CONFERENCE COVERAGE SERIES
Society for Neuroscience Annual Meeting 1999
Miami Beach, FL, U.S.A.
23 – 28 October 1999
Society for Neuroscience Annual Meeting: β-Secretase Further Confirmed
Sukanto Sinha, departing from his abstract (122.1), described the purification and partial characterization of a novel 501 amino acid long aspartyl protease. The data were entirely consistent with the β-secretase activities reported by SmithKline Beecham (240.11) at this meeting and by Amgen in their recent Science publication (Science 1999;286:735-741). That is: 1) the protein sequences appear identical, 2) overexpression results in an increased secretion of both Aβ1-40 and A-beta1-42, 3) the activity has an acidic pH optimum and 4) it appears to be the dominant β-secretase in neuronal cells. In the Elan study β-secretase activity was assessed by cleavage of a fusion protein that contained the C-terminal 125 amino acids of APP (APP570-695) wild-type or Swedish (K595M596 to NL, APP695 numbering) sequence. The purification regime was classical biochemistry at its best and involved: detergent extraction, lectin affinity chromatography, cation exchange chromatography and inhibitor affinity chromatography and resulted in a 55,000-fold enrichment with only a single ~70 kDa gylcoprotein detected by silver staining. Interestingly, Sinha indicated that Elan already had potent non-peptidomimetic inhibitors of BACE. The independent identification of a single protein with β-secretase activity by three different pharmaceutical companies may mean that an effective therapy for Alzheimer's disease will soon be realized.-Dominic Walsh.
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Society for Neuroscience Annual Meeting: Another APP Secretase?
Just when it appeared as though the mechanisms of the proteolytic processing of APP were finally becoming apparent, Andreas Weidemann presented an elegant study demonstrating the existence of a different processing event (122.2). Using SH5Y and Cos-7 cells expressing C-terminally tagged APP and SPACT (C99) he isolated C-terminal fragments (CTFs) from membrane preparations and by radiosequencing demonstrated the presence of a CTF generated by cleavage between Leu645 and Val646 (APP695 numbering). Cleavage at this site (termed epsilon) appeared to occur late in the secretory pathway and was not affected by γ-secretase inhibitors and did not appear to be a precursor to γ-secretase cleavage.—Dominic Walsh
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Society for Neuroscience Annual Meeting: Imaging Amyloid Plaques in the Living Mouse
One of the limitations in studying the pathogenic role of amyloid plaque deposition is the fact that conventional methods allow microscopic analysis only of postmortem tissue, which can provide only a single snapshot of a process that is clearly dynamic and evolves over a long time span. An approach to overcoming this constraint was presented by Rich Christie (120.11), who reported on the use of two-photon imaging to visualize plaque formation in Tg2576 mice while they are still alive. The technique involves labeling plaques with thioflavin S and then imaging the brain of the mouse by placing the animal on the microscope stage and visualizing the plaques through multiphoton microscopy. (To protect the brain, the skull is thinned down to provide a "window" for imaging without a complete craniotomy.) The images are quite impressive and the evidence indicates that it is possible to reimage the same field in successive sessions over a period of several months.
Once the feasibility of the method was established, the first question addressed was whether plaques are stable or increase in size over time. The evidence so far indicates that the majority of plaques are remarkably stable in size. Although occasional "outliers" show evidence of increases in size, these are in the minority. In addition, Christie provided a good example of new plaque formation within a field that had been imaged on several occasions. Although this evidence appears to suggest that amyloid plaques form precipitously and then remain static, previously published work from this laboratory, led by Bradley Hyman, indicates that the relatively stable size of plaques results from an equilibrium state between an aggregation and degradation process. The in vivo two-photon technique should provide additional insights into the dynamics of plaque formation that, in turn, could reveal aspects of AD. What is more, they could offer a literal window onto the effects of "plaque-busting" therapies in transgenic mouse models.—Keith Crutcher
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Soc for Neuroscience Ann Mtg: One γ-Secretase, Two Different Activities?
Using difluoroketone peptidomimetics previously shown to reversibly inhibit γ-secretase activity in CHO cells, Michael Wolfe reported that increasing the bulkiness of the P1 substituent of these compounds increased their potency, thus, suggesting that γ-secretase has a large S1 pocket (122.3). Moreover, analysis of the effects of these compounds on Aβ40 and Aβ42 secretion indicated that the activity responsible for Aβ40 production is more susceptible to inhibition than the activity responsible for Aβ42 generation. Intriguingly, at low inhibitor concentrations the amount of Aβ42 in conditioned media was actually increased. Wolfe also found that Aβ42 production was selectively increased after removal of reversible γ-secretase inhibitors and he cautioned that inappropriate pharmacokinetic profiles for drugs that work at this level may ironically lead to the opposite of the therapeutic goal, which is to lower Aβ42. These data suggest either that A-beta40 and Aβ42 are produced by discrete enzymes or that they are produced by the same enzyme with two distinct pharmacological activities. Wolfe favors the latter and is currently undertaking studies using irreversible bromoacetyl difluoroalcohols inhibitors to definitively identify the γ-secretase activities responsible for Aβ40 and Aβ42 production.—Dominic Walsh
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Society for Neuroscience Annual Meeting: Caspase-mediate Production of Aβ
In testing the hypothesis that unscheduled cell death may alter APP processing, Andrea Le Blanc (122.4) provided compelling evidence that caspase 6 can directly cleave APP and cause an increase in Aβ levels in serum deprived human primary neurons. She is currently examining the p10 active fragment of caspase 6 in AD brain and non-AD age matched controls.—Dominic Walsh
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Soc for Neuroscience Ann Mtg: APP and Intracellular Aβ Balance Critical
Recent studies have demonstrated that wild-type APP can protect against p53 mediated apoptosis, whereas FAD mutant APP (FADAPP) cannot. N3 rat neuroblastoma cells which are deficient in APP were transfected with wtAPP or FADAPP and then irradiated with UV light to induce apoptosis. In this paradigm inhibition of Aβ restores the anti-apoptotic function of FADAPP, leading Li Gan (122.5) to conclude that the balance between APP and Aβ is critical in determining whether or not a cell will proceed down the cell death pathway. Interestingly, extracellular Aβ did not have a significant effect indicating that intracellular Aβ is the "bad guy" and that it either activates p53 or causes it's nuclear translocation, whereas APP prevents p53 activation.—Dominic Walsh
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Society for Neuroscience Annual Meeting: α1-Antichymotrypsin
Yamin (122.9) presented evidence that the zinc metalloproteinase EC 3.4.24.15 (E24.15) plays a role in neuronal mediated degradation of Aβ. In fact transfection of neuroblastoma cells with antisense E24.15 allows the accumulation of Aβ, whereas overexpression of E24.15 causes a dramatic reduction in Aβ levels. α-1-antichymotrypsin (ACT), a plaque-associated protein and member of the serpin protease inhibitor family causes a significant reduction in Aβ degrading activity. ACT may operate either directly as a protease inhibitor or by binding to Aβ and protecting it from proteolysis. Complexes of ACT and E24.15 were found in conditioned media from neuroblastoma cells treated with ACT confirming that it can bind and inhibit E24.15.—Dominic Walsh
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Society for Neuroscience Annual Meeting: Making the Right Mouse Model?
