitis was never reported in mouse studies of AD immunotherapy, but later developed in humans. That said, upregulating Aβ-degrading enzymes is unlikely to provoke adverse immune reaction. Finally, genetically manipulated mice frequently appear fine until a specific challenge (e.g., oxidative, neurotoxic, or metabolic) reveals more subtle vulnerabilities, so the IDE- or neprilysin-overexpressing mice might yet turn up other phenotypes, once challenged.

In fact, age-related oxidative stress does appear to affect these enzymes, though not in a way that would contradict the general gist of this therapeutic approach. Instead, neprilysin and IDE may be means through which oxidative byproducts fuel amyloid pathology. This October, scientists led by Dennis Dickson at the Mayo Clinic in Jacksonville, Florida, reported that 4-hydroxynonenal (HNE) adducts form on NEP proteins in AD brains in greater proportion than in control brains (Wang et al., 2003). And at the Neuroscience conference in New Orleans, Frank LaFerla’s group at the University of California, Irvine, showed preliminary data suggesting that IDE also is a substrate for HNE and is oxidized disproportionately in the hippocampus of AD brain (202.11).

More on Neprilysin
Also in New Orleans, Nobuhisa Iwata, working with Takaomi Saido at RIKEN Brain Sciences Institute in Saitama, Japan, and colleagues elsewhere, presented the latest findings of that group’s longstanding research on neprilysin. Previously, this group had shown that this enzyme localizes to presynaptic sites and axons (Fukami et al., 2002). They also generated neprilysin knockout mice that have elevated brain Aβ levels Iwata et al., 2001). At the conference, Iwata presented data on an experimental gene therapy approach to test the notion that increasing neprilysin activity can stem the amyloid component of AD and the synaptic damage associated with it. Using adeno-associated virus to ferry the neprilysin gene into cells, Iwata et al. showed that neprilysin transfer decreased Aβ levels in the brains of both neprilysin knockout mice and APP-transgenic mice (525.6). (Last March, Eliezer Masliah’s group at University of California, San Diego, reported that overexpressing neprilysin from a lentiviral vector lowered amyloid deposition; see Marr et al., 2003; also see ARF related news story and scroll to Masliah).

What’s Age Got to Do with It?
Last year, Saido’s group had shown that neprilysin expression wanes with age in the perforant pathway connecting the entorhinal cortex to the hippocampus and on mossy fibers, suggesting local reductions of neprilysin in areas known for synaptic damage in AD Iwata et al., 2002), while Antonella Caccamo and colleagues in LaFerla’s lab picked up the question whether age-related changes in IDE and NEP could account for amyloid buildup in sporadic AD. Last month at the Neuroscience conference in New Orleans, Caccamo presented initial data comparing steady-state levels of IDE and neprilysin in brain extracts of normal mice at two and 18 months of age. She found that both proteases fell with age in the hippocampus; in the cortex, neprilysin was unchanged and IDE appeared to increase slightly. In cerebellum, a brain region largely spared by AD, neprilysin increased with age and IDE stayed unchanged. When compared directly, cerebellum was the brain region with the highest steady-state levels in old mice. Likewise, human brain extracts had higher IDE levels in cerebellum than in hippocampus or cortex (202.11). In a separate study, Zhongmin Xiang, working with Giulio Pasinetti and colleagues at Mt. Sinai School of Medicine in New York, assayed IDE activity in postmortem brain samples of people with AD and correlated it with clinical progression. With advancing dementia, these investigators reported in New Orleans, IDE activity appeared to decrease in the hippocampus but not in the visual cortex, an area unaffected by AD (202.10).

What Have Genes Got to Do with It?
As yet, the jury is still out on the question of whether IDE is an Alzheimer’s risk gene. As occurs frequently in AD genetics, an initial report (Bertram et al., 2000) was thrown into doubt by failures to repeat (Abraham et al., 2001; Boussaha et al., 2002). However, more recent case-control studies (Edland et al., 2003) and haplotype analysis (Prince et al., 2003) keep finding hints that it might contribute significantly, after all. For a news summary on the insulin connection, scroll down in ARF ISOA meeting report. Lars Bertram, Rudy Tanzi, and colleagues of Massachusetts General Hospital presented data in New Orleans that appear to strengthen the candidacy of IDE.

No neprilysin mutations or independently confirmed polymorphisms predisposing to AD have been found to date (but see Clarimon et al., 2003). Last January, however, Saido’s group implicated neprilysin functionally, if not genetically, in some familial forms of AD. The Dutch, Flemish, Italian, and Arctic mutations of APP are unusual in that they lie within the Aβ peptide sequence of APP (see ARF related news story). They also, it turns out, make the mutant Aβ more resistant to proteolysis by neprilysin, the scientists reported (Tsubuki et al., 2003).

While this report focused on IDE and neprilysin, there are clearly more enzymes capable of chewing up the Aβ peptide, chief among them endothelin-converting enzyme and plasmin. And new candidates are cropping up, for example, a yet-unidentified serine protease reported by Carmela Abraham of Boston University last month in New Orleans (524.15). To date, scientists do not have a firm understanding of the relative roles of neprilysin, IDE, or the other Aβ-degrading proteases in the normal regulation of brain Aβ levels. What’s more, the field has not shown whether these Aβ-degrading proteases actually play a significant role in causing the disease. In other words, except in some forms of familial AD, it is unclear whether the spigot is turned up or the drain is clogged, or both. However, many researchers believe that, to the extent that Aβ accumulation contributes to Alzheimer’s, opening the drain makes intuitive sense, and perhaps by now this idea has garnered enough experimental support to warrant attention from drug developers.—Gabrielle Strobel.

Q&A with Malcolm Leissring—Posted 24 December 2003.

Q: The premature mortality of the APPSwInd mice was news to me. Has it been described somewhere?

A: As mentioned in the paper, other APP transgenics, specifically Karen Hsiao's (1995 Neuron paper), have been reported to die early, prior to plaque deposition. The speculation at the time was that mechanisms unrelated to amyloid deposition might be involved.

Q: Do the dying mice have neurodegeneration as in AD?

A: I don't know, but based on other APP transgenics, I doubt it. Lennart Mucke, in collaboration with Roger Nicoll and Robert Malenka, reported that there were electrophysiological changes in these mice at an early age (Hsia et al., 1999), as well as sharp reductions in synaptophysin (Mucke et al., 2000). The synaptophysin changes might count as a form of "neurodegeneration," but I know of no mouse model that shows degeneration of the type seen in AD.

Q: If not, what kills them?

A: Soluble oligomer-induced desynchronization of respiratory neuronal circuits during sleep??? Your guess is as good as mine. But this is an equally interesting question to ask of clinicians: What ultimately does kill AD patients? Neurodegeneration, per se? Probably not. Whatever does kill them, the results from our study suggest that it is likely a soluble species of Aβ, rather than aggregates, since the effects are seen prior to plaque deposition.

Q: Your neprilysin or IDE transgenic mice seem normal. But Roberto Malinow and other scientists are suggesting a physiological role for Aβ in synaptic function (Kamenetz et al., 2003). With Aβ presumably decreased in your mice right from the get-go, were you able to detect an LTP or a learning/memory phenotype in them?

A: We did not specifically address this question in our mice, but there is a strong inference that the LTP deficits present in Mucke's APP transgenics would be reversed by coexpression of either Aβ-degrading protease. We are currently crossing these mice to Frank LaFerla's triple-transgenic AD mice (Oddo et al., 2003), and they might have the resources to do this. It would certainly be interesting to find out.

Q: Did you assess behavior in the IDE+PDAPP and neprilysin+APP double-transgenics? If the described deficits improved only partially, this would be a way to distinguish between the roles of Aβ and some other APP cleavage products in learning and memory.

A: I agree that the double-transgenics are a nifty model in this respect: no Aβ, but lots of every other APP metabolite (except AICD in the IDE transgenics). I am actually hoping someone will use our mice to look at just this, as this would be too much work for our small IDE group (three people, counting Dennis).

Q: Therapeutically, how would one boost IDE or neprilysin?

