Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/bredesenLab © 2011 Buck Institute, All Rights Reserved The single methionine residue of amyloid peptide (A), at position 35 of the 42mer, has important relevance for Ainduced oxidative stress and neurotoxicity. Recent in vivo brain studies in a transgenic Alzheimer disease (AD) mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) referred to as the J20 Tg mouse demonstrated increased levels of oxidative stress. However, substitution of the Met631 residue of APP to leucine M631L in human APP numbering referred to as M631L Tg and corresponding to residue 35 of A142, resulted in no significant in vivo oxidative stress levels, thereby supporting the hypothesis that Met35 of A contributes to oxidative insult in AD brain. It is conceivable that oxidative stress mediated by Met35 of A is important in regulating numerous downstream effects leading to differential levels of relevant biochemical pathways in AD. Therefore, in the current study using proteomics, we tