CONFERENCE COVERAGE SERIES
International Conference on Alzheimer's Disease 1998
Amsterdam, Netherlands
18 – 23 July 1998
Sixth Int. Conference on AD: Mechanisms of Neurodegenerative Conditions I
Perhaps one of the most striking features of the conference this year, reflecting the state of research on AD in general, is the continued absence of a clear consensus on what is the most relevant neuropathological change in AD. The first of three symposia on Mechanisms of Neurodegenerative Conditions reflected this ambivalence. Cochaired by D.M. Michaelson (Tel Aviv, Israel) and S.D. Yan (New York), the session included seven presentations (out of eight originally scheduled) that were largely unrelated to each other except, as the title indicated, that each addressed possible means of causing or preventing neuronal degeneration.
On the importance of understanding how neurons die, at least, there does appear to be some agreement. The loss of basal forebrain cholinergic neurons in AD, for example, has previously been proposed to be due to loss of neurotrophic support, i.e., NGF, but the simplest hypothesis, that there is a decrease in NGF in the AD brain, is not supported by published studies. However, Hock (abstract 287) presented evidence for a decrease in mRNA for the high affinity NGF receptor (trkA) in AD parietal cortex samples (with no change in cerebellar levels). This decline appears to be quite specific since no changes were found for mRNAs of other neurotrophin receptors or for the neurotrophins themselves. Dr. Hock suggested that the reduction in trkA might contribute to basal forebrain neuron degeneration by reducing the uptake of NGF. He also provided data implicating microglial cells as a potential source of NGF. A cell line sharing properties with human microglial cells was found to increase NGF synthesis in vitro in response to IL1-beta and TNF, two cytokines that have previously been implicated in AD. This effect appears to be mediated, at least in part, by NFkB.
NFkB, in turn, has been implicated in the cellular response to oxidative injury. The role of oxidation in AD pathology was addressed by three speakers, each using a different approach. C. Behl (Abstract 289) reviewed two lines of evidence implicating oxidative injury in neuronal degeneration. The first relates to the apparent protective effect of estrogens against AD. A variety of estrogens, and estrogen derivatives, were tested for antioxidant activity and their ability to protect neurons from glutamate or Aβ cytotoxicity. Only compounds with a hydroxl group were effective, consistent with the antioxidant effects of other phenolic compounds. The activity does not seem to be mediated by classical estrogen receptors. The second line of research involved the role of NF-kB. A mutant PC12 cell line that is resistant to Aβ toxicity shows high levels of NFkB. Dexamethasone treatment, or expression of the super-repressor of NFkB, reverses this resistance, presumably through down-regulating NFkB. Thus, NFkB is implicated in conferring resistance to oxidative insults.
F. Van Muiswinkel (Abstract 291) also addressed the role of oxidative injury with specific attention to changes in the expression of NADPH-oxidase in AD. This enzymatic complex is implicated in the respiratory burst associated with microglial activation. An antibody raised against one of the subunits of this enzyme (p22-phox) was found to stain microglial cells in AD brain tissue, but rarely in control tissue. Clusters of microglia associated with plaques exhibit immunoreactivity, but so do microglia throughout the rest of the neuropil and in perivascular locations. This suggests that production of free radicals is likely to be occurring on the part of microglial cells throughout the tissue, not just in plaque areas.
Still within the theme of oxidative injury, the possible contribution of advanced lipid peroxidation end products to neurodegeneration in AD was addressed by Dr. L. Sayre (Abstract 292). A number of previous studies have demonstrated markers of oxidative change in AD tissue. One product of lipid peroxidation, 4-hyroxy-2-nonenal (HNE), has also been shown to exhibit cytotoxic effects. The approach to studying the possible contribution of HNE-modified proteins to AD pathology involved raising antibodies to the more stable complexes of HNE, e.g., HNE-pyrrole or HNE cross-linked proteins, and screening AD tissue using immunostaining. Neurons that are positive for Alz-50, a suspected marker for neurons that will ultimately exhibit tangles, are stained with the HNE antibodies. This led to the speculation that HNE modification of tau might contribute to alterations that alter tau folding, e.g., apposition of the carboxy and terminal ends of the protein, thereby providing exposure of the Alz-50 epitope. This, or the association of lipophilic and/or amphiphilic molecules with tau, may subsequently lead to altered tau function, thus providing a potential route by which oxidative injury is coupled to tangle formation.
Other hypotheses of neurodegenerative mechanisms were also presented in this session. R. Itzhaki (Abstract 290.) reviewed her group's work on the role of HSV-1 in AD. HSV-1 transcripts are present in a majority of both AD and control brain tissue samples and an interaction between ApoE genotype and HSV-1 was found such that the presence of viral transcripts and the E4 isoform of ApoE confer a significantly higher risk of AD. In addition, E4 individuals are much more likely to suffer from "cold sores" with reactivation of HSV-1. Interestingly, a similar risk was not found for herpes keratitis, perhaps, Itzhaki suggested, due to reactivation of the virus in non-neuronal cells in the cornea rather than in neurons, as occurs in AD and in herpes labialis.
E. Masliah (Abstract 293) outlined a hypothesis implicating aggregation of α-synuclein (also known as NACP) in neurodegeneration. Mutations in this protein, which is abundant in synapses, have been implicated in a subset of patients with Parkinson's disease. Transgenic mice expressing α-synuclein under the control of the PDGF promoter exhibit high levels of the protein in the hippocampus, neocortex, and olfactor bulb. NACP aggregates were found within cortical and hippocampal neurons with a morphology highly reminiscent of Lewy bodies. No obvious glial changes were found. Ultrastructurally, inclusions were found within the nuclei and cytoplasm of neurons, although filamentous morphology was lacking. Masliah proposed that these aggregates might ultimately be toxic although no data were presented on this point. He suggested that crossing of these transgenic lines with other transgenic models of AD (currently under way) would provide a useful means of examining the interaction of α-synuclein with other components thought to play a role in neurodegenerative diseases.
Turning from the dark side of the force, so to speak, I. Gozes (Abstract 294) brought the audience up to date on activity-dependent neurotrophic factors (ADNF) and a homologous mouse peptide (ADNP) that exhibit remarkably potent (femtomolar) neuroprotective effects in vitro. These peptides show homology to hp60, a heat shock protein that is upregulated in neurons in response to VIP. Gozes provided a model involving two-way interactions between neurons and astrocytes whereby the release of VIP leads to release of ADNF from astrocytes which, in turn, protects neurons by up-regulating hp60. The peptides protect against Aβ toxicity and also show the ability to compensate for a number of the deficits in apoE-deficient mice, including retarded development, decreased cholinergic activity, and learning and memory deficits. The mechanism of action of these peptides remains to be established. If the peptides hold up as the potent trophic agents they appear to be, they provide a novel avenue for developing therapeutic strategies to promote neuronal protection in AD and other neurodegenerative diseases.
Overall, one left the session with the feeling that many questions, mostly non-trivial, remain to be answered before the puzzle of neuronal degeneration is solved.—Keith A. Crutcher
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Sixth Int. Conference on AD: Genetics I
This session was disappointing, offering nothing really new. What new data were presented appear to be very controversial.
Farrer et al. (Abstract 280) tried to confirm the linkage described by Duke University, North Carolina. They described a linkage with chromosome 12 for familial late-onset Alzheimer's disease (LOAD) (mean age at onset: 70.5 + 10.7 years). This association appears to be independent of ApoE4 status and not related to LRP1 and α2 macroglobulin loci.
Rebeck et al. (Abstract 281) analyzed the LRP exon 3 polymorphism (766) and the α2-macrogobulin V1000I polymorphism in sporadic AD. Regarding the LRP, they found a previously published polymorphism linked to AD. Concerning the α2-macroglobulin, GG frequency is overrepresented in AD. The combined data show a strong association (odd ratio 4.1 (pLilius et al. (Abstract 282; Lannfelt gave the talk) described 11 polymorphisms, all in strong linkage disequilibrium, on the tau gene, including five missense mutations on exons 4A and 6 (not expressed in brain). The 11 polymorphisms segregated together in a Mendelian way as two alleles. Cases with at least one ApoE4 allele and the AA tau genotype showed an increase risk for AD.
Premkumar et al. (Abstract 283): Bleomycin hydrolase (BH) is a Cys protease from the papain family. BH is a good β-secretase candidate. By immunoflurescence, it co-localizes with APP in transfected CHO cells. The BH gene has 12 exons and is localized on chromosome 17q11.1-11.2. There is a potentially functional polymorphism V442I. In their population, the authors did not confirm the increased frequency of the V/V genotype in AD patients previously described by Montoya et al. However, in Ashkenazi Jews and African-American populations, cases with I/I/ genotypes have a higher risk for AD.
Dermaut et al. (abstract 284) demonstrated that the PS1 mutation Glu318Gly is a neutral mutation (polymorphism) and is not related to dementia.
Sandbrink et al. (Abstract 285), confirmed an association of butyrylcholinesterase K variant (BCHE-K with LOAD, as previously described by Lehmann et al. The authors showed that there is no major synergy of BCHE-K and ApoE4.
Casadei et al. (abstract 286), studied three polymorphisms related to IL1 metabolism:
IL-1α -889 promoter polymorphism; IL-1β -511 promoter polymorphism; IL1ra intron 2 polymorphism.
IL1α polymorphism is associated with EOAD; IL1β is less clearly ass0ciated with EOAD, and finally, the question: Is IL1ra intron 2 polymorphism a disease susceptibility trait?