A: One promising way would be disinhibition, that is, finding a small molecule that displaces an endogenous inhibitor. There are a few known endogenous inhibitors of IDE (e.g., ATP and fatty acids) and others for NEP (spinorphin, sialorphin) so this might not be impossible. Wes Farris in our lab discovered that IDE naturally exists as a homodimer, and possibly a trimer or tetramer, and Lou Hersh recently published the same result ( Song et al., 2003). Intriguingly, Lou found that low (nanomolar) levels of other substrates could increase the hydrolysis of Aβ, and we have seen similar effects resembling cooperativity with different substrates. Lou's idea is that the dimer is the more active form, and substrate binding might favor the dimer form, thus activating the protease. In this regard, a small molecule that would disrupt tetramer formation, or stabilize dimer formation, could conceivably activate the molecule.

Another way is to just screen as many compounds as possible, and hope to get lucky and find some molecule that lodges itself into the enzyme and in some way alters the Km or Vmax. Note that activators have been found for other enzymes, including the red wine constituent resveratrol activating the sirtuins Howitz et al., 2003).

There are other ways, including identifying compounds that upregulate the expression of the proteases. I'm sure you have heard Saido talk about somatostatin upregulating neprilysin, and estrogens have been reported to do this, as well. Interestingly, in our paper we found that IDE was actually downregulated in the NEP transgenics. This suggests that some common substrate might regulate the expression of IDE, and if you can identify what is regulating it, you can conceivably exploit that to activate expression.

For our own part, we are actively pursuing compounds that might enhance the activity or expression of IDE and other Aβ-degrading proteases. Over the past few years, I have used a newly devised high-throughput Aβ-degradation assay (Leissring et al., 2003) to screen over 130,000 compounds against naked recombinant IDE (i.e., with no endogenous inhibitor). While we have found a few activators, none are particularly impressive, showing only subtle effects at rather high concentrations. But there is much more to do, such as repeating the screens with endogenous inhibitors present. The nice thing about our assay is that it is universal: it can be used with any Aβ-degrading protease. So we've got a lot of screening lined up for this assay in the future. Please keep your hopes high but your expectations low!

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New Orleans: Rudy Tanzi on What’s Brewing in AD Genetics

13 Jan 2004

This report summarizes some of the genetic findings for late-onset Alzheimer's disease presented last November at the 33rd Annual Meeting of the Society for Neuroscience in New Orleans. It covers recent progress in identifying the genes responsible for the two well-established AD linkage peaks on chromosomes 9 and 10, as well as recent studies of several other candidate genes.

Chromosome 9
UBQLN1: Several genome screens for late-onset AD loci have agreed upon genetic linkage on chromosome 9. It is fair to say that after chromosome 10, chromosome 9 is one of the best confirmed locations for a novel AD gene (for review, see Tanzi and Bertram, 2001; Myers and Goate, 2001). The gene, UBQLN1, encoding the presenilin-interacting protein ubiquilin (Mah et al., 2000), maps close to the apex of the linkage peak on chromosome 9q22 (e.g., see Blacker et al., 2003). At the conference, Lars Bertram and Rashmi Menon from our group at Massachusetts General Hospital in Charlestown reported the results of analyses of five single nucleotide polymorphisms (SNPs) in the UBQLN1 gene. All five belong to the same haplotype block spanning about 50 kb; more importantly, two of the five SNPs were significantly associated with AD in the NIMH AD family sample (n = 437 families; p-values = 0.018 and 0.046). The 27 best UBQLN1-associated and 9q22-linked AD families from the NIMH sample had an average onset age of 71.7±7.3 years (range: 59-93); 20 of these carried at least one copy of the ApoE ε4 allele, while 7 families did not carry any ApoE ε4. Bertram went on to report that eliminating the 27 families that exhibit the strongest association with UBQLN1 (n = 27, i.e., about six percent of the full NIMH sample) reduced the linkage evidence on chromosome 9q22 by about 50 percent; this corroborates the association of UBQLN1 with AD. Moreover, linkage analysis with these 27 families alone enhanced the linkage peak on 9q22 and shifted it in the direction of UBQLN1.