Several groups have reported a lack of phenotype in α-synuclein (α-S) transgenic mice, but it appears Lennart Mucke (122.10) has got it right. Mucke reported that inclusion bodies were obvious in PDGF promoter-driven α-S Tg mice as early as three-four months, and that these inclusions were most obvious in the higher expressing lines. Moreover, bigenic APP/α-S mice showed an increase in the number of Lewy body-like inclusions and displayed memory and learning deficits greater than mice carrying a single transgene. These observations suggest that interactions between Aα and α-S contribute significantly to dementia with Lewy bodies.—Dominic Walsh
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Society for Neuroscience Annual Meeting: Enzyme Mediates Aβ Degradation
Previous studies on the purification and characterization of the predominant extracellular Aβ degrading activity in the murine BV-2 microglial cell line demonstrated that insulin-degrading enzyme (IDE) or a novel protease indistinguishable from IDE was responsible for this activity. In an extension of this work Kostas Vekrellis (122.11) reported that differentiated PC-12 cells and primary rat cortical cultures also possessed an Aβ-degrading activity that could be competitively inhibited by addition of insulin or glucagon, and that this activity was cell associated. Moreover, transfection of CHO cells (already overexpressing APP) with wtIDE caused a dramatic decrease in the Aβ present in the conditioned media of these cells, whereas transfection with mutant IDE had no such effect. Furthermore, immunohistochemical studies of AD brain indicated that IDE was increased within neurons proximate to amyloid plaques.—Dominic Walsh
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Soc for Neuroscience Ann Mtg: Novel Assay for in Vivo Catabolism of Aβ
The N- and C-terminals of synthetic Aβ1-42 were differentially labeled with 3H and 14C and then used as a probe to measure degradation by injection into rat hippocampus. Effects of various protease inhibitors and two dimensional HPLC analysis of degradation products lead Saido (122.12) to conclude that neprilysin (NEP) or an NEP-like protease is responsible for the degradation of A-beta in vivo.—Dominic Walsh
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Society for Neuroscience Annual Meeting: APP Binds to Fibrillar Aβ
Treatment of human cerebovascular smooth muscle (HCSM) cells with Aβ1-42 or AβQ22 (a mutant associated with hereditary cerebral hemorrhage with amyloidosis-Dutch type) leads to the formation of amyloid fibrils on the surface of HCSM cells within 24 hours. After three days cell associated APP is increased and cell viability decreased. Using monoclonal antibodies which recognize both FLAPP and APPs or FLAPP alone, Melchor (122.13) was able to demonstrate that most of the increase in cell associated APP was due to APPs. Binding of APPs to fibrillar Aβ was demonstrated by electron microscopy of biotinylated APPs and by a solid phase binding assay akin to the synthaloid assay developed by John Maggio's group.—Dominic Walsh
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Soc for Neuroscience Annual Meeting: Novel Receptor Interaction for Aβ
That β-amyloid plays a central role in the pathogenesis of Alzheimer's disease is doubted by few investigators. However, pinning down the exact contribution amyloid makes has been remarkably difficult to accomplish. Novel hypotheses are becoming rarer but the series of reports from investigators at the R.W. Johnson Pharmaceutical Research Institute and collaborators at UMDNJ certainly falls into this category. Two of the presentations today provided evidence that amyloid may bind to α7-nicotinic cholinergic receptors (a7nAChR). The first, given by Hoau Yan Wang (12.2), noted that a7nAChR are present in plaques, co-localizing with Aß42. Aß42 also shows high affinity binding to cells transfected with a7nAChR, binding that is inhibited by bungarotoxin. In addition, co-precipitation experiments demonstrate that the level of a7nAChR/Aß42 complex is over ten times greater in AD tissue as compared with control tissue. Aß40 has similar properties but with lower affinity.
The second talk, given by Daniel Lee (12.3), provided evidence that the interaction of amyloid with 7nAChR leads to tau phosphorylation. Three different phosphorylation sites were examined, serine 202, threonine 231 and threonine 181. The latter two sites appear to be phosphorylated more rapidly than the serine site. Generally similar results were obtained with guinea pig hippocampal synaptosomes. The phosphorylation was inihibited by bungarotoxin but not by α -conotoxin or macamylamine. In addition, there is some indication that amyloid toxicity can be partly blocked by bungarotoxin. Collectively, these results provide an interesting connection between the amyloid hypothesis and tau pathology. If additional studies confirm the high affinity binding of amyloid to nicotinic receptors and the phosphorylation effects, this provides a new framework for understanding the role of amyloid in AD.
A third presentation, by Michael D'Andrea (120.10), referred to the earlier talks demonstrating the binding of amyloid to nicotinic receptors but focused on the question of plaque origin. He noted that plaques are distributed in specific cortical laminae and have restricted sizes. He also noted that amyloid staining can be found in some pyramidal neurons, leading to the hypothesis that intracellular amyloid may serve as a nidus for the development of dense-core plaques. Using the same transfected cell line described by Wang and Lee, fluo-Aß42 was used to follow the intracellular accumulation of amyloid (presumably via the nicotinic receptors). There appears to be distortion and eventual disruption of cells in which the amyloid accumulates. This observation led to further consideration of the evidence that disrupted neurons may serve to seed plaque formation. A variety of neuronal markers, as well as nucleic acid, are found in the plaque cores. Interestingly, a fairly good correlation was also found between the size of plaques and the size of the surrounding pyramidal cells. Although the studies are largely based on correlations, the possibility that intraneuronal amyloid (perhaps accumulated through binding to nicotinic cholinergic receptors) may ultimately seed plaques is an intriguing suggestion.—Keith Crutcher
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Society for Neuroscience Annual Meeting: Evidence for Additional Genetic Risk Factors for AD
Papassotiropoulos (1201) began his presentation by reviewing the evidence for a role of cathepsin D, an aspartyl protease, in AD. Previous work has implicated catD as a secretase in the cleavage of APP. It is found in plaques and exhibits increased expression in AD. It has also been shown to degrade tau. However, there is no evidence that overall catD activity is increased in AD tissue, and A-beta is still produced in catD knockout mouse, indicating that the enzyme is not required for amyloid production. In the present study, the T allele (causing a valine at position 224 of the catD gene in exon 2) was found to be overrepresented in a population of 102 AD patients versus 191 age-matched controls (odds ratio of 2.6). This effect was independent of the ApoE4 genetic association. However, having both ApoE4 and the catD T allele resulted in a 10-fold greater risk of AD. The catD effect did not influence age at onset.