Finally, a poster session by Lambert et al. (Abstract 127), reported on polymorphisms in ApoE promoter that are good candidates to modulate ApoE expression.—Luc Buee
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Sixth Int. Conference on AD: Monkey Brain Injections
Bruce Yankner took time out from his talk on Down's syndrome (Abstract 7) to discuss his recent injection studies in young versus aged rhesus monkeys, published in the July issue of Nature Medicine. His key findings were that injections of fibrillar Aβ at a "plaque equivalent dose" resulted in cell loss, the proliferation of microglia and tau hyperphosphorylation in aged animals, but not young ones. These findings are in contrast to earlier studies by Podlisny and Selkoe (where "adult" but not "aged" monkeys were used).
Yankner showed an interesting example of immunocytochemical staining for Aβ revealing an "injected plaque" near a natural plaque in an aged monkey. The injected Aβ was more lightly stained and diffuse than the natural plaque, and was oblong in shape, as if oriented along the needle tract. Without the natural plaque present for comparison, one would have assumed the artificial plaque was real based on morphology. Yankner used several antibodies to hyperphosphorylated tau, including Ser-262, PHF-1 and AT8 to demonstrate dystrophic neurites in response to the injection. He indicated that Ser-262 yielded the strongest staining, with PHF-1 next and weak staining by AT8. This reaction was not seen in the brains of young rhesus monkeys. In the brief time allotted, he was unable to show slides of vehicle injections in neither aged monkeys nor Aβ injections in young monkeys for the audience to evaluate. Given Selkoe's observations that the neuropil response to vehicle injections was indistinguishable from Aβ injections and Bishop et al's poster (Abstract 535) which shows that there is neuronal damage and loss surrounding saline control injections into rat brain this is an important question. The curious will have to examine Yankner's Nature Medicine paper for the details.
Yankner favors the position that extracellular Aβ is the bad guy compared to intracellular Aβ, pointing out that the intracellular Aβ "load" is "minuscule" compared to the extracellular "load." Thus, the toxic effects of extracellular Aβ are probably more important. He hypothesizes that intracellular Aβ from a cell which lyses could lead to a seeding process which accumulates extracellular Aβ, propagating the problem.—Brian Cummings
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Sixth Int. Conference on AD: Cellular/Animal Models I
Soto (Abstract 295) presented data documenting the ability of their 11 and 5 amino-acid inhibitors to inhibit initial Aβ peptide aggregation and its ability to dissociate already formed Aβ fibrils in vitro. In addition to the assays mentioned in the abstract, this inhibition of fibril formation is also evident by EM analysis and protection via live cell/dead cell staining has been extended to six days in culture where 50 micromolar concentrations of inhibitor alone were not toxic to cells. He also presented additional data on the effects of Aβ injections into rat amygdala. Injections alone (still an N=9) resulted in deposition of Congo positive deposits of Aβ as well as detection of fibrils at the EM level. Cresyl violet staining shows hypertrophied cells (and frank cell death) and staining for microglia (IL1beta) and astrocytes (GFAP) revealed both local and more distant increases resulting from the injection. When inhibitor was co-injected with Aβ, the amyloid deposits were 50 percent smaller and Congo red and thioflavine negative. (It was not clear whether the glial response was also reduced.)
While it is already counterintuitive that any peptide could actually reverse already formed fibrils (as suggested by their in vitro data), this effect was also demonstrated in vivo! Additional animals were injected with Aβ alone and eight days later, the iAβ5 inhibitor was injected into the original site. Even under these conditions, there was an extensive reduction in the area occupied by Aβ. Soto concluded with the tantalizing suggestion that this approach might also by applied to other protein conformational folding disorders (e.g., prions) and hinted that work in progress is promising. During the Q&A, he clarified that to be effective in vitro, the inhibitors were used in a 1 to 1 molar ratio with Aβ while in vivo they used a 20 molar excess. Dr. Soto also indicated that these inhibitors can cross the blood-brain barrier and that there are some modifications to the peptide that can be made (have they been made?) to increase its influx. So my question is: What's the name of your startup company and when is the IPO?
Xu (Abstract 296) reported that 17β estrodiol was effective in reducing both Aβ40 and Aβ42 levels as measured via mass spectrometry, and that the effects of estrogen were time-dependant (over the course of many days). He also reported that pretreatment over many days resulted in increases in APP/Aβ within vesicles in the TGN and hypothesized that this "sequesterization" of APP/Aβ may reduce the amount of available substrate for β- and c-secretases, thus explaining estrogen's protective effect. Dennis Selkoe asked about a dose response effect (these data were not shown) and the level of effect, because his group has experienced high variability in Aβ levels between different neuroblastoma cell lines. From the data Dr. Xu described, this issue appears to be unresolved. Another person asked if 17α estradiol has a similar effect. The answer with regards to APPs is yes, but the investigators have not measured Aβ yet.
Suzuki (Abstract 297) presented evidence that phosphorylation of APP plays a role in neurite development. I missed the first part of his talk concerning the role of APP770/Arg672 in regulating the metabolism of APP, but returned to hear that the T668E mutation's effects on neurogenesis were demonstrated by measuring neurite number and neurite length via immunocytochemistry for MAP5 (both of which were reduced compared to controls). Someone asked if the Arg672 site is close to the motif for trafficking via the clathrin pathway. The short answer was that in nonneuronal cells, internalization of APP is normal.
Baumeister (Abstract 302) presented data on the effects of mutations in sel-12 on cellular function, morphology and behavior. Sel-12 is the C. elegans homologue to human presenilin. Since there is a caspase cleavage site on PS, it is a potential target site for therapeutic intervention. Sel-12 mutant animals have a lethal egg-laying deficit which results in the eggs hatching inside the mother. Expression of human PS1 or PS2 in sel-12 mutant C. elegans can block this lethal effect, but if mutant (FAD) human PS was used, there was no rescue effect. This also indicates that C. elegans is capable of processing human PS (and PS mutations) despite sequence differences. They also made mutants to block the caspase cleavage site on PS1 and PS2 and found no effect on the function of PS in C. elegans. Immunocytochemically, sel-12 was shown to be expressed in a variety of cell types, including the CNS (if you can call 302 neurons a CNS). There were no substantial differences within the CNS of sel-12 mutants, but subtle differences were observed in the neurite branching pattern of specific neurons. Since theses neurons are known to play a role in "temperature memory", they tested mutants and found they were deficient on this task. They were also impaired in the reversal response to noxious stimuli. They have not looked for synaptic changes yet.
Following Baumeister's talk, there was a spirited discussion between Christian Haass and Rudy Tanzi on whether caspase cleavage plays a role in the generation of Aβ. Haass argued that caspase cleavage plays no role while Tanzi remained unconvinced. Perhaps at basal levels of expression, cells might not need caspases to generate Aβ, but the story might be different in stressed cells (whose opinion?). They finally agreed to jointly state that "caspase cleavage is not required for basal production of Aβ in healthy cells" and concurred that "the caspase derived c-terminal fragment of PS1 is definitely BAD for cells." Stay tuned to the two labs for details (further developments?).—Brian J. Cummings
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Sixth Int. Conference on AD: Mechanisms of Neurodegenerative Conditions II
Alonso et al. (Abstract 590) reviewed the role of tau proteins in tubulin polymerization. There are six tau isoforms in the human brain. Three have three microtubule-binding domains (3R) and the three others have four microtubule-binding domains (4R). In Alzheimer's disease (AD), all six tau isoforms are found abnormally phosphorylated in paired helical filaments (PHF) and referred to as AD-P-tau. 4R isoforms bind better to microtubules than 3R isoforms. AD-P-tau induces microtubule depolymerization. AD-P-tau binds to normal tau isoforms (2+3+10+>2+3-10>2-3-10+>2+3+10->2+3-10->2-3-10-). AD-P-tau inhibits microtubule polymerization induced by normal tau isoforms by binding to them. This may be useful in developing new therapeutic strategies.
By immunoblotting using specific tau antibodies, Buée et al. (Abstract 591) demonstrated that all six hyperphosphorylated tau isoforms aggregate into PHF. Tau isoforms without the exon 10 sequence (3R) are hyperphosphorylated in Pick's disease, with the exception of the 12E8 epitope, and aggregate into Pick bodies in particular subsets of neurons. In corticobasal degeneration and progressive supranuclear palsy, 4R tau isoforms are hyperphosphorylated and aggregate into straight filaments. Similar conclusions were obtained using tau cDNAs with or w/o exon 10 and COS cell transfection followed by OA treatment. These data suggest that different subsets of neurons expressed tau isoforms at different levels.
Brion et al. (Abstract 592) obtained transgenic mice for the shortest tau isoform (fetal). They used the promoter of 3-hydroxy-methyl-glutaryl CoA reductase gene. The transgene was expressed preferentially in the brain. Somatodendritic domain in neurons was strongly labeled by AT180 and AT270 antibodies. Astrocytic structures were labeled by PHF-1 and AD2 antibodies. No labeling was obtained with Ubiquitin, TG3, AP22, AT100 and AT8 antibodies. These mice display a neuronal pathology similar as that described for pretangles.
Wang et al. (Abstract 593) studied tau phosphorylation and its effects on tubulin polymerization. Tau isoform was phosphorylated using CK-1, GSKß and pKA. Phosphorylated tau isoform inhibited tubulin polymerization. The weakest effect was observed when tau was phosphorylated by CK-1. The inhibition was much more severe with GSK3, then pKA, CK-1 + GSK3 and dramatic with pKA +GSK3. The phosphorylation sites dscribed in the present work are different from those found by Zheng-Fishhöfer (Abstract 914). Vincent et al. (Abstract 594) nicely demonstrated the importance of mitotic mechanisms in AD. Cdk2 activity slightly increases in AD. Cyclin A is not detected in controls and is present in AD. Cyclin A complex activity increases in AD. Upstream regulators of cdc2 (cdc25 A and B isoforms, cdk7) were analyzed. As an example, cdc25A data were presented. Cdc25A is expressed in NFT and dystrophic neurites but also present in neurons in controls. Equivalent amounts are found in AD and C. Surprisingly, cdc25A is constitutively active in differentiated neurons of human brain. Expression of mitotic kinase/cyclin is an initiating step in NFD.