Kristina Mullin of our group reported further support for the candidacy of UBQLN1 as a novel AD gene. She obtained independent confirmation of association of the same two UBQLN1 SNPs with AD (and the same risk alleles from the NIMH sample) in a second independent set of families, the Consortium on Alzheimer’s Genetics (CAG) sample. CAG has enrolled 314 families, with current genotyping being done on 155 families consisting mainly of discordant sibships. Collectively, these data strongly suggest that UBQLN1 is the one gene responsible for the well-established AD linkage peak on chromosome 9q22. At this time, it remains unclear whether this region harbors additional AD loci.

APBA1: Menon and Bertram presented evidence for single-locus association with one out of the seven SNPs analyzed in the gene encoding X11-α [MINT1] in the NIMH AD family sample. However, conditional logistic regression analysis did not reveal a significant increase in risk for this variant. Further, the best APBA1-associated families (n = 37) did not account for a significant proportion of the 9q22 linkage signal. Finally, the association was not confirmed in the CAG sample, suggesting that genetic variants in APBA1 most likely do not make a major contribution to the overall risk for AD.

ABCA1: Michele Parkinson et al. from our group reported that, contrary to previously published findings (Wollmer et al., 2003), analyses performed on 7 SNPs in ABCA1 showed no association with disease risk or onset age in the NIMH families (see also Sun et al. 2003).

Chromosome 10
In December 2000, three groups simultaneously reported the first evidence for significant genetic linkage of AD to the long arm of chromosome 10. Efforts to isolate the gene responsible have been encumbered by the fact that the linkage region implicated by the three papers spans more than 40 megabases. This has raised the possibility that there may be two different AD genes on chromosome 10, one in the more proximal region, reported by the Younkin and Goate groups (Ertekin-Taner et al., 2000 and Myers et al., 2000, respectively), and the other located more distally, reported by our group (Bertram et al., 2000).

Chromosome 10 distal linkage region
IDE: In our initial report of linkage of AD to chromosome 10, we had focused on the gene for insulin-degrading enzyme (IDE) as a candidate because of IDE’s capacity to degrade Aβ. We presented evidence for significant genetic linkage and association of AD with IDE. In New Orleans, Bertram presented the results of our analyses of 14 IDE SNPs in the large NIMH AD family sample, which comprises 437 uniformly ascertained and evaluated AD families. Bertram showed the IDE gene to reside next to the KIF11 gene in a region of approximately 200,000 base pairs, which was found to contain three haplotype blocks. The middle haplotype block encompassing the 5’UTR and promoter region of IDE was shown to contain a 4-SNP haplotype that revealed significant family-based association of IDE to late-onset AD (p = 0.0009). The association of AD with this haplotype block accounts for roughly half of the observed genetic linkage of IDE to AD in the NIMH family sample.

Bertram also presented a single SNP in the IDE promoter region (IDE-U4) that was associated with AD in the NIMH sample. This association was confirmed (with the same risk allele) in the CAG sample. Collectively, these data provide additional evidence for the candidacy of IDE as a late-onset AD locus on the long arm of chromosome 10.

In his presentation, Bertram also mentioned that five other labs have observed association of various IDE SNPs or haplotypes with increased risk for AD. Along these lines, also in New Orleans, Nilufer Ertekin-Taner, Steve Younkin, and colleagues at the Mayo Clinic in Jacksonville, Florida, presented evidence that IDE SNPs (from Anthony Brookes’s group at the Karolinska Institute in Stockholm; Prince et al., 2003) were associated with both increased risk for AD and increased plasma Aβ42 levels. This result concurs with the recent publication of positive association of AD with other SNPs in IDE in the Mayo samples by Steve Edland et al. (Edland et al., 2003; see also Edland section in ARF related news story). In addition, at the 53rd Annual Meeting of the American Society of Human Genetics held in Los Angeles just prior to the Neuroscience meeting, Alison Goate and colleagues at Washington University in St. Louis, Missouri, reported genetic association of AD with two independent SNPs in IDE; this is in contrast to her group’s previous report of negative association (see ARF related news story).
Thus, since our original report of IDE linkage and association, converging evidence from our own lab and several others now strongly suggests that IDE is at least one of the AD genes residing on the long arm of chromosome 10. It should be emphasized, however, that it remains unknown which, if any, of the AD-associated IDE SNPs may be pathogenic for AD, as opposed to being in linkage disequilibrium with unidentified pathogenic DNA variants or mutations. Thus, further studies are required.