Altstiel (120.2) revisited the controversy surrounding the reported genetic association of α2-macroglobulin (a2M) with AD (see previous news report). His group, as is the case for several other groups, has not found increased disease risk with the a2M deletion. However, in the results reported at the meeting today, it was found that this deletion is associated with a higher density of neuritic plaques. This retrospective analysis was carried out on sections from five different brain regions from archived autopsy material from approximately 160 patients. Plaque number was found to be greater in tissue that was hemizygous for the deletion and even higher for homozygous deletions. A similar effect was found for E4, confirming previous studies. The effect of the a2M deletion was not age-specific, leading Altstiel to speculate that the impact of the a2M allele is on disease progression. Defining the interactions between the various genetic risk factors will no doubt keep investigators busy for a while yet.—Keith Crutcher
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Soc for Neuroscience Annual Meeting: Lipoproteins Reduce Clearance of Aβ
Using exquisite human primary cortical cultures containing neurons, astrocytes, and microglia, Nadeau (319.1) demonstrated that addition of VLDLs or HDLs increased media levels of both Aβ40 and Aβ42. Moreover, addition of ApoE3, Apo4 or ApoA1 caused elevations in Aβ similar to that induced by HDL, whereas other plaque associated proteins such as C1q had no effect. To determine if the increase was due to altered production he examined FLAPP and APP CTFs and found no effect. He then added 35S-labeled cell-derived Aβ back to unlabeled cortical cultures and found that addition of HDL prevented clearance of Aβ. Interestingly, addition of insulin at concentrations less than or equal to 10 micromolar also reduced Aβ degradation, leading Nadeau to speculate that IDE (also see 122.11) was the principal Aβ degrading activity in these cultures and that Aβ could escape IDE-mediated degradation by binding to lipoproteins.
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Soc for Neuroscience Ann Mtg: Reduced Cholesterol Levels Inhibit Aβ
Using primary hippocampal neurons transfected with APP by means of the Semliki-forest-virus expression system Christine Bergmann (319.2) demonstrated that lovastatin (which inhibits de novo cholesterol synthesis) and methyl-b-cyclodextrin (which can form inclusion complexes with plasma membrane cholesterol) cause a dramatic decrease in both intracellular and secreted Aβ40 and 42 and an increase in C99. By fluorescence microscopy of filipin-cholesterol complexes, she nicely demonstrated that even when cholesterol levels were reduced by 60%-70% that the neurons were still viable and that the Golgi network managed to retain some cholesterol. To determine if γ-secretase processing was also affected by cholesterol she transfected neurons with SPA4CT (C99, an APP construct that does not require beta-secretase cleavage) and assessed the effect of lovastatin and β-cyclodextrin. In contrast to her results using FLAPP she found that Aβ42 generation is resistant to extracellular cholesterol depletion, whereas Aβ40 is dramatically reduced. This differential effect on Aβ40 production probably results from Aβ40 being produced in a compartment (the TGN) which is more accessible to the effects of cyclodextrin than the IC/ER where Aβ42 is generated. Together these results demonstrate that both β- and γ-secretase are strongly influenced by their lipid environment and that modulation of cholesterol levels may be a useful therapeutic strategy.
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Society for Neuroscience Annual Meeting: Intraneuronal Aβ
Until very recently, Aβ accumulation and aggregation were thought to be exclusively extracellular phenomena. However, a number of abstracts presented at this meeting (see 120.10 and 720.10) suggest that this notion must be revisited. Gunnar Gouras (319.5) presented beautiful immunostaining of human brain demonstrating the presence of intraneuronal Aβ. This material did not bind Congo red and did not require formic acid for extraction and was visible in young Down's syndrome brain and in brains from non-demented elderly. Moreover, he also showed very nice images of plaques that bore a remarkably morphological similarity to pyramidal neurons (some of which were found to contain neuronal mRNA) suggesting that some neurons may die as a result of intraneuronal Aβ accumulation and subsequently give rise to extracellular plaques.
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Society for Neuroscience Annual Meeting: DIGs—a Site of Aβ?
Kawarabayashi (319.10) presented compelling data for a time-dependent accumulation of Aβ in the detergent-insoluble glycolipid enriched membrane domains (DIGs) of Tg2576 mouse brain. He also showed that Aβ accumulated in DIGs from human brain and the extent of accumulation was greater in AD brain than in matched controls. These data suggest that Aβ accumulation in DIGs maybe a harbinger of amyloid fibril formation and lend further support to the thesis that membrane environments plays a central role in both the generation and aggregation of Aβ.
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Society for Neuroscience Annual Meeting: The TGN—Where It's Happening?
In an exhaustive study, Iwata (240.1) presented evidence that the trans Golgi network was the major site of Aβ42 production in both neural and non-neural cells. Cos-1 and N2a cells were transfected with C100 bearing retention signals for the ER, CGN or TGN. Intracellular and extracellular production of Aβ were then determined by Western blotting or ELISA. Aβ production was wiped out in cells containing C100 which were tagged for ER or CGN retention, whereas Aβ production from cells transfected with C100 bearing a TGN retention signal was indistinguishable from untagged C100 transfectants. Moreover, co-transfection with mtPS2 and TGN-tagged C100 caused a marked increase in intracellular and secreted Aβ, suggesting that PS2 and C100 normally interact at this locus.—Dominic Walsh
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Society for Neuroscience Annual Meeting: Endocrepsin is BACE
At times the scene around this poster (240.11) was more like a football scrimmage or a rugby scrum than the normally genteel atmosphere associated with a scientific event. By the time I made my way to within sight of the poster, the presenter, Ishrut Hussain was nowhere to be seen, presumably too exhausted to face further cross-examination! Although the data were somewhat scant it was obvious that what SB call endocrepsin 2 is what Martin Citron and colleagues at Amgen call BACE. The sequence as far as I could tell was identical to that of BACE, and differed in only one respect, the open reading frame appeared to encode a protein of 500 AA and not 501 as reported by Amgen. The molecular weight of the deglycosylated protein was ~55.4 kDa and it appeared to localize to the ER and Golgi when transfected into Cos 7 cells and as expected caused an increase in beta-secretase processing.—Dominic Walsh
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Soc for Neuroscience Ann Mtg: Proteasome Inhibition Boosts Aβ
Janet Johnston (240.16) presented a detailed study of the effect of proteasome and prolendopeptidase (PEP) inhibitors on Aβ production by SH SY 5Y human neuroblastoma cells stably transfected with SPA4CT (C99). Both the proteasome and PEP have been reported to directly alter Aβ production. However, Johnston presented compelling evidence that PEP does not influence γ-secretase processing, whereas specific inhibitors of the chymotrypsin-like activity of the proteasome elevate secretion of both Aβ40 and 42. The mechanism by which this elevation is mediated is currently under investigation.—Dominic Walsh
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Society for Neuroscience Annual Meeting: TACE, TAPI, and ADAM10
The mechanisms of regulated α-secretase cleavage were thoroughly examined in two excellent companion posters from Virginia Lee’s lab (240.2, 240.3). The authors demonstrated that TNFalpha converting enzyme (TACE) knockout mice show reduced sensitivity to PMA and consequently secrete less α-cut APPalpha in response to treatment with phorbol esters. Although Moore was careful to point out that non-TACE activities were capable of α-secretase-like cleavage, transfection of TACE KO mice increased both constitutive and regulated APPa secretion.—Dominic Walsh
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Society for Neuroscience Annual Meeting: Be Wise—Immunize!