Arendt et al. (Abstract 595) brought more arguments about the high degree of plasticity in neurons predispose to tangle formation by studying cdk inhibitors. Cdk inhibitors are elevated in AD and are closely associated with neurofibrillary degeneration. It is true for p15 INK4b, p16INK4a, p18INK4c and p19INK4d. However, no alteration of altered expression of p21Cip1 and p27Kip1. Finally, INK4 and NFD have similar regional distribution patterns.
Takashima et al. (Abstract 596). confirmed that PS1 mutations increased Aß42 formation. They have also analyzed the interactions Tau-PS1 and GSK3ß-PS1. They found that both GSK3ß and Tau bind to the same region on PS1 [250-298]. PS1 mutations (C263R and P264L) increase by about three times the association PS1-GSK3ß. They do not increase the association PS1-Tau. However, tau hyperphosphorylation was increased. These data suggest that there may be a physiological connection between tau, GSK3 and PS1.
Lee et al. (Abstract 597) demonstrated in a very complete study that tau proteins bind to SH3 (Src homology 3) domains of Fyn. SH3 (from Fyn, Lek and src) usually binds PXXP proline-rich motives. Fyn is expressed in brain and thymus. In brain, it is localized in developing axonal tracts and promotes neurite growth in response to specific cell adhesion molecules. Fyn is also required in cytokinesis in B cells. They found that a subpool of tau binds Fyn. When Tau and Fyn are co-transfected, they co-localized in 3T3 cells. Furthermore, cell morphology is completly modified: cells are spherical. After Fyn transfection, Tau is found Tyr phosphorylated. Tau protein is more than a MAP...—Luc Buee
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Sixth Int. Conference on AD: Towards the Next Generation of AD Therapeutics
This roundtable session, supported by a grant from SmithKline Beecham and cochaired by G. Wilcock (Bristol, UK) and R. Kumar (Essex, UK), sought to provide a glimpse of future directions for the development of new therapeutic agents for AD. The speakers provided a rather broad range of perspectives on this topic. The first two presentations, by Grundman (Abstract 312) and H. Feldman (Abstract 313) dealt primarily with the design of clinical trials. Grundman reviewed the theoretical basis for different study designs, emphasizing the need to use designs that will reveal differences between agents that only treat symptoms (as is thought to be the case with existing cholinesterase inhibitors) as opposed to modifying the course of the disease (the ideal goal for new therapeutics). Candidates in the latter category are likely to include anti-inflammatory drugs, estrogen, neurotrophic agents and inhibitors of amyloid deposition or tangle formation (yet to be developed). Grundman reviewed two types of designs: randomized withdrawal and randomized start. Each model has its own advantages and disadvantages, depending on the extent to which the drug affects symptoms versus modifying progression. Results may be especially difficult to interpret if a drug has both types of effects. It is also possible that rates of progression may vary depending on the stage of disease. He also emphasized the ability to reduce the required sample size by extending the duration of a trial.
These theoretical issues were given practical consideration by Feldman, who provided examples of actual clinical trial data. The results of a clinical study with donepezil (Aricept), for example, showed clear alleviation of symptom progression but a subsequent regression to the status of patients receiving placebo following drug withdrawal (Rogers et al., 1998). A study of Exelon effectiveness (Rogers & Friedhoff, 1998), however, did not show a similar "catch-up" following drug withdrawal, suggesting that the underlying disease course might have been slowed. But Feldman cautioned that this interpretation assumes a linear rate of clinical progression. Evidence for an effect on disease progression has been obtained with studies of propentofylline, which exhibited effectiveness beyond the treatment period. Similar effects were not found with a delayed start trial, probably, Feldman suggested, due to limited sample size. Trials with deprenyl and vitamin E showed positive effects on some measures but not others. Feldman urged caution when relying on outcome measures that are not directly anchored to the disease process.
The session became even more focused on specific approaches to developing new treatments for AD beyond inhibiting cholinesterase activity. Kumar (Abstract 314) reviewed the rationale for developing drugs that would effectively replace lost cholinergic function without relying on residual cholinergic neurons (as is the case with AChE inhibitors). A variety of muscarinic agents have been examined. Compounds that show little discrimination between different muscarinic receptor subtypes (e.g., arecoline, bethanecol, oxotremorine) show some beneficial effects on cognitive measures but also give rise to undesired effects such as sweating and nausea. More selective compounds, partial muscarinic agonists such as Memric, have been developed that show fewer side effects. Unfortunately, they also show limited, nonsignificant, benefit on cognitive measures. Xanomeline, on the other hand, appears to be effective on cognitive measures but is poorly tolerated. Some of these agents also show effectiveness on behavioral measures other than cognitive performance. Kumar suggested that there may yet be hope for agents of this type to treat other symptoms of AD.
J. Ghiso (Abstract 315) brought amyloid to center stage with a discussion of proteins that are likely to be "molecular chaperones" of the Aβ peptide. The best candidates for chaperones in serum are apolipoproteins such as ApoJ (also known as clusterin), which shows high affinity binding to the Aβ peptide. The ApoJ/Aβ complex is quite stable and ApoJ prevents aggregation of the peptide as well as amyloid fibril formation. Prior interaction with ApoJ does not appear to modify the peptide, however, since it is still capable of aggregating after being isolated from the ApoJ/Aβ complex. This interaction also has functional consequences in that ApoJ protects cultured rat hippocampal neurons from Aβ toxicity. Since ApoE shows a relatively high affinity for aggregated Aβ, Ghiso suggested that ApoE and ApoJ may serve antagonistic functions as amyloid chaperones, the former promoting amyloid deposition and the latter preventing it. He noted that agents that promote ApoJ/Aβ complex formation or stability would potentially be useful in preventing amyloid deposition.
K. Iqbal (Abstract 316), tauist defender, reviewed evidence for the primacy of neurofibrillary degeneration in AD. The most likely mechanism leading to tangle formation involves hyperphosphorylated tau (p-tau), which is much more abundant (approximately 25-fold) in AD brain tissue than in control brain tissue. Iqbal believes that p-tau plays a pivotal role in destabilizing microtubules (which then leads to neuronal degeneration) and in forming tangles (not a cause of neuronal death itself). This hypothesis is supported by biochemical studies in which p-tau from AD brain was found to inhibit microtubule formation, an effect that could be reversed by dephosphorylation of tau. P-tau does not form paired helicial filaments, which is thought to be due to interactions between p-tau and normal tau. A role for tau is supported by the fact that tau is elevated in AD CSF and by recent studies demonstrating that mutations in the tau gene give rise to some forms of neurodegeneration. Iqbal proposes that AD drugs could be developed that would prevent hyperphosphorylation of tau.
The final speaker, D. Price (Abstract 317), provided an overview of the use of transgenic mouse lines as models of AD pathology. He noted that a recent panel had recommended the use of C57BL/6J mice as founders due to their good performance on a variety of behavioral tasks and that other strains are less optimal in this regard. Transgenic APPswe mice show Aβ deposits, an increased ratio of Aβ42/Aβ40, dystrophic neurites, gliosis and behavioral deficits. By 12 months, these animals show impaired performance on the water maze and the eight-arm radial arm maze. They also show extensive Aβ deposition in the outer portion of the dentate molecular layer and, at 26 months of age, numerous plaques in the cortex. Crossing this line with mutant presenilin transgenic mice gives animals that show accelerated plaque development, suggesting that PS1 mutations may enhance Aβ deposition. Price suggested that putting mutant tau into this mix may well provide the key to the ultimate AD mouse line.
The session concluded with several questions from the audience. Perhaps the most interesting question was put as a general one to all speakers asking for their opinion on what the future holds for new drugs. The answers were interesting, if not surprising, in light of the presentations. Ghiso-inhibitors of amyloid deposition; Price - inhibitors of the c-secretase that cleaves APP; Iqbal-agents that inhibit degeneration or promote microtubule stability; Grundman-cocktail approach; Feldman-ditto; Kumar-ditto. Taken as a whole, one might conclude that none of the current alternatives to AChE inhibitors for treating AD should be abandoned yet.—Keith A. Crutcher
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Sixth Int. Conference on AD: Mahley Plenary Lecture
B. Mahley (Abstract 303) reported new data on transgenic mice that could help elucidate the role of ApoE isoforms in neurodegeneration. He began his lecture with an overview of the structure and function of apolipoprotein E (ApoE), a lipid transport protein that exhibits an isoform-specific association with the risk of AD (the E4 isoform being linked to greater risk). Much is known about the structure of ApoE, which includes an N-terminal portion that contains the receptor binding domain (residues 136-150) and a C-terminal portion that includes the major lipid-binding region (residues 244-272). Studies designed to uncover isoform-specific differences in ApoE function suggest that the arginine at position 61 is more available for interaction with the C-terminal region (glutamic acid at position 255) in E4 as compared to the E2 and E3 isoforms. This suggests the possibility of somehow modifying this interaction to make the E4 isoform function more like the E3 isoform.
Regarding the function of different isoforms, early studies demonstrated isoform-specific effects of ApoE on neurite outgrowth such that the E3 isoform promotes outgrowth whereas the E4 isoform inhibits outgrowth, leading to the hypothesis that E4 may not provide effective neuronal repair or protection. As a test of this hypothesis, transgenic mice were generated in which human E3 or E4 are expressed under the control of the NSE (neuron-specific enolase) promoter in ApoE knockout mice (ApoE -/-). The level of ApoE in the brains of the transgenic mice is comparable to that in the human brain. The mice were evaluated in terms of neuronal and behavioral changes. Synaptophysin and MAP-2 immunoreactivity are reduced in ApoE -/- mice and in E4 transgenic mice as compared with wild-type or E3 transgenic mice (more pronounced declines at seven to nine months than at three to four months). A similar result is obtained with kainic acid injections, which elicit greater reduction of these neuronal markers in the ApoE -/- and E4 transgenic lines.