Malcolm Leissring, Dennis Selkoe, and colleagues at Brigham and Women’s Hospital in Boston presented their analyses of parallel sets of transgenic mice overexpressing IDE or neprilysin (NEP) in the same genetic background, using a CaM kinase II promoter, crossed with APPSwe/Ind transgenic mice. Soluble and insoluble Aβ40 and Aβ42 brain levels were reported to be decreased by more than half in IDE/APP and NEP/APP transgenic mice, relative to age-matched six- to 10-month-old APP transgenic controls. Plaque burden was also found to be decreased in the double-transgenic mice, as was the rate of premature death prior to eight months of age, which is normally elevated in the APP transgenic mice. Interestingly, overexpression of IDE by only twofold over physiological levels still significantly reduced β-amyloid burden in these animals. Frank LaFerla and colleagues at University of California, Irvine, presented evidence that in regions of the human brain that are vulnerable to AD pathology, e.g., hippocampus, and also in muscle fibers sensitive to inclusion body myositis pathology (IBM is a human model for AD-like amyloidosis), steady-state levels of IDE and/or NEP diminish as a function of age. In contrast, muscle and brain regions not associated with significant Aβ accumulation exhibited an age-dependent increase in these catabolic enzymes. The authors suggest that differences in the steady-state levels of these enzymes with aging may account for susceptibility to Aβ-related pathology (see also ARF conference story).

GSTO1/GSTO2: In 2002,Yi-Ju Li, Peggy Pericak-Vance, and colleagues reported linkage of a locus affecting age-at-onset (but not “risk”) for AD and PD on the distal long arm of chromosome 10 (see ARF related news story) in a region telomeric of IDE. Based on expression profiling in AD brains, the same group (Li et al., 2003) reported four genes (Stearoyl-CoA desaturase; NADH ubiquinone oxidoreductase 1 β complex 8; protease, serine 11; and glutathione S-transferase, omega-1, GSTO1) to differ significantly in their expression between AD and control brain samples, and to reside in the AD/PD age-at-onset linkage region on chromosome 10. This group later reported genetic association for two members of the GST omega family (GSTO1 and GSTO2), both of which play roles in the inflammation process (see also ARF related news story).

One of the main findings in this recent study was association of age-at-onset of AD and PD with an amino acid substitution (Ala140Asp) in the GSTO1 gene. At the Neuroscience meeting, however, Bertram presented the absence of association of this same polymorphism with either “age-at-onset” or “risk” for AD in the NIMH sample. Clearly, more work in independent samples is necessary to elucidate whether these genes play a role in AD and PD pathogenesis.

CH25H-LIPA: In a poster presentation, Parkinson and colleagues from our group reported that four SNPs in the CH25H-LIPA genes located within a 50 kb interval on chromosome 10q23 (near our linkage region in the vicinity of the IDE-gene) revealed a marginally significant effect in the NIMH AD sample, with one variant located in intron 2 of LIPA, and only in ApoE ε4/4-positive families (p-value = 0.034, OR 3.4 [1.2-9.8]). However, no other variants in this region showed evidence for association, and the haplotype analyses performed on this set of polymorphisms yielded results that were inconsistent with the single SNP findings. We are currently investigating whether these findings are specific for the CH25H-LIPA region, or whether they are perhaps the result of linkage disequilibrium effects with the IDE association about 3 Mb further distal. (CH25H encodes cholesterol 25-hydroxylase; LIPA is an atherosclerosis candidate gene.)