In the relatively short time allotted, Dale Schenk (519.5) managed to describe most of the data from his group’s recent Nature paper (Nature 1999;400:173-177), and a little more. Schenk demonstrated that Aβ immunization of young (six-week-old) mice, in which treatment had begun before the occurrence of plaque pathology, essentially prevented the development of plaque formation and associated changes. Moreover, immunization of older (11-12 month) mice which already had signs of plaque pathology markedly reduced the extent and halted the progression of the Aβ-mediated pathology typically seen in these mice. As to how immunization produces this effect, Schenk speculated that since these mice have high titer antibodies to Aβ in their serum (and ~0.15% of any given circulating antibody can cross the blood-brain barrier) and immunohistochemistry shows Aβ within microglia in regions where remaining plaques are evident, an Fc-mediated uptake and clearance mechanism is probably responsible both for preventing amyloid plaque formation and for mediating plaque removal.
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Papers
- Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R, Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, Walker S, Wogulis M, Yednock T, Games D, Seubert P. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 1999 Jul 8;400(6740):173-7. PubMed.
Society for Neuroscience Annual Meeting: A Sniff of Aβ
Chronic mucosal administration of certain proteins is known to decrease organ-specific autoimmune processes in several autoimmune models, including those affecting the CNS. In AD, local inflammatory responses frequently occur in the vicinity of plaques; thus, Cindy Lemere and colleagues reasoned that mucosal administration of Aβ could prevent or reduce the deleterious effects associated with plaque-induced inflammation (519.6). To test their hypothesis, Aβ peptide or control proteins were administered either orally or intranasally to PD-APP tg mice on a weekly basis from ~5 to 12 months of age, and the animals' brains were analyzed immunohistochemicalyl and biochemically. Oral administration of Aβ failed to suppress inflammation and did not alter plaque burden, whereas nasal administration of Aβ1-40 at 25 mg/week caused a dramatic 50-60 percent decrease in plaque burden. Activated microglia were found in those plaques that remained, indicating that the peri-plaque inflammatory response was not suppressed. Serum anti-Aβ antibodies were found in some oral, and eight out of nine nasally treated animals, with antibodies being highest in the nasally treated mice which showed reduction in plaque burden. These results together with those of the Elan group (519.5) confirm that immunization with Aβ can reduce plaque pathology and suggest that optimization of mucosal delivery may offer an attractive therapeutic route.
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Society for Neuroscience Annual Meeting: Presenilins: An Overview
Research on presenilins presented at this year's meeting centered around two major questions: 1) What is the role of presenilins in Aβ production and APP processing, and 2) what are the other biological actions of presenilins? A major debate in the presenilin field revolves around the suggestion by Wolfe, Selkoe and colleagues that PS1 is a γ-secretase. Different presentations addressed this issue by investigating three different subjects: inhibitors of APP processing, the effect of mutations on presenilin function and the distribution of presenilins.
Inhibitors of APP Processing: Wolfe and colleagues (122.3) have designed a series of peptides that mimic the site on APP cleaved by γ-secretase. These peptides contain difluoro ketone moieties embedded in the peptide which are known to inactivate aspartyl proteases. Wolfe tested these peptides against the cysteine protease calpain, and observed no effect on calpain activity. Each of these APP-mimetic peptides inhibited Aβ production. Based on this data, Wolfe and colleagues suggested that the γ-secretase is an aspartyl protease. The data are quite appealing but the conclusion contradicts a paper by Robakis' group [Figueiredo-Pereira, 1999 #1362] showing that three other cysteine protease inhibitors, Z-VF-CHO, Z-VL-CHO and E-64d all inhibit Aβ40 production, although they were less effective on Aβ42 production. An essential question is the specificity of the different inhibitors used by Wolfe's and Robakis' groups. Each group of peptides ostensibly acts specifically on the classic aspartyl of cysteine proteases, but if PS1 is a protease, it is clearly not a classic protease. The activities of these inhibitors against proteases that don't fit the classic structural definitions is unknown.
Murphy, Golde, and colleagues (219.10 and 219.11) addressed this issue by examining the action of peptide inhibitors on mutant APP constructs. They have observed that the aspartyl protease inhibitor, pepstatin, inhibits Aβ40 and 42 production in cells expressing wild-type APP. However, when they introduce mutations into APP that shift the predominant cleavage of APP from Aβ40 to other sites, such as Aβ43, they observed that pepstatin does not inhibit Aβ43 production from the mutant APP constructs. The fact that pepstatin does inhibit γ-secretase with one substrate (wild-type APP) but does not inhibit it with another substrate (mutant APP) suggests that there are some proteases that can cleave Aβ that are not classic aspartyl proteases. However, this approach does not rule out the possibility that the mutant APP outcompetes the pepstatin for the enzyme or that the mutations cause the APP constructs to be processed by a different set of proteases than normally carry out γ-secretase activity.
Mutations affecting PS function: An important part of the argument that PS1 is γ-secretase lies in the discovery that mutation of the aspartates at positions 257 and 385 (to alanine) reduces the production of Aβ. Many groups replicated this finding. Haass and colleagues have now extended this work to PS2 (219.5) by showing that mutation of the homologous aspartates in PS2, at positions 257 and 366, also reduce Aβ production. Selkoe and colleagues also examined the PS2 mutations (219.6). Previously, they had observed that expressing the PS1 D385A construct reduced Aβ expression by about 65%, but did not block Aβ production. Now Selkoe and his team have shown that cells expressing both PS1 D385A and PS2 D366A constructs produced no Aβ. Their interpretation of this data is that PS1 is either a direct regulatory cofactor for γ-secretase or the actual γ-secretase. Most of this work has been performed using autosomal cells, such Cos7 cells. Kim, Sisodia and colleagues evaluated the function of the D257A mutation in N2A cells and in primary neurons from PS1 -/- mice (291.8). They observed no loss of Aβ production in the N2A cells, and a small amount of rescue Aβ production in the primary PS1 -/- neurons (~30%). However, the expression level of the mutant construct in the N2A cells was not sufficient to completely replace wild-type PS1 function, which leaves open the possibility that the remaining Aβ activity resulted from residual wild-type PS1 activity. In contrast to Kim and Sisodia's findings, we observe that the D257A and D385A mutations inhibit APP processing, as judged by impaired stimulation of APPs secretion by protein kinase C and elevated levels of APP C-terminal fragments, although we have not yet examined Aβ production (29.10).