Behavioral analysis included water maze performance and measures of exploratory activity. For these studies analysis was restricted to six-month-old female mice, which showed greater effects than male mice or younger female mice. Again, the same general group pattern was obtained as for the immunohistochemical studies; wild-type and E3 mice showed similar performance levels but ApoE -/- and E4 mice showed deficits. Decreases in exploratory behavior were due to declines in vertical motion (rearing events). Interestingly, on the water maze task, the E4 mice showed greater deficits than the ApoE -/- mice. There were no deficits in any of the mouse lines in visual or motor skills.
Dr. Mahley concluded that ApoE -/- and E4 transgenic mice have greater "neurodegeneration," although no direct evidence for degeneration of neurons (as opposed to altered development of these markers) was provided in this presentation. These mouse lines may prove useful in ultimately clarifying the normal function of ApoE in brain as well as its potential role in neurodegenerative diseases.—Keith A. Crutcher
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Sixth Int. Conference on AD: Mechanisms of Neurodegerative Conditions III
Aβ's fibril-formation processes were revealed in a tour de force of atomic force microscopy by Harper (Abstract 909). The initial species to be detected are four nm globular assemblies which range in size from 1.4 to 14 nm. He indicated that these globules are similar in size to that reported for "ADDLs." Following the appearence of the globular assemblies, they see protofibrils which do not exceed 200-300 nm in length. Finally, they see the traditional 8-10 nm in diameter and up to one micron in length Aβ fibrils. This transformation is very rapid and globular and protofibrils rapidly disappear from the samples. Using Aβ1-40, it takes four days for the globules to first appear. Fibrils are first observed around four weeks and when they first appear, they are already very long. Comparing the rate of assembly between Aβ40 vs. Aβ42, the Aβ42, as others have already indicated, fibril formation is accelerated. If they divide a sample and seed half with one percent fibrils, they see very rapid fibril growth but no fibers in the other half even after seven days. Using protofibrils as seeds does not produce as rapid a growth into fibrils. Harper also presented data that low temperature inhibits the formation of fibrils and keeps the peptide in the globular (ADDL-like) state. How small can these ATM studies go? The current tip size is 10 nm, but using new single wall carbon tips of one nm (available at K-Mart in aisle 4), Harper indicated that we are close to imaging in the two to five nm range in biological samples. Not bad.
The take-home conclusions from Walsh's presentation (Abstract 910) on the process of fibril formation: LMW Aβ shows little order by CD, but upon incubation LMW Aβ can aggregate. Protofibrils can dissociate, but this pathway is minor. Protofibrils have electrophysiological effects on cells (although Walsh is careful to point out that this doesn't mean that this is physiological). Protofibrils are toxic to cells, but not as toxic as full fibrils.
Greg Cole gave a nice presentation (Abstract 911) on the possible presence of "synapoptosis" in both the AD brain and the Hsiao mouse. He has generated an antibody to a caspase cleavage fragment of actin he calls "Fractin" which stains plaques, dystrophic neurites and microglia (see Am J Pathol, Feb 1998). He showed several examples of apoptotic blebbs coming off of dystrophic neurites and synaptic clusters and synapses degenerating and being phagocytosed by microglia. He proposed the term "synapoptosis" for this stripping away of synapses. In double and triple confocal imaging Fractin labelling was sometimes co-localized with APP and AT8, but the majority was independent in both the AD brain and Hsiao mouse.
There was also co-localization with synaptophysin. Cole also reported a decrease in synaptophysin staining around amyloid plaques in entorhinal cortex of the Hsiao mouse. Quantification via Western blot indicates a strong trend for decreased synaptophysin in 12-month vs. two-month mice, but due to an outlier, the difference is not yet significant. (Hsiao pointed out that Irizarry's report of no synaptophysin decreases in the molecular layer of the dentate gyrus not the entorhinal cortex). In the AD brain, whole microglia around plaques were Fractin immunopositive while positive microglia within the Hsiao mouse were almost never seen. Cole hypothesized that when reactive oxygen species damage mitochondria within the synapse, the release of cytochrome C activates a caspase cascade and degradation of the synapse. The question remains, however, why there is little neuron loss in the Tg mouse compared to man and Cole's suggestion is that there is more caspase labeling in terminals and far less activation of caspase in the microglia of Tg mice. Someone asked if Cole was suggesting that the synapoptosis was presynaptic and not postsynaptic. He replied that he has looked for co-localization of MAP-5 and Fractin and sees little evidence of co-localization. However he also pointed out that his Fractin antibody may not recognize epitopes within dendrites as well as those within axon terminals.—Brian Cummings
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Sixth Int. Conference on AD: Pathology I
Three abstracts presented during this session, as well as those by Vincent et al. (Abstract 594) and Arendt et al. (Abstract 595), indicate that cell cycle and mitotic mechanisms may be a key factor in the understanding of tau phosphorylation. Illenberger et al. (Abstract 912) analyzed tau phosphorylation in LAN-5 neuroblastoma cells and CHO cells stably transfected with tau protein. In both cells nonsynchronized, the phosphorylation pattern using 32P and 2D phosphopeptide mapping and sequencing was similar: phosphorylation of Ser-Pro motifs and Ser214 and 262. CHO cells were also blocked in anaphase with nocodazole. In this situation, tau proteins are free and do not bind to microtubules. They are highly phosphorylated, especially at Ser214. Finally, Ser214 phosphorylation by PKA prevents microtubules nucleation. However, AT100 is not found in mitotic cells (see Abstract 914).
Stem cells are potential precursor cells for both neurons and glial cells. Tatebabayashi et al. (Abstract 913) developed this approach. First, they characterized the cells: they are MAP2 negative (except for MAP2c), NF negative, GFAP negative, NSE positive and also tau-positive (except for tau-1 epitope). Tau expression and phosphorylation increases in a dose-dependent manner with FGF2.
Zheng-Fishhöfer et al. (Abstract 914) demonstrated that in vitro AT100 epitope generation needs the sequential phosphorylation of GSK3ß and PKA. In fact, they used two approaches, one way using tau constructs, kinase activity from rat brain and specific kinase inhibitors and the second way using activated kinases and tau constructs. They nicely showed that first a PHF-like conformation is required (that can be mimicked by heparin or RNA), second GSK3ß phosphorylation at AT8 sites and Thr212 and finally Ser214 pKA phosphorylation.
Mutations on the tau gene suggest that when tau proteins do not bind to microtubules they are free to aggregate (see news summary of Hereditary Fronto-Temporal Dementia and Pick's disease). However, it does not explain why they are hyperphosphorylated when aggregated into filaments in all neurodegenerative disorders (Abstract 591). After tau release from microtubules, cell cycle mechanisms may be reactivated and lead to tau phosphorylation. It is part of the new challenge to understand neurofibrillary degeneration: What is the role of phosphorylation?
The two following abstracts may be useful for illuminating this question. One indicates that tau phosphorylation is also linked to the cholinergic system, and the other demonstrates that nonphosphorylated tau proteins may aggregate in to PHF.
Forlenza et al. (Abstract 915) confirmed the study by Sadot et al. in Journal of Neurochemistry showing that muscarinic agonists reduce tau phosphorylation through PKC activation of m1 and m3 receptors. They demonstrated that this decrease in tau phosphorylation is mediated through GSK3 inhibition. In conclusion, 100 µM carbachol induces a decrease in tau phosphorylation and an increase in the formation of microtubules bundles.
Mandelkow et al. (Abstract 916) reviewed their in vitro experiments on tau aggregation. One of the first step is the dimerization of tau isoforms through a disulfide bridge (Cys322). Presence of polyanions including RNA and glycosaminoglycans enhances tau assembly into PHF. Effects of glycosaminoglycans on tau aggregation into filaments were also described by Avila et al (Abstract 619) and Goedert et al. (Abstract 926). Moreover, N- and C-terminal truncation of tau isoforms further increase the rate of in vitro PHF formation. The role of truncation was also emphasized by the two next speakers, Novak (Abstract 917) and Cattaneo (Abstract 918).
Mitro et al. used the monoclonal antibody MN423 that recognizes tau isoforms cleaved at Glu 391 (-DHGAE) to demonstrate that truncated tau proteins are found within neurofibrillary tangles (Abstract 917). Tau truncation seems to be an early event in tau pathology. Furthermore, a significant population of MN423-positive neurons was apoptotic. To further address this problem of apoptosis, Fasulo et al., from the same group, (Abstract 918) used cell transfection to demonstrate that truncated tau isoform (residues 151-391) induce apoptosis in about 50 percent of COS cells. Interesting preliminary results seemed to indicate that induction of apoptosis may also lead to truncated tau proteins (Abstracts 1120 and 1121).
Arawaka et al. (Abstract 919) described new monoclonal antibodies that recognizes sequence boundaries of regions encoded by alternatively spliced exons. They developed specific antibodies against sequences encoded by exons 2, 3 and 10 but also against sequences at the boundaries between exons 1 and 4, 2 and 4, 9 and 11. All antibodies labeled NFT by immunohistochemistry. They are highly specific when tested on recombinant tau proteins. However, there was no data on phosphorylated tau proteins or on total brain homogenates. If these antibodies are still specific for hyperphosphorylated tau proteins, they may be useful tools for studying neurofibrillary degeneration.—Luc Buee
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Sixth Int. Conference on AD: Molecular Interactions in AD Pathogenesis
The "great amyloid debate" appeared to resolve at least one issue in the rather contentious arena of defining the critical neuropathological events in AD. Amyloid per se, i.e., "extracellular, fibrillar, congophilic deposits" (as stated by Robert Terry), is unlikely to be central to the disease process (not implying that APP or the Aβ peptide are not important). Of course this doesn't narrow the possibilities down very much, but it does give those who have not devoted their lives to the study of amyloid some hope that their efforts are not in vain. (It wasn't that long ago that one of the leaders in AD research quipped that "if you weren't working on amyloid, you weren't studying Alzheimer's disease"). In this workshop, half of the presentations related to the potential role of ApoE (reviewed in the plenary lecture by Dr. Mahley on Sunday), two others focused on Aβ peptide effects and one reported on tau-microtubule interactions.