Chromosome 10 proximal linkage region
VR22: Recently, Nilufer Ertekin-Taner, Steve Younkin, and colleagues reported evidence that the locus responsible for increasing plasma Aβ42 on the more proximal linkage peak on chromosome 10 was the gene encoding α-T catenin, a binding partner of β-catenin (Ertekin-Taner et al., 2003). Specifically, they reported that two intronic VR22 SNPs (4360 and 4783, in strong linkage disequilibrium with one another) were associated with higher plasma Aβ42 levels in 22 late-onset AD families. In New Orleans, Younkin reviewed these findings and also reported that the association with VR22 accounted for a significant portion of the genetic linkage that his group originally reported for this region (Ertekin-Taner et al., 2000).

Bertram presented the results of our analysis of these and three other SNPs in the VR22 gene in the NIMH AD sample. While the overall results (including for SNP4360) were negative (p = 0.34), it should be noted that a trend toward association with risk for AD (p = 0.08) was observed in the subset of families with onset after 65 years. Thus, it may still be worthwhile to search for potentially pathogenic SNPs/mutations in VR22. While at this point it is unlikely that the tested VR22 SNPs represent pathogenic variants, they may still be in linkage disequilibrium with other unknown pathogenic variants or mutations.

PLAU: The urokinase-type plasminogen activator (PLAU) can degrade Aβ aggregates by generating plasmin (Tucker et al., 2000). The PLAU gene resides on 10q21-22 at about 95 cM within the 1-lod support interval of the proximal linkage peak at about 80 cM. Younkin and colleagues reported on analyses of various SNPs and haplotypes in PLAU, particularly the P141L polymorphism. The CT/TT genotype of P141L was reported to be associated with an age-dependent increase in plasma Aβ42. Interestingly, Younkin added, plasma Aβ is elevated in PLAU KO mice. Younkin went on to review the data of 10 different studies performed in collaboration with Brookes—Matthias Riemenschneider at the Technical University, Munich, and Steve Estus at the University of Kentucky in Lexington. (Estus also reported negative data on several other genes related to the plasmin pathway.) While the overall findings supported an association of PLAU with risk for AD, the results of these studies revealed inconsistent risk alleles across cases and families. Thus, the data suggest that P141L is most likely in linkage disequilibrium with unknown pathogenic variants and mutations that are yet to be identified. Younkin also emphasized the need to subdivide samples genetically when searching for mutations.

Other AD candidate genes
CYP46: Parkinson et al. reported analyses of two noncoding SNPs flanking exon 3 in the cholesterol 24-hydroxylase gene, CYP46. While neither revealed overall association with risk for AD, stratification by ApoE ε4/4-genotype was found to reveal strong but opposite associations with AD in the NIMH sample. Specifically, the minor alleles of both variants (i.e., C-allele in CYP46-intron 2 and T-allele in CYP46-intron 3) exhibited significant overtransmission to affected subjects in the ApoE ε4/4-positive samples (p = 0.0009 and 0.007, respectively), but this did not translate into a significantly elevated risk for AD in these families. On the other hand, the opposite alleles were overtransmitted in the ApoE ε4/4-negative sample (p =0.017 in both cases), and this elevated the odds for AD by about twofold. This latter finding is in agreement with previous studies that have reported association of the major alleles of both of these variants with increased risk for AD (Koelsch et al., 2002; see also ARF related news story).
In considering the candidacy of CYP46 as an AD gene, some caveats should be mentioned. First, Desai et al. found no association in their case-control sample (Desai et al., 2002). Second, in New Orleans, Parkinson and Bertram reported no evidence of association between these two variants and AD in the independent CAG family sample. Third, to date, there is no reported evidence for genetic linkage of AD to the region of chromosome 14q32 where the CYP46 gene is located. Thus, the potential contribution of CYP46 to AD genetics may turn out to be genuine, but with limited effect in the general population.

Ben Wolozin and colleagues at Loyola University in Maywood, Illinois, reported on the distribution of CYP46 in the brain and found signal in neurons in both AD and control brain. In AD brain, an increase in the levels of cholesterol 24-hydroxylase was also reported. Additionally, CYP46 co-localized with neuritic plaques along with microglia and astrocytes. CHO cells overexpressing APP that were either transfected with CYP46 or treated with 24(S)-hydroxycholesterol were reported to lead to decreased PMA-stimulated secretion of APP and decreased APP-CT. The authors proposed the hypothesis that CYP46 levels may be elevated near neuritic plaques to remove cholesterol from degenerating neurites, and that increased production of 24(S)-hydroxycholesterol increases Aβ secretion. (See also Rebeck section of ARF conference story.)