It appears that the D257A and D385A mutations in PS1 are not the only mutations capable of blocking PS1 activity. Golde and colleagues (219.11) produced two novel dominant negative PS1 constructs. One construct has a 3 amino acid displacement of the aspartate at position 385 (achieved by inserting three amino acids ahead of the aspartate), while the other PS1 construct lacks transmembrane domains 1 and 2 (but maintained the N-terminal signal region). Expression of either construct inhibited Aβ production and inhibited the ability of protein kinase C to stimulate secretion of APPs. Haass and colleagues (219.5) showed that a naturally occurring PS2 variant that lacks exon 8 inhibits NOTCH processing but does not inhibit Aβ production, although it does increase the levels of APP C-terminal fragments in a manner similar to the PS2 D366A dominant negative construct. This indicates that there are multiple different mutations in PS1 that can lead to losses of function.
Signal transduction: One of the most exciting findings in the field of presenilin research is discovery of the importance of presenilins in cell biology, which is exemplified by the lethal phenotype of the PS1 knockout. The discovery that PS1 is required for cleavage of Notch has captivated the field. Several groups in the meeting replicated this finding. For instance, De Strooper and colleagues (219.1), Ray Goate and colleagues (219.2) and Song, Yankner and colleagues (219.3) showed that expression of the D257A PS1 construct inhibits Notch cleavage in a dominant negative manner and is unable to rescue Notch cleavage in fibroblasts from PS1 knockout fibroblasts. Over-expression of PS2, however, could rescue Notch cleavage in the PS1 knockout cells, showing that PS2 can complement PS1 function.
In contrast to the work on Notch, which appears to be very consistent among laboratories, studies of PS1/beta-catenin interactions appear to be more variable. It appears that interaction of PS1 with β-catenin changes with the state of the cell and, possibly, the type of cell. This issue is exemplified by studies investigating the cellular localization of PS1. De Strooper and colleagues (219.1) showed that most of the PS1 localizes to the golgi/endoplasmic reticulum in dispersed cells. Ray Goate and colleagues (219.2) showed that surface biotinylation of cells did lead to detection of PS1 at the cell surface. The distribution is important because the PS1/ γ-secretase hypothesis requires that PS1 co-localize with all sites of γ-secretase activity. Because Notch is cleaved at the cell surface, the identification of PS1 at the plasma membrane fulfills an important prediction of this hypothesis.
Work by Marambaud, Efthimiopoulos, Georgakopoulos, Robakis, and colleagues (523.3 and 523.4) presented one of the most striking observations about PS1 function. They observed that PS1 localized to the surface of cells at points of contact with other cells. They immunostained cells with anti-PS1 antibodies. In dispersed cells they observed that PS1 was localized mainly in the Golgi and endoplasmic reticulum, however in confluent cells, anti-PS1 antibodies produced bright staining at the plasma membrane at sites of cell to cell contact. Immunocytochemistry with E-cadherin and β-catenin produced a similar pattern in confluent cells, and Robakis' team followed up this work by showing that PS1 co-immunoprecipitated with E-cadherin, α- and β-catenin, which have all previously been implicated in regulating cell to cell adhesion. By quantitating cell adhesion, they were also able to demonstrate a functional phenotype. Cells overexpressing PS1 exhibited increased cell adhesion while the D257A mutant PS1 constructs inhibited cell adhesion.
This work validates a prior observation by Dewji and Singer showing that PS1 can localize to the plasma membrane and regulates cell to cell adhesion. They also found examples in vivo in which PS1 localized to points of cell to cell contact, including synapses in the brain and corneal epithelial cells. Robakis speculates that PS1 might exist as a large complex that includes Notch in addition to E-cadherin and α-/β-catenin. Although extremely interesting, the localization of PS1 to sites of cell to cell contact does not directly agree with prior in vivo studies of PS1, which show that most PS1 primarily localized in the endoplasmic reticulum/Golgi compartment-despite the fact that most of the cells in our body are in contact with other cells.
Other studies showed that PS1 can regulate β-catenin signaling. Koo and colleagues report that PS1 increases beta-catenin signaling in PC12 cells (309.3). In contrast, we observe that both wild-type and the D257A and D385A PS1 mutants inhibit β-catenin signaling. Expression of these mutant constructs in N2A cells inhibited β-catenin signaling using a hTcf4 luciferase reporter or by measuring nuclear translocation of β-catenin (29.10). The difference in results between the two laboratories might lie either in the methods or in the cell types. Koo uses PS1 expressed transiently from an ecdysone-inducible promoter in PC12 cells, while we use NSA cells stably transfected with the PS1 constructs.
Several reports were also presented examining the interaction of presenilins with other proteins. One interesting protein being investigated is calsenilin. Choi, Buxbaum, Wasco and colleagues (523.2) showed that calsenilin binds to the C-terminal fragment of PS2 that is generated by caspases during apoptosis but not to the C-terminal fragment generated by the basal processing of PS2. Leissring, LaFerla and colleagues (641.7) showed that overexpression of PS1 increases calcium fluxes into the endoplasmic reticulum and that calsenilin inhibits the PS1-dependent calcium fluxes. Tezapsidis and colleagues (219.9) identified another protein that binds to PS1, CLIP-170, which is a protein that links membranous organelles to microtubules or intermediate filaments. As with calsenilin, the interaction appears connected with calcium metabolism because binding to PS1 is calcium-dependent. PS1 and PS2 have also been shown to regulate proteins involved in apoptosis. Alberici, Binetti, and colleagues showed that PS1 binds to Bcl-2 during apoptosis (641.4) and Sato, Takashima and colleagues (641.6) showed that PS1 binds to JNK, an important kinase in apoptosis and the stress response.
In Vivo Studies: Clarification of the functions of PS1 might come from in vivo studies. Shen and colleagues (426.1) showed that brains from mice lacking PS1 show a reduced neural progenitor cell population, region-specific neuronal loss and disrupted cerebral laminar organization. They focused on the stem cell defects and observed that the reductions in neural progenitor cells was due to premature differentiation of the neural progenitor cells. In situ studies implicated Notch in this process because the brains from PS1 knockout mice showed decreased expression of Hes5, a downstream effector of Notch, and increased expression of the Notch1 ligand DII1. Notch1 transcript and protein were unchanged. These results suggest that loss of PS1 interferes with Notch signaling. One of the challenges in studying PS1 function in vivo is the lethal phenotype of the PS1 knockout. Zheng, Qian, and colleagues (426.2) approached this issue by rescuing PS1 knockout mice with PS1 constructs driven by a Thy1 promoter which restricts PS1 expression to the brain in adults. Interestingly, these animals develop epithelial hyperproliferation and tumors consistent with increased activation of β-catenin. This suggests that PS1 might normally inhibit activation of cell growth by bet-catenin.