ApoE is a bit like an unwelcome dinner guest, not exactly a pariah, but, rather, someone who is well-known but just doesn't belong at the event. Its well-documented role in lipid transport seemed to secure a comfortable place in biology before it barged into the field of AD research. But that's exactly what it did when the group at Duke University (Roses et al.) showed the greater epidemiological risk of AD with the ApoE4 genotype.
The three presentations on ApoE were all based on in vitro models of ApoE function. D. Holtzman (Abstract 876) provided an update on studies of the effects of ApoE on neurite outgrowth using cultured astrocytes from transgenic mice expressing human ApoE4 or ApoE3 under the control of the GFAP promoter. This leads to high levels of ApoE in the brain, primarily localized to astrocytes, which also secrete high levels of ApoE in culture. The secreted ApoE is found in association with HDL-like particles and cells secreting E3 promote more neurite outgrowth than E4-secreting cells, outgrowth that is blocked by antibodies to LRP. Entorhinal cortex lesions in ApoE-deficient mice reveal slowed clearance of degenerating fibers, suggesting a role for ApoE in this process. More recent unpublished data reveal that the ApoE3 particles look similar to the mouse wild-type particles but have reduced lipid content. ApoE-deficient astrocytes do not appear to secrete HDL-like particles. Holtzman summarized with a suggestion that ApoE may be able to deliver or remove cholesterol from neurons, thereby accounting for possible roles in neurite outgrowth and clearance of degenerating fibers, respectively.
M. LaDu (Abstract 877) continued the ApoE theme but was more focused on the interaction between ApoE and Aβ. She reviewed published data demonstrating that ApoE2 and ApoE3 form stable SDS-resistant complexes with Aβ whereas ApoE4 does not. This isoform difference in complex formation is dependent on the presence of lipid. This suggests that the type of lipid particle is important in determining the activity of the ApoE. In fact, lipid-associated ApoE3 prevents Aβ neurotoxicity, but ApoE4 does not (in fact, ApoE4 exhibits slight neurotoxicity by itself). Furthermore, both isoforms show the ability to block the activation of astrocytes by Aβ. This is a transient effect and may involve a signaling pathway. LaDu suggested that the interaction of ApoE with Aβ might ultimately lead to differences in the extent to which Aβ is deposited as plaques (along with ApoE). In the presence of ApoE4, higher levels of soluble Aβ may result in greater activation of astrocytes and cytotoxicity. She emphasized the importance of characterizing the lipid environment of ApoE since this seems to be critical in explaining the isoform-specific differences.
The third ApoE-related talk was presented by W. Strittmatter (Abstract 879), who, as part of the Duke group, was one of the first to propose that ApoE may play a critical role in AD through interactions with the cytoskeleton. He noted that isoform influences of ApoE are not restricted to AD since there is considerable evidence in the literature for greater deficits following a variety of neurological insults in the presence of the ApoE4 allele. In order to uncover possible biological functions of ApoE, ApoE-deficient mice have been studied for alterations in peripheral nerves. In fact, nerves from such mice exhibit more irregular profiles of unmyelinated axons (decreased circularity) as well as a decrease in their number. This may potentially relate to the fact that ApoE is normally expressed in nonmyelinating Schwann cells. These mice also exhibit decreased nociceptive function. Another alteration is an increase in the type of tau ("big tau") normally found in peripheral nerve. Strittmatter suggested that this may reflect alterations in the cytoskeleton arising from the loss of a normal interaction of ApoE with tau or MAP2c.
E.-M. Mandelkow (Abstract 878) presented a fast-paced overview of the "tau hypothesis" and the potential role played by MARKs (microtubule-affinity-regulating-kinases). She reviewed evidence for the importance of phosphorylation sites on tau (especially serine-214 and serine-262), which appear to regulate the interaction of tau with microtubules. Overexpression of MARKs in CHO cells, for examples, leads to disruption of microtubules. Mandelkow presented an interesting hypothesis regarding the role of MARKs in maintaining neuronal polarity. A homologue of MARK, par-1, appears to play a critical role in establishing polarity in the C. elegans zygote and in epithelial cell polarity. By analogy, Mandelkow suggested that the primary alteration in AD neurons is also a loss of polarity. In fact, an antibody that detects tau phosphorylated at serine-262 reveals otherwise apparently-healthy neurons prior to the development of neurofibrillary changes.
The final two talks related to effects of the Aβ peptide. D. Small (Abstract 880) presented data on the effects of this peptide on AChE expression. He noted that although there is a general decline in AChE in the AD brain, there is an increase around plaques. Aβ peptides that form fibrils cause increased AChE expression in a carcinoma cell line (P19 cells), an effect that is mediated by L-type calcium channels. In transgenic mice expressing CT100 there is also high production of Aβ, providing a model for examining the effects of increased levels of Aβ on AChE in the brain. In fact, these mice have higher AChE levels when extracted under non-detergent conditions. Since there are different forms of AChE, depending on the extent of glycosylation, the possibility of differential effects on AChE type was studied. A greater amount of abnormally-glycosylated AChE is present in both the transgenic mice and in AD brain tissue. Furthermore, elevated levels of this form of AChE are present in postmortem CSF samples from AD patients, but not controls or patients with non-AD neurological disease, suggesting potential utility for diagnostic purposes.
S. Yan (Abstract 881) reviewed evidence for possible mechanisms mediating Aβ toxicity. She noted that at low levels of Aβ, its effects may be mediated by different mechanisms than when present at high, fibrillar levels, the latter perhaps causing more nonspecific changes. One receptor candidate for mediating extracellular effects of Aβ is RAGE (Receptor for Advanced Glycation End-products), a member of the immunoglobulin superfamily that is expressed at high levels in the AD brain and is expressed on cells that are affected by Aβ. Expression of dominant-negative RAGE in neuron-like cells blocks the activation of ERK1/2 and apoptosis. A candidate for mediating intracellular effects of Aβ is ERAB (Endoplasmic Reticulum Aβ-Binding Protein), which appears to promote Aβ toxicity, as evidenced by increased apoptosis in COS cells expressing mutant ERAB. Future studies are directed to clarifying the role of these candidate mediators of Aβ toxicity through transgenic models.
Unfortunately, the subsequent discussion period, in which a number of questions from the audience were fielded by the panel of speakers, was not much more illuminating than the individual presentations, suggesting that ApoE, tau, and Aβ, all of which deserve to be invited dinner guests, still have not revealed their true identify and remain somewhat enigmatic players in a very complicated drama.—Keith A. Crutcher
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Sixth Int. Conference on AD: Development of Aβ Peptide Inhibitors
If you came to Amsterdam hoping to hear a lot of new data on Aβ peptide inhibitors, you might have learned more by window shopping in the red-light district. This doesn't mean that the session was without merit. Barbara Cordell (Abstract 937) gave an excellent presentation on issues surrounding our understanding of c-secretase(s). Previous reports of a compound that inhibits the production of Aβ40 but not Aβ42 have been interpreted as supporting the existence of more than one C-secretase. However, Cordell noted that in these experiments, the decrease in Aβ40 is accompanied by an increase in Aβ42. She suggested that the compound is not a specific C-secretase inhibitor but rather an inhibitor of a carboxy-terminal exopeptidase, which is responsible for converting long Aβ to shorter forms.
Cordell also pointed out that the "theoretical" transmembrane domain of Aβ is precisely that: "theoretical." For Aβ, it is possible the cleavage occurs outside the membrane, because we really don't know where Aβ sits. Further supporting her hypothesis is the fact that one rarely sees Aβ peptides shorter than 39 amino acids. Because carboxy-terminal exopeptidases "don't like to chew glycines," Cordell proposed, they stop before a.a. 37 and 38.
Dennis Selkoe (Abstract 940) summarized his work on Aβ processing, emphasizing that all known PS1 and PS2 mutations increase Aβ42. He also suggested that the fact that PS mutations influence Aβ40 to Aβ42 ratios in non-neuronal cells from a variety of peripheral systems and cause rapid stabilization of oligomeric forms of Aβ further supports the Amyloid Casade Hypothesis. If it happens in systems so far removed from brain, and it happens in neuronal systems too, it is most likely a central process. In response to Cordell's suggestion that the c-secretase does not need to cleave Aβ within the membrane, Selkoe favored the hypothesis that c-secretase binds with normal presenilin so that it can cleave APP within the membrane.—Brian Cummings
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Sixth Int. Conference on AD: Roundtable C: Metabolism and Alzheimer's
Over a decade ago, John Blass and his colleagues proposed that AD was caused by compromised metabolism. Today, this theory has gained support from a convergence of numerous therapeutic, pathological and biochemical studies pointing to a metabolic dimension to Alzheimer's disease (AD)-the subject of this roundtable.
Siegfried Hoyer (Abstract 15) focused on glucose, the primary energy source of the brain. He found that glucose utilization initially drops during the course of the disease, with a compensatory increase in use of other substrates such as fatty and amino acids. Increased amino acid metabolism is supported by increased ammonia secretion from the brain in AD. Hoyer feels that the switch from glucose is a result of deficiencies in the insulin receptor, actually suggesting that AD may be a kind of cerebral diabetes. However, while the insulin receptor is reduced in normal aging, its level is not substantially further reduced in AD. Nonetheless, the theory gains support from the observation that changes in insulin receptor activation can lead to tau phosphorylation. What's more, its expression level is high in hippocampus and hypothalamus-areas affected in AD.