OLR1: The OLR gene encoding the lipoprotein receptor for oxidized proteins resides on chromosome 12, close to the α2-macroglobulin (A2M) gene. In a poster presentation, Parkinson et al. reported the results of testing a 3’UTR SNP (rs1050283) that was previously reported to be associated with AD risk in two studies (Luedecking-Zimmer et al., 2002; Lambert et al., 2003). In the NIMH AD family sample, this same SNP was not found to be associated with AD. This negative result for OLR1 contrasts with the consistently positive findings that have been previously reported in the NIMH sample for multiple polymorphisms in the gene encoding α2-macroglobulin (Saunders et al., 2003), which maps within 1 Mb proximal of OLR1. Parkinson et al. also reported no evidence for significant linkage disequilibrium between variants in the OLR1 and A2M genes, suggesting that the previously reported associations of AD with OLR1 are most likely not due to linkage disequilibrium with A2M. Thus, the DNA variant in OLR1 does not appear to substantially affect AD risk independently of A2M in the NIMH AD family sample.

TNFA: After testing two SNPs in the tumor necrosis α (TNFA) gene in an age- and gender-matched case-control study, E. M. Pfeiffer and colleagues at University of Washington Medical Center in Seattle presented evidence that the TNF-863 polymorphism, which leads to decreased production of TNFa protein, is associated with a reduced risk for developing AD. Interestingly, TNFA resides in a region of chromosome 6 shown in genome screens by multiple groups to contain a novel AD locus. These results provide further support for the hypothesis that genetic variability in the production of TNFa might affect the inflammatory response in AD pathogenesis. Further study is warranted.

MTHFR: Given that epidemiological studies have recently revealed elevated plasma homocysteine levels in AD (see ARF related news story), Yosuke Wakutani, Kazuhiro Nakashima, and colleagues of Tottori University in Japan performed a case-control study in a Japanese population to test three polymorphisms (C677T [Ala222Val], A1298C [Glu429Ala], and A1793G [Arg594Gln]) in the gene encoding methylenetetrahydrofolate reductase (MTHFR) for association with late-onset AD. They found MTHFR to contain four major haplotype alleles, one of which, haplotype-C (677C-1298C-1793G) was less represented in cases than in controls. This protective effect was stronger in patients who lacked the ApoE4 allele. These results suggest that this haplotype of MTHFR may protect against the development of LOAD, perhaps by regulating homocysteine levels (see also ARF Live Discussion.

STH: The Saitohin (STH) gene resides in the intron between exons 9 and 10 of the human tau gene on chromosome 17 (ARF related news story, see also Conrad et al., 2002). In New Orleans, Chris Conrad, Peter Davies, and colleagues at Albert Einstein College of Medicine in the Bronx, New York, reported that the STH is expressed similarly to the tau gene. Moreover, the STH SNP, Q7R, was also reported to be overrepresented in the homozygous state in late-onset AD cases. Based on this association, the potential for a pathological role of this amino acid change in STH was suggested. However, it will also be important, in parallel, to test whether the association of AD with STH might involve possible linkage disequilibrium of Q7R with polymorphisms/mutations in the tau gene, especially those associated with frontotemporal dementia.

DLD: Abe Brown, John Blass, and colleagues of Weill Medical College of Cornell University in White Plains, New York, presented evidence for association of the mitochondrial-ketoglutarate dehydrogenase complex with AD (see also ARF Live Discussion with Blass). Four SNPs in the DLD gene, which resides on chromosome 7, were genotyped in a case-control series of 297 Caucasians from New York City, which included 229 Ashkenazi Jews. Association with AD was reported specifically for the male population independently of ApoE status. Interestingly, this result was noted to be consistent with a previous report of association of AD with markers from this same region of chromosome 7, exclusively in paternal families (Bassett, et al., 2002).—Rudy Tanzi, Massachusetts General Hospital, Charlestown.

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