Summary: After much confusion in the presenilin field, a clearer picture of PS1 and PS2 is emerging. PS1 is a transmembrane protein that binds to a group of molecules known to regulate cell contact. These contact-related molecules include Notch, GSK-3, α-, β- and γ-catenin and E-cadheren. PS1 might bind to these molecules as part of one large complex that mediates cell to cell contact or signaling. Part of the function of PS1 in this complex might be as a scaffold, holding these proteins together, while another part of the function is to facilitate cleavage of Notch in response to engagement of Notch by ligands. PS1 might either bind the critical protease or be the protease.
PS1 exists both in the endoplasmic reticulum/golgi and at the plasma membrane. Its function might depend on its localization. In the endoplasmic reticulum PS1 might function in regulating calcium metabolism in a process that is regulated by calsenilin. PS2 appears to have functions that overlap PS1.
The cell appears to have borrowed PS1 function and used it to regulate APP processing, possibly because APP also plays a role in regulating cell to cell contact. It is clear that PS1 is required for APP and Notch proteolysis, PS1 co-localizes to sites of APP proteolysis and that inhibiting PS1 function inhibits APP and Notch proteolysis. This suggests that PS1 is either a γ-secretase or is a scaffold protein required for γ-secretase activity. A definitive answer to this question can really only be shown by demonstrating that recombinant PS1 can catalyze APP cleavage in vitro or by identifying a protease that binds to PS1 carries out the cleavage of APP. Because several groups are actively prusuing these questions, it seems quite possible that this question will be answered within the year.—Benjamin Wolozin
Presentations Cited
29.10: Presenilin 1 and presenilin 2 are necessary for the regulation of amyloid precursor protein secretion. - J.J. PALACINO*, B.E. BERECHID, C. ECKMAN, S. YOUNKIN, J.S. NYE AND B. WOLOZIN Loyla Univ. Chicago.
122.3: Peptidomimetic probes suggest a large S1 pocket for Alzheimer's γ-secretase. - M.S. WOLFE*, C.L. MOORE, T.S., DIEHL, T. RAHMATI, W. XIA AND D.J. SELKOE Univ. of Tennessee, Memphis, Brigham and Women's Hosp. and Harvard Med. Sch.
219.1: Notch and APP proteolytic processing controlled by presenilin 1 and 2. - B. DE STROOPER*, W. ANNAERT, P. CUPERS, D. HARTMANN, P. SAFTIG AND R. KOPAN K.U. Leuven, Belgium, Christian Albrechts Univ. Kiel and Molec. Cell Biol., Göttingen, Germany and Washington Univ.
219.2: The presenilin-1-Notch1 complex forms in the secretory pathway and is transported to the cell surface. - W.J. RAY, M. YAO, J. MUMM, E. SCHROETER, M. WOLFE, D. SELKOE, P. SAFTIG, B. DE STROOPER, R. KOPAN AND A. GOATE* Washington Univ. Med. Sch., Harvard Med. Sch., Univ. of Gauottingen, Germany and KU Leuven, Belgium.
219.3: Proteolytic release and nuclear translocation of Notch-1 are induced by presenilin-1 and impaired by pathogenic presenilin-1 mutations. - W. SONG*, P. NADEAU, M. YUAN, X. YANG, J. SHEN AND B.A. YANKNER Children's Hosp. and Brigham and Women's Hosp., Boston.
219.4: Differential effects of the presenilin-1Δexon 8 splice variant on amyloidogenesis and notch signalling. - A. CAPELL*, H. STEINER, S. KECK, H. ROMIG, B. PESOLD AND C. HAASS Ludwig Maximillians Univ., Munich and Boehringer Ingelheim KG, Germany.
219.5: A functional role of presenilin-2 in amyloidogenesis and notch signaling. - C. HAASS*, A. CAPELL, J. WALTER, H. ROMIG, M. CITRON, K. FECHTELER, U. LEIMER, J. HARDY, R. BAUMEISTER, K. DUFF AND H. STEINER Univ. of Munich, Boehringer Ingelheim, Germany, Amgen Inc., Mayo Clin. Jacksonville and Nathan Kline Inst., Orangeburg, NY.
219.6: Evidence that presenilins 1 and 2 mediate intramembranous cleavage of amyloid precursor protein rather than acting via a protein trafficking role. - D.J. SELKOE*, W.T. KIMBERLY, B.L. OSTASZEWSKI, T. RAHMATI AND W. XIA Brigham and Women's Hosp., Harvard Med. Sch.
219.8: Evaluation of a role of presenilin-1 in the proteolytic processing of amyloid precursor protein. - S.H. KIM*, Y. ZHANG, H.H. SLUNT AND S.S. SISODIA Univ. of Chicago and Johns Hopkins Univ.
219.10: FAD-linked presenilin: effects on pepstatin sensitive and insensitive γ-secretase activities. - M.P. MURPHY*, R. WANG, S.N. ULJON, T.E. SMITH, H.A. LOOKINGBILL, K. FINDLAY AND T.E. GOLDE Mayo Clin. Jacksonville and Rockefeller Univ.
219.11: Presenilins function as regulators of multiple γ-secretase activities. - T.E. GOLDE*, R. WANG, S. ULJON, T.E. SMITH, H.A. LOOKINGBILL, K. FINDLAY AND M.P. MURPHY Mayo Clin. Jacksonville and Rockefeller Univ.
309.3: The Neurobiology of Presenilins and Associated Signaling Molecules in Development and Disease. - E. H. KOO
426.1: Studies of presenilin-1 function in neurogenesis and the adult brain. - J. SHEN*, H. YU, M. HANDLER, X. YANG AND J. KESSLER Brigham and Women's Hosp. and Harvard Med. Sch.
426.2: Reduced expression of presenilin 1 leads to epithelial hyper-proliferation in transgenic mice. - H. ZHENG*, Y. WU, A. FLETCHER AND S. QIAN Baylor Col. of Med. and Merck Res. Labs., Rahway, NJ.
523.2: Calsenilin preferentially interacts with the caspase-derived presenilin 2 C-terminal fragment and cleaved by caspase during apoptosis. - E.K. CHOI*, N. ZAIDI, A.C. CROWLEY, J. MILLER, C. LILLIHOOK, J.D. BUXBAUM AND W. WASCO Harvard Med. Sch., Charlestown and Mount Sinai Sch. of Med.
523.3: Presenilin1 forms Ca++-dependent complexes with the E-cadherin/catenin cell adhesion system. - P. MARAMBAUD, J. SHIOI, A. GEORGAKOPOULOS, S. EFTHIMIOPOULOS AND N.K. ROBAKIS* Mount Sinai Sch. of Med.