George Perry (Abstract 16) presented the extensive evidence that he, Mark Smith, and others have amassed showing that increased oxidative damage specifically involves vulnerable neurons in AD. Since no cytopathology has been ascribed to these neurons (i.e., no neurofibrillary tangles), the increased radicals cannot be lesion-based. To understand whether mitochondria might be that source, they performed in situ hybridization for mtDNA, both wild-type and with the 5kb common deletion, in AD and control samples. Perry reported a two- to threefold increase in mtDNA specific for vulnerable neurons. Cytochrome oxidase immunoreactivity is similarly increased while cytochrome oxidase activity is unchanged. These findings suggest a profound alteration in mitochondrial metabolism leading to mtDNA proliferation in AD. Perry also presented the first evidence of oxidative damage to RNA in AD and further showed that its level was positively correlated with mtDNA during normal aging but not during AD. Instead, in AD cases, the best predictor of RNA oxidation was amyloid-β, and it was inversely related suggesting amyloid-β may be playing a protective, rather than a toxic, role.
Christian Pike (Abstract 17) presented cell culture models of amyloid-β toxicity to analyze whether amyloid-β-related death is mediated by oxidative stress. He compared iron, a true oxidative insult, with amyloid-β and found that while both yield similar profiles of death in ~15 hours, iron, but not amyloid-β, can be blocked by probucol. Pike proposes that amyloid-β leads to reactive oxygen production, and interprets amyloid-β-related oxidative stress as a feature, but not the critical feature, of neuronal death in his model of amyloid-β toxicity. Several questions were raised about the relevance of culture studies, because compartmentation and redox state will play key roles in amyloid-β toxicity if it acts as a pro/antioxidant. Certainly in vivo models have shown no amyloid-β toxicity.
Ahmad Salehi (Abstract 18) presented a compelling argument that metabolism is vastly reduced for neurons in AD. His assessment is based on the finding of Nicholas Gonatas that there is a reduction in the area occupied by Golgi apparatus in AD versus controls. A further premise of their studies is that apolipoprotein E plays a role in protecting neurons from death in AD, and that that role is to protect neurons by increasing their metabolism. Consistent with this view is the finding that the reduction in the Golgi appartus is greatest in cases with apolipoprotein E4. Salehi further suggested that Trk4 abnormalities may underlie the metabolic abnormalities he has noted. Not discussed is that vulnerable neurons in AD are heterotypic regarding growth factors, making an argument for a single growth factor abnormality difficult to understand.
Leon Thal (Abstract 20) discussed the recently completed trial of vitamin E (1000U/day) and selegiline (5mg). The results show a clear delay of deterioration in milestones of daily living for both vitamin E and selegiline treated patients, e.g., 25 percent reduction in institutionalization, while there was little difference in mental improvement between any group. Interestingly, the group receiving both vitamin E and selegiline showed no improvement beyond either agent administered separately. These findings clearly present the value of two weak antioxidants as therapeutics. While it might be criticized that all cases to enter the study were already moderately demented, this is essential to detect changes over a two-year study period. Significantly, no toxicity was noted for either vitamin E or selegiline.
Hans Gutzmann (Abstract 19) presented the results of a clinical trial with idebenone, a coenzyme Q derivative with strong antioxidant activity specifically related to its reduction in the mitochondria. As such, idebenone protects cells from oxygen radicals at a primary source of production. Idebenone significantly slowed the decline of patients, particularly those less who were demented. Switching patients receiving placebo from the first year to idebenone in the second year also slowed their decline, but never resulted in a "catching-up" to the group receiving idebenone for both years. Significantly, patients responded rapidly to idebenone, seemingly protecting them from further deterioration. These promising findings suggest that carefully targeted antioxidants can be efficacious in the treatment of AD.
Masaomi Miyamoto presented the pharmacology of idebenone, stating that it rapidly passes the blood-brain barrier. It is the reduced form of idebenone that terminates damaging radicals. However, because idebenone can only be reduced in the mitochondria, between sites I and III coupled with complex II, it does not participate in redox cycling-a problem associated with most other strong antioxidants. Redox cycling would lead to increased radicals rather than radical quenching. The low toxicity of idebenone as well as strong antioxidant properties are ascribed to its ability to prolong the lifespan of rats, a property that vitamin E, a weak antioxidant, does not share.
Idebenone administration has been shown to increase cerebral blood flow and glucose utilization in models of infarction and basal forebrain lesions. In consideration of the major reduction in cerebral blood flow in AD, one can wonder if the clinical benefit of idebenone to patients might involve increased brain perfusion. In summary, idebenone, by acting as an antioxidant and increasing electron transport and cerebral blood flow, improves brain metabolism.
Ken Nagata presented intriguing positron emission tomography studies that show that cerebral blood flow is reduced by 30% in AD while the extracted oxygen fraction is reduced by only 10%. The sum of these findings is that brain perfusion is not reduced as a result of lowered oxygen demand but rather that brain perfusion is limiting in AD. These findings are particularly important in light of recent studies showing strong relationships between arteriosclerosis and hypoperfusion and AD, and certainly gives pause to the importance of apolipoprotein E genotype in AD, since apolipoprotein E genotype is a major risk factor not only in AD but also in arteriosclerosis.
In summary, the roundtable made a convincing case for metabolic insufficiency and subsequent compensation playing a central role in AD. Further these findings not only supported the clinical efficacy of antioxidants, but suggest that greater effects will be noted with agents that also improve electron transport and increase cerebral blood flow, leading to enhancement of cerebral energy metabolism.—George Perry, Institute of Pathology, Case Western Reserve University (Note: Dr. Perry was also a speaker at this roundtable.)
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Sixth Int. Conference on AD: Satellite Symposium on AD7c-NTP
21 July 1998. The focus of this symposium was the biology and diagnostic value of a recently discovered protein, termed AD7c-neuronal thread protein (NTP), in Alzheimer's disease (AD). Dr. Suzanne de la Monte presented the discovery of AD7C-NTP. The gene/protein was isolated from an AD cDNA library (7th clone) based on similarities to pancreatic NTP. AD7C-NTP is distinct from the pancreatic protein and is primarily expressed in neurons and shows a pronounced increase in vulnerable and degenerating neurons in AD. Interestingly, the cellular distribution of AD7C-NTP is distinct from the neurofibrillary tau pathology, because it is diffusely localized within the neuronal cytoplasm and is actually reduced in neurons with extensive neurofibrillary pathology. This latter aspect argues that AD7C-NTP is an earlier marker than tau of neuronal degeneration and, in support of this, AD7C-NTP occurs in the earliest neuritic abnormality and its introduction into cells results in increased tau phosphorylation. Of interest, AD7C-NTP levels show no relationship to amyloid-β deposits.
Antibodies to AD7C-NTP, both mono- and polyclonal, show about a 2.5-fold increase in immunoreactivity in cortical tissue in AD compared to controls. In contrast, in immunoblot analysis, AD7c-NTP can only be found in AD brain, suggesting that the antibodies may be cross reacting with related brain proteins that are distinct from AD7c-NTP, but that in immunoblot and sandwich ELISA assays of body fluids, the antibodies are instead specific for AD7C-NTP. In ELISA analysis of cerebral spinal fluid (CSF) samples, 84% of AD cases showed higher levels than controls. Further, AD7C-NTP levels correlated with the Blessed dementia score (r2 = 0.6) suggesting it can serve as a surrogate marker of neurodegeneration.
Dr. Philipp Kahle presented findings showing that AD7c-NTP increases as neurodegeneration proceeds. Dr. Kahle compared the assay for tau from Innogenetics with the AD7C-NTP assay in analyzing CSF samples. He found that the values produced from either test were highly correlated, r = 0.435, p Dr. Hossein Ghanbari presented work on the specificity and sensitivity of the AD7C-NTP test. In early testing, he found that 87 percent of clinically diagnosed cases of early AD show AD7C-NTP levels over two ng/ml while only 11 percent of controls show these levels. The average values for AD is 4.6 ng/ml while in controls, it is 1.2 ng/ml. Other neurological diseases were also considered. Multiple sclerosis showed one ng/ml while Parkinson disease averages 1.8 ng/ml. In non-AD demented cases, the mean value for AD7C-NTP levels was 1.1 ng/ml, with only 6 percent above two ng/ml. While this overlap might be cause for concern, it must be kept in mind that the clinical or even pathological diagnosis of AD is only 72-90 percent specific for clinical, and 84-92 percent with an autopsy, diagnoses, and therefore a 100 percent specificity would not be expected as long as the diagnosis of AD is based on traditional clinical protocols. Significantly, levels of AD7C-NTP do not increase during aging.
More recent studies show that testing urine yields results similar to the CSF test, which would greatly increase the potential utility of the AD7C-NTP test. Dr. Ghanbari presented a concordance of 82 percent between urine and CSF levels of AD7c-NTP, a comparison of 4.2 ng/ml for CSF versus 3.3 ng/ml for urine in AD cases. Importantly, AD7C-NTP in both CSF and urine is stable under frozen storage for at least a year, thereby opening an opportunity for noninvasive assessment of biochemical abnormalities in AD.
During discussion it was brought out that AD7C-NTP may have the greatest value in diagnosing "classic or pure" AD and show the greatest fall out when other confounding factors intervene, such as multi-infarct dementia. Nonetheless, it can be argued that AD7C-NTP provides not only a defined diagnostic test but also enables the collection of better data to rigorously define "AD" heterogeneity.—George Perry
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Sixth Int. Conference on AD: Aging Astrocytes: A Role in AD?
Yamaguchi (Abstract 1245) presented his theory that very diffuse plaques within nondemented individuals are cleared by astrocytic phagocytosis. He showed many examples of very diffuse Aβ-positive staining within the brains of 40-50 year olds. Within the boundaries of these plaques were dense Aβ immunopositive granules which were co-localized with GFAP. The punctate granules were positive with antibodies to several C-terminal and midregions of Aβ, but were usually not labelled with N-terminal antibodies. Preadsorption with Aβ peptides completely abolished the staining of these granules. At the EM level, these appeared to be polycystic electron dense structures; probably secondary lysosomes.
The occurrence of these intra-astrocytic accumulations of Aβ were inversely age-dependent. While the apparent phagocytosis of Aβ was observed in nearly half of 40-50 year olds, the older the individual, the less frequently these structures were observed. Interestingly, the presence of phagocytosis was not observed in AD brains. Yamaguchi proposed that there is a dynamic balance between diffuse plaque formation and resolution in normal aging, and suggested that in AD, the clearance mechanism is overloaded and neuritic plaques form.
H. Wiswiewski, who was chairing the session, agreed that diffuse plaques can be removed, but also thinks that neuritic plaques can also be resolved and that astrocytes are involved in the process. On the contrarian side, I see no evidence that the phagocytosis of Aβ is actually "successful" at clearing an entire plaque. I only saw one or two Aβ-positive astrocytes per plaque; it doesn't look like they could possibly eat all that amyloid. Yamaguchi's presentation raises the novel possibility that AD is due to the failure of astrocytes to clear Aβ. News flash: Is AD caused by astrocyte aging?—Brian Cummings
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Sixth Int. Conference on AD: Fibril-busting Mystery Herb
While a number of drug companies have been pumping big bucks into creating novel molecules capable of inhibiting formation of Aβ fibrils, along comes Alan Snow and his colleagues at the University of Washington and his Seattle-based company, ProteoTech, Inc., with a commercially available "plant extract" that appears to do the trick (Abstract 1070). This substance can apparently dissolve pre-exisiting amyloid fibrils in an in vitro Thioflavin T fluorometry assay. Snow presented data that this compound can "dissolve" both AWhile a number of drug companies have been pumping big bucks into creating novel molecules capable of inhibiting formation of Aβ fibrils, along comes Alan Snow and his colleagues1-42 fibrils and islet amyloid fibrils.
Snow indicated that this substance was discovered by screening off-the-shelf health food supplements and finding one that yielded positive results in the Thioflavin assay. He also stated that this food supplement is readily available and produced by several different manufactures, although he declined to identify the compound. In response to the question about adsorption in the gut and whether this compound can cross the blood brain barrier, Snow said that those tests have not been run. He added that the extract is a low molecular weight lipophilic compound, so he has hopes that it will get into the brain.
Snow's company is currently negotiating with a major supplier of the extract to run a human clinical trial, although it might be more appropriate to first test the compound in a transgenic mouse. Snow said such tests have not yet been done, and he would be interested in such a study. Got mice? Give him a call. I'll hold off on buying stock until data shows that compound X can cross the blood-brain barrier in sufficient quantities to actually reduce AWhile a number of drug companies have been pumping big bucks into creating novel molecules capable of inhibiting formation of Aβ fibrils, along comes Alan Snow and his colleagues plaques in a transgenic animal.—Brian J. Cummings
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Sixth Int. Conference on AD: Presenilin-binding Proteins Abound
Because presenilin was discovered in positional cloning experiments involving large familial Alzheimer's disease kindreds, there were no previous data concerning its biological function. As a result, many groups have used a variety of techniques to garner clues regarding the function of this novel gene, which bears a key relationship to Alzheimer's disease.
Since the cloning of presenilin, one of the important functional clues to emerge was the critical role that presenilin plays in development. This insight derived from presenilin 1 "knockout" animals. The next step in further understanding the function of presenilin is the identification of the verification of binding partners. The lengthy procedures involved in discovering these interactors and more importantly in authenticating them accounts for the two-year interval between the discovery of presenilin and the reporting of several interactors. Before this meeting the putative interactive PS1 proteins were β-catenin, δ-catenin, actin-binding protein, and the amyloid precursor protein. All of these interactors have proponents and nay-sayers. Described in the Molecular Pathology II session, the new interactors were CLIP-170 (with PS1) from the Robakis group, and calsenilin (with PS1 and PS2) from the Wasco group. Other presentations not in this session described an interaction between Notch and PS1 by Alison Goate's group and an interaction between sorcin and PS2 by Tae-Wan Kim in the Tanzi group. Together these proteins constitute a thicket of new clues with few if any clear directions concerning which of the interactions, if any, will play a role in the disease pathogenesis. All of them are worthy of further pursuit.
Tezapsidis N et al. (Abstract 1257) reported that one PS1 mutation (E280A) showed an increased binding to CLIP170. This protein, first discovered by Tomas Kreis in the early nineties, has 1390 amino acids with a large coiled coil α helical domain in the central part of the protein. Fifty-seven amino acids at the amino terminus bind to vesicles and an acidic carboxy terminus binds to microtubules. Thus, CLIP170 lies at a most interesting interface between endocytosis and the microtubules. In addition to the in vitro interaction, the authors described co-localization of a myc-CLIP 170 transfectant and PS1 in 5YSY cells. This cell line is a human neuroblastoma line.
Choi E et al. (Abstract 1258) described how calsenilin was discovered in a two-hybrid screen using the carboxy terminus of PS2 as the bait. Confirmation of the interaction was by coimmunoprecipitation both ways in transfected COS cells. The authors were also able to coimmunoprecipitate calsenilin and PS1. The high degree of homology between the PS1 and PS2 amino termini probably accounts for the ability of both proteins to form a complex with calsenilin. In contrast the lack of homology between the PS1 and PS2 loop regions has made the identification of common interactors with this region more problematic. When transfected, calsenilin shows a cytoplasmic distribution, but following co-transfection with PS2 the complex gets recruited to the endoplasmic reticulum and the Golgi. Although a novel gene, calsenilin is a member of the recoverin family. One characteristic of the family is the presence of EF hand motifs; there are four in calsenilin. The authors demonstrated that calsenilin binds calcium by showing a mobility shift with and without EGTA and calcium 45 overlay techniques. Interestingly, the protein is expressed specifically in the brain by Nothern blots and in both neurons and glia by in situ hybridization.
Together, these new interactors all offer the potential for new insights into presenilin biology and possibility.—Kenneth S. Kosik
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Sixth Int. Conference on AD: Search for Late-Onset AD Genes
Note: This article was revised on 10 August, 1998, to accommodate some factual corrections. See also R. Tanzi's comment, appended below.
22 July 1998. A study published last year by Margaret Pericak-Vance et al. (JAMA 1997;278) reported a putative locus for late-onset Alzheimer's disease (LOAD) at chromosome 12p11-12, and set off a race to confirm and nail down the gene. The high interest in the search for LOAD genes even attracted a TV news camera crew to this session.
C. Van Broekhoven (Abstract 957) underscored the importance of searching for genetic factors for late-onset AD, pointing out that the genes on chromosomes 1, 14 and 21 linked to early onset Alzheimer's (EOAD) collectively account for ~15% of all EOAD, or less than 5 percent of all AD cases. She reviewed the APP mutations which affect APP processing and the importance of Aβ42 in AD pathology, as well as the role of PS mutations in EOAD. Out of the 40 or so scattered mutations in PS1, she observed, most mutations are linked to increases in Aβ42 secretion. However, she added, PS1 δ4 mutations do not affect Aβ42 secretion. Van Broekhoven also reviewed presenilin polymorphisms-many more in PS2 than in PS1-and noted PS2 polymorphisms are not associated with AD. In her studies, the candidate locus at chr 12p11 was not totally reconfirmed in LOAD, reflecting the (still) common fnding of false positives or genes which have ony a minor effect in LOAD.
Rudy Tanzi (Abstract 961B, not available online) presented his group's evidence linking the α2 macroglobuling gene to late-onset AD. Three hundred fifty sibling pairs from the NIMH genetics screening initiative were screened and showed hits on chromosomes 3,4,6,9 and 12. Chromosome 12 studies focused on the LRP receptor (for secreted APP, α2 microglobulin and ApoE). The α2 macroglobulin (A2M) gene product is particularly interesting because it is a pan-protease inhibitor which tightly binds β-amyloid, preventing fibril formation and neurotoxicity, possibly facilitating β-amyloid clearance and degradation. D12S391 is near A2M and near the tip of chromosome 12p. Using sib-TDT or SDT, the linkage of A2M to AD had an odds ratio of 3.56 with a p-value of 0.00009, compared to an odds ratio of 3.45 and a p-value of 0.00006 for the ApoE4 allele. Thus, A2M in this study has as strong an association with AD as does ApoE4. Tanzi proposed that A2M mutations may lead to lifelong increased accumulations of β-amyloid. He also noted that LRP and A2M are at the same locus at chromosome 12p and both may be involved in LOAD.
Tanzi's finding received support from Alison Goate's group, which reported modest evidence of linkage to a region of chromosome 12 containing the A2M gene (abstract 959). Goate's study involved 230 families consisting of 510 affected and 97 unaffected individuals using multipoint sib pair analysis (MAPMAKER). Although data points were obtained indicating involvement to the same general area at 12p, it must be significantly smaller than the locus for ApoE on chr 19. Goate thought it was encouraging that all groups were detecting linkage to chromosome 12, and said the differences in location were probably due to inaccuracies in pin-pointing a gene using this methodolgy in a disease like late onset AD. "The gene we are detecting could be A2M, LRP or another gene in the region," she said.
In her review of current research, M. A. Pericak-Vance pointed out problems and new approaches in identifying genes associated with late-onset Alzheimer's disease (Abstract 956). Individual case /control studies are easy to perform when large data sets are available, and were instrumental in the identification of APOE-e4, for example. However, case/control studies are much less successful when anomalies in population selection are factored in. Positive associations can be missed due to population heterogeneity, population-specific effects and chance selection. The transmission disequilibrium test (TDT) can often, but not always, avoid such sampling bias by using internal parental controls. The major drawback is that it can't be used for late-onset diseases, because parents are usually deceased and not available for genotyping. Using unaffected siblings as controls (sib-TDT; as delineated by Spielman and Ewens (1998) and others) compares marker allele frequencies in pairs of affected and unaffected siblings. Such studies to test linkage and association more thoroughly are needed to dissect the inherent complexity.in AD and other late-onset diseases.