523.4: Presenilin 1 forms complexes with cell surface E-cadherin and regulates cell-cell aggregation. - S. EFTHIMIOPOULOS*, W. CUI, A. GEORGAKOPOULOS, J. SHIOI, P. MARAMBAUD AND N.K. ROBAKIS Mount Sinai Sch. of Med.
641.4: Modulation of presenilin-1 and Bcl-2 interaction during apoptosis. - A. ALBERICI, L. BENUSSI, D. MORATTO, L. GASPARINI, R. GHIDONI, J.H. GROWDON, R.M. NITSCH AND G. BINETTI* IRCCS Ctr. San Giovanni di Dio, FBF, Brescia, Italy,
641.5: Presenilin 1 and nerve growth factor modulation in cultured hippocampal neurons. - S. DI LORETO, R. MACCARONE, A. CRESTINI, L. MALVEZZI CAMPEGGI AND A.M. CONFALONI* CNR, L'Aquila, Italy and Inst. Superiore di Sanità, Rome.
641.6: Regulation of JNK signal transduction by presenilin-1. - S. SATO, X. SUN, O. MURAYAMA, K. KAMINO*, Y. SAKAKI AND A. TAKASHIMA Brain Sci. Inst., RIKEN, Wako and Nippon Med. Sch., Japan.
641.7: Presenilin-2 modulates intracellar Ca2+ release and clearance.-M.A. LEISSRING*, I. PARKER AND F.M. LAFERLA Univ. of California, Irvine.
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Society for Neuroscience Annual Meeting: α-Synuclein: A Review
Hashimoto, Masliah and colleagues (27.14) reported that cytochrome C stimulates α-synuclein aggregration in vitro and that cytochrome C can be observed in Lewy bodies, in postmortem brain tissue. Whether cytochrome C actually plays a role in α-synuclein aggregation in vivo remains to be seen, but one could imagine a scenario in which cytochrome C release during apoptosis could promote α-synuclein aggregation. Since cytochrome C release is regulated by Bad, the link between cytochrome C and α-synuclein complements our observation that α-synuclein binds to Bad, as well as other proteins known to bind 14-3-3 including PKC, 14-3-3 itself and ERK (Ostrerova et al., 27.8; and Hanin, Petrucelli et al., 27.7).
One of the keys to understanding α-synuclein pathology will be to develop appropriate animal models. Forloni and colleagues (27.16) injected the central portion of α-synuclein, termed NAC, into substantia nigra of rats. They observed no effects associated with injecting soluble NAC, but did observe that injecting 5 ul of 500 uM pre-aggregated NAC induced neurodegeneration in the substantia nigra. Given the difficulty in showing toxicity with Aβ, the apparent toxicity of NAC is quite interesting. It is important to note that α-synuclein accumulates intracellularly in Lewy body diseases, rather than extracellularly, so the relevance of the model to disease remains to be demonstrated.
Two studies described promising results in mice over-expressing wild-type and mutant α-synuclein. Mucke and colleagues (122.10) reported on transgenic mice over-expressing wildtype α-synuclein from a PDGF promoter. Several different lines of PDGF-synuclein mice developed age-dependent neuronal inclusions resembling Lewy bodies found in Parkinson's disease and some variants of Alzheimer's disease (AD). Loss of dopaminergic terminals in the basal ganglia of these mice was associated with behavioral alterations that improved with anti-Parkinson drug treatment. Interestingly, other transgenic mice expressing high levels of wild-type α-synuclein from a Thy-1 promoter did not develop inclusions.
Yang, Hsiao and colleagues (120.13) reported on transgenic mice overexpressing α-synuclein from a PrP promoter. These mice express α-synuclein throughout the brain, and by nine months develop a progressive loss of motor function that culminates in a lack of mobility and death. Surprisingly, they do not observe any evidence of inclusion bodies in these animals, which suggests either that if α-synuclein aggregates, then they must be dispersed in the neuron rather than coalesced into inclusions. Taken together, these two sets of mice appear promising because they suggest that it will be possible to develop transgenic mice that develop Lewy body pathology, exhibit neurodegeneration and show a behavioral phenotype.
Several groups confirmed the observations by Spillantini and Trojanowski that α-synuclein accumulates in Lewy bodies. Perrin, Trojanowski, and colleagues (27.10) now report that the two homologues of α-synuclein-β- and γ-synuclein also accumulate in Lewy bodies and axonal spheroids.—Benjamin Wolozin
References:
16.3: Towards a transgenic model for Lewy body disorders. T. GOMEZ-ISLA, J. SONDEL, A. MARIASH, M. IRIZARRY, M. VAN BEUSEKOM, K. EYER, B. CHEUNG, H.B. CLARK, B.T. HYMAN AND K. HSIAO. Univ. of Minnesota, Menniapolis and Massachusetts General Hospital.
27.7: Alpha-Synuclein attenuates ERK/Elk signaling. - I. HANIN*, L. PETRUCELLI, N. OSTREROVA, M. FARRER, N. MEHTA, J. HARDY AND B. WOLOZIN Loyola Univ. Sch. of Med. and Mayo Clin. Jacksonville
27.8: Alpha-Synuclein binds to multiple proteins that regulate signal transduction pathways. - N. OSTREROVA*, L. PETRUCELLI, N. MEHTA, M. FARRER, J. HARDY AND B. WOLOZIN Loyola Univ. Med. Ctr. and Mayo Clin. Jacksonville.
27.10: Degenerating axon terminals in hippocampus of Parkinson's disease and dementia with Lewy bodies contain alpha, beta and gamma- synuclein. - J.E. GALVIN, K. URYU, V.M.-Y. LEE* AND J.Q. TROJANOWSKI MCP Hahnemann Univ. and Univ. of Pennsylvania.
27.14: Role of cytochrome c as a stimulator of amyloid-like fibril formation of alpha-synuclein/NACP. - M. HASHIMOTO, A. TAKEDA, L. HSU, T. TAKENOUCHI, A. SISK AND E. MASLIAH* Sch. of Med., UCSD.
27.16: Specific neurotoxic effect of alpha-synuclein fragment (NAC) on dopaminergic neurons. - G. FORLONI*, I. BERTANI, A.M. CALELLA AND R. INVERNIZZI Inst. of Pharmacol. Res., Mario Negri, Milan.
120.13: Amyloid-associated alpha-synuclein (NACP) pathology in aged amyloid precursor transgenic mice. - F. YANG*, G.M. COLE, K. UEDA, W. BEECH, P.-P. CHEN AND K. HSIAO VA Med. Ctr., Sepulveda, CA, UCLA, Tokyo Inst. of Psychiat. and Univ. of Minnesota.