Several presenters discussed other candidate genes for LOAD. M.-C. Chartier-Harlin et al. (Abstract 958) described their studies examining polymorphisms in ApoE gene promoter. The Th1/E4cs polymorphism and the Th1/E47cs polymorphism are highly significant in modulating the ApoE4 allele impact on LOAD (each ANOVA E. Tourneier-Lasserve et al. (Abstract 960) discussed recent findings relating to CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), an adult-onset autosomal dominant condition that involves recurrent ischemic strokes, vasculature abnormalities and associated subcortical dementia. EM reveals dense granular osmophilic deposits in the vasculature. CADASIL has been linked to the human homolog of Notch 3, which was just recently cloned: a 33-exon, 30-kb monster gene. Screening of 55 unrelated CADASIL patients showed that at least 70 percent of all mutations were located in the 5' end of the Notch 3 gene in exons 3 and 4, corresponding to the first five EGF-like repeats. Nearly all of these mutations led to creation or destruction of a cysteine residue in the EGF-type repeats The resultant mutations may produce an aberrant dimerization of Notch proteins into granual, osomophilic and aggregated forms and may underly the pathophysiological mechanism which leads to the CADASIL phenotype.
The most novel finding was presented by Fred van Leeuwen et al. (Abstract 961C, not available online), who described his theories on "gene information assembly line slippage" generating mutant RNA forms which are subsequently translated into mutant dysfunctional proteins. Loss of two bases (GA) garbles the RNA message.(see TINS 1998;21:331-335) ultimately generating "+1" proteins. Altered "GAGAG" motifs are found in the RNA messages for β-APP, tau, ubiquitin B, ApoE4, PS-1, PS-2, MAP 2b, NF-L, NF-M, and NF-H (neurofilaments light, medium and heavy chain proteins, respectively), indicating potential involvement of all of theses species in the "molecular misreading" hypothesis. Van Leeuwen's theory offers an explanation of a wide range of "pathological" molecules in the pathophysiology of neurodegeneration. The molecular mechanism for "molecular misreading" is still not known.
To address the question whether the +1 proteins a cause or a consequence of AD neurodegeneration, the following data were reported. In young Down's syndrome cases, APP+1 is present when amyloid deposition is not apparent, suggesting that "molecular misreading" is an early event in neurodegeneration. Data suggest that mRNA surveillance may be a common mechanism in which the accuracy (of mRNA surveillance) may decrease during aging and hence, account for the accumulation of aberrant proteins in the aging brain. The biological basis for +1 protein generation, accumulation and degradation are currently unknown, but should fuel abundant future research. This intriguing, refreshingly new and welcome hypothesis could explain a lot of "nongenetically linked" or sporadic/idiopathic neurodegenerative disease. (See also Van Leeuwen On-line Journal Club.)—Walter Lukiw
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Papers
- Pericak-Vance MA, Bass MP, Yamaoka LH, Gaskell PC, Scott WK, Terwedow HA, Menold MM, Conneally PM, Small GW, Vance JM, Saunders AM, Roses AD, Haines JL. Complete genomic screen in late-onset familial Alzheimer disease. Evidence for a new locus on chromosome 12. JAMA. 1997 Oct 15;278(15):1237-41. PubMed.
Sixth Int. Conference on AD: Scientists Smitten by Mad Tau Disease
A new epidemic of "mad tau disease" swept Amsterdam at the workshop on Hereditary Fronto-Temporal Dementia and Pick's disease. Fronto-temporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) has been linked to mutations on the tau gene, and, in the words of Peter Davies, most of speakers themselves exhibited symptoms of the disorder: euphoria, disinhibition and impulsivity. (One wonders about other FTD symptoms such as hyperactivity, hyperorality and hypersexuality....)
In addition to the preannounced program, three speakers were added: Peter Heutink, Gerry Schellenberg and Maria Grazia Spillantini. In the first part of the workshop, clinical and neuropathological characteristics of fronto-temporal dementia were described. The subgroup of FTDP-17 was then introduced and description of the mutations found on tau were reported. Finally, biological effects on tau proteins were also presented.
Cummings briefly described clinical chracteristics of FTD versus Alzheimer's disease (Abstract 1232). AD increases with age whereas FTD is a typical presenile dementia. In FTD, behavior is particularly affected and there is a severe disinhibition. In contrast to AD, visuospatial ability and calculation are preserved. Some variants may be observed: a left temporal lobe variant characterized by aphasia and right temporal lobe variant showing irritability and impulsivity. Some sub-syndromes may be also distinguished in progressive nonfluent aphasia and semantic aphasia. Hodges also emphasized the role of semantic dementia in FTD (Abstract 1233). Van Swieten (Abstract 1235) described the different subgroups of FTD and introduced the history behind FTDP-17. He indicated that Pick's disease (characterized by the presence of Pick bodies) is likely to be nonfamilial. In other FTD, hereditary accounts for probably between 20% and 38%. Neuropathology and PET studies in sporadic FTD were described by Forster (Abstract 1237). Neuropathologically, brains of FTD patients display cortical gliosis, severe neuronal loss and superficial laminar spongiosis. There is a relative sparing of hippocampus. By imaging, atrophy and hypermetabolism are observed in anterior temporal lobe.
Michael Hutton kicked off the genetics part of the session (Abstract 1234). Tau mutations were always found in FTDP-17 patients with tau pathology. The presence of tau-immunoreactive intraneuronal and glial inclusions was very common. Among the families studied, three mismutations were found in coding regions G272V, N279K and R406W. Three other mutations affecting the splicing region after exon 10 were also reported at +13, +14 and +16. G272V and P301L affect the microtubule-binding domain of tau protein. On Tuesday, Michel Goedert reported the effects of both mutations in an in vitro system of microtubule assembly (Abstract 926; See Audio Recording). Mutated tau isoforms do not bind microtubule and induce microtubule disassembly.
Regarding the mutations in the splicing region, Hutton et al. indicated that they affect the ratio of tau mRNA with exon 10 (E10+)/tau mRNA w/o exon10 (E10-). For these mutations, they demonstrated by RT-PCR that the ratio E10+/E10- was between two and six. For patients with the P301L mutation and control cases, this ratio was between 0.57 and 1.25, suggesting that the mutations in the splicing site induce an increase in E10+ tau isoforms. By exon-trapping, they confirmed these observations and demonstrated that the most striking mutation is at +14 and induces almost a sixfold increase in E10+ tau isoforms. Mutations in the splicing site disturb a stem loop that may stabilize this region of the pre-mRNA and decrease access of U1snRNP to this RNA region. Without this stem loop, access of U1snRNP may be facilitated and increase the formation of E10+ tau isoforms. Hutton et al. tested this hypothesis by a rescue method in which they added two new nucleotides to the mutated sequence in order to form a new stem loop. This new structure was tested in their exon-trapping experiment and they showed that there is a decrease in E10+ tau isoforms compared to non-modified mutated sequence. Finally, sequence analysis of this splicing in different animals indicates that if there is no stem loop structure, there is an increase in E10+ tau isoforms.
Heutink focused on FTD in The Netherlands (no abstract available). 38% of FTD cases have a family history. Three families were analyzed by Hutton's group (HFTD I, II and III). Only families I and II showed mutations (P301L and G272V), and the affected individuals are clinically characterized by a disinhibition. Neuropathologically, they exhibited a tau pathology. In family III, no mutation was found on the tau gene, there was no tau pathology and clinically, the characteristics were loss of initiative, rather than disinhibition.
Schellenberg (no abstract available) briefly summarized his group's data (P301L, Seattle families D and F, and Oregon EL; and V337M, Seattle A) and described a new mutation N279K in PPND. He noted that Virginia Lee had described aggregation of E10+ tau isoforms in PPND (Abstract 617). Schellenberg also emphasized that the change in nucleotide for the P301L mutation may also create an exon-splicing enhancer sequence.
He reported that no coding mutation was found on the tau gene in PSP patients. He suggested that PSP may exhibit a polymorphism that increases susceptibility.
Wilhelmsen (Abstract 1236) reported that FTDP-17 families are highly heterogeneous. In Sweden, the most common mutation in FTDP-17 families is the G272V one. Sequencing of tau gene in exons 9-13 was performed in FTD, tauopathies, PSP and CBD: no other mutation was found.
Finally, Spillantini made a summary of the biochemical analysis of phosphorylated tau isoforms in FTDP-17. In Dutch HFTD II (G272V mutation), tau-immunoreactive neuronal glial structures are found. The neuronal structures are very close to Pick bodies. In Dutch HFTD I (P301L mutation), both neurons and glia are tau-immunoreactive. At the electron microscopy level, tau aggregates into twisted ribbons and to a much lesser extent into PHF. Biochemically, phosphorylated aggregated E10+ tau isoforms and also the E10- isoform with the 29 aa insert (exon 2) are found. In MSTD (intronic mutation described by Spillantini in PNAS, abstract 1294), phosphorylated E10+ tau isoforms aggregate into twisted ribbons filaments and appear by immunoblotting as a major tau doublet and a minor 72 kDa tau variant. Tau-immunoreactivity is found in neurons and glia. In Seattle family A (V337M mutation), neurons are tau-immunoreactive. All six tau isoforms are phosphorylated (with a PHF-like electrophoretic profile) and aggregate into PHF and to a lesser extent into straight filaments. Finally, soluble tau proteins are always similar to those found in control cases, except in MSTD where an increase in E10+ tau isoforms was observed.—Luc Buee
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