122.10: Potential roles of alpha1-antichymotrypsin and alpha-synuclein in Alzheimer's pathogenesis assessed in bigenic mice expressing human amyloid precursor proteins. - L. MUCKE*, G.-Q. YU, C.R. ABRAHAM, L. MCCONLOGUE, E.M. ROCKENSTEIN AND E. MASLIAH UCSF, Boston Univ., Elan Pharm., South San Francisco and UCSD.
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Society for Neuroscience Annual Meeting: ApoE Affects Pattern of Amyloid Deposition in Tg Mice
Irizarry and collaborators (626.5) provided intriguing new results from transgenic mouse lines expressing human APP (the PDAPP mice) in the presence or absence of endogenous mouse ApoE. The PDAPP mice lacking ApoE have previously been reported to have diminished amyloid deposition (Nature Genetics 17:263) and no thioflavin S-positive plaques (indicating the absence of fibrillar amyloid deposits). The reduction in amyloid deposition in the cortex is striking but the focus of this presentation was on the pattern of amyloid staining in the hippocampal formation. In the PDAPP mice, amyloid is deposited as compact plaques distributed throughout the CA1 and CA3 regions of the hippocampus (mainly stratum radiatum and stratum oriens) and as a dense band of staining in the outer molecular layer of the dentate gyrus. In the absence of ApoE, however, a very different staining pattern is obtained. In these mice, the amyloid diffusely fills the CA1 and CA3 regions of the hippocampus but is almost absent in the dentate gyrus. In addition, the astrocytic response (increased GFAP staining) that is found around plaques in the PDAPP mice also seems to be less prominent and more diffuse in the ApoE-deficient line. This difference in distribution does not appear to be due to altered expression of the APP transgene, different levels of APP or Aβ, or to alterations in LRP protein or mRNA (a receptor that may serve to bind both ApoE and APP). In other words, there is no clear explanation for this effect. However, this striking alteration in amyloid deposition emphasizes the importance of the interaction between ApoE and amyloid in the dynamics of amyloid deposition. The transgenic mice are proving to be fruitful sources of new insights on the development of AD-like pathology.—Keith Crutcher
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Society for Neuroscience Annual Meeting: Which Aβ Species Is the Culprit?
Dean Hartley (12.5) presented data on the deleterious effects of "protofibrillar" Aβ on neurons. Several other groups have reported that amyloid protofibrils of one sort of another are toxic to neurons (e.g., Oda, Aksenova, Roher or Lambert). And Hartley and Selkoe have previously reported that 30-40 kD, 5-10 nanometer fibrils are toxic and can increase electrical activity of cultured neurons (J Neuroscience;19(20):8876-8884). Low-molecular weight amyloid peptide (e.g., non-fibrillar) does not alter the firing rate (normally 60 action potentials/min) in their preparations while protofibrillar amyloid increases the firing rate to 350 action potentials/min. If the protofibrils are washed out within 30 minutes, the increase in firing rate will return to baseline. The addition of protofibrillar amyloid (1 micro molar) with 100 micromolar D-APV (an NMDA antagonist) resulted in a partial block of the effect (69 action potentials/min) which was not further reduced by the addition of the competitive NMDA antagonists NBQX. The addition of higher molecular weight fibrils yielded similar results. However, with the higher molecular weight fibrils, additional activity was blocked by the NBQX. Following exposure of mouse neuroblastoma cells to protofibrils (15 micromolar for two days), they observed a significant loss of processes. In fetal progenitor cells, exposure for one hour caused a 70 percent reduction in primary processes and a 90 percent reduction in secondary processes. To determine if exposure to protofibrils induced apoptosis, they labeled exposed cells with Annexin-V, which binds to the phosphatidyl-serine present on the internal side of the plasma membrane. Under normal conditions, Annexin labeling should be negative, but if the membrane flips, as during apoptosis, labeling would be detected. They observed strong Annexin labeling on the cell body and in patches along specific processes (akin to Greg Cole's "synaptosis"). Further evidence that toxicity may be along an apoptotic pathway was the accumulation of caspase-3 within the exposed cells. While the detection of Annexin suggests a rapid activation by protofibrils, Hartley pointed out that the signal to noise ratio was such that at least 12 hours was necessary to see a strong enough signal in their system.—Brian Cummings
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Society for Neuroscience Annual Meeting, Miami: Tau Update
Toward a neurofibrillary degeneration animal model, Michael Vitek's group (317.1) reported on their tau knockouts. Homozygous mice demonstrated the lack of tau at both mRNA and protein levels. Embryos (-/-) were smaller than wt animals. It should be noted that axonal diameter was not determined. This group also developed tau gene transgenic mice that express all of the six human tau isoforms at mRNA level. Both KO and transgenic strains of mice were crossed and mice exhibiting both changes were analyzed. Human tau-immunoreactivity was found in somato-dendritic domains of some neurons but no NFT structure was observed. Similarly, transgenc mice expressing the entire human tau gene (including its promoter) were obtained in Karen Duff's lab (317.2). Tau proteins were mainly found in neurons. They were hyperphosphorylated but no neurofibrillary degeneration was visualized. It should be noted that overexpression of 3-repeat tau isoforms was observed. Other transgenic mice were also described including those expressing the longest human tau (htau40, 441 amino-acid) and GSK3β (wild-type or constitutively active ([Ser9Ala] mutant)). Double transgenic mice expressing both htau40 and [Ser9Ala]-GSK3 exhibited hyperphosphorylation of tau protein (both murine and human) as revealed by phosphorylation-dependent antibodies (317.3).
Tau mutations and haplotypes and consequences in tauopathies: Numerous studies have reported both new mutations and their effects in animal and cell models (317.4; 317.5; 446.5; 446.6; 446.7; 446.9-446.15; 447.1; 447.2; 447.4;447.5; 448.5). Transgenic mice have been developed in Hutton's lab (317.4; 447.1). However, it is too early to observe any changes. Hutton and Schellenberg groups (317.4; 446.10) emphasized the role of intronic tau mutations whereas Mandelkow's group developed some cell models to analyze tau mutations (317.5). Here is the most recent list of tau mutations found in FTDP-17 (P189A, K257T, I260V, G272V, N279K, DK280, L284L, P301L, P301S, S305N, intronic (+3, +12?, +13, +14 and +16), V337M, G389R, R406W). Tau mutations may change the degree of phosphorylation, the ratio of 3-repeat/4-repeat-tau isoforms, their binding to microtubules and their aggregation ability.
Tau cell biology: Mandelkow's group (317.6) nicely showed that some phosphorylation events may have an inhibitory effect on tau aggregation (especially at Ser262 and 214). These sites are involved in the regulation of tau binding to microtubules. They developed a kinetic model of PHF assembly and also demonstrated that tau overexpression could directly influence cell trafficking. Other studies also analyzed the effects of 4-repeat-tau overexpression as observed in FTDP-17 in animal and cell models (446.6; 446.9; 446.15; 447.2; 447.4).—Luc Buee, INSERM, France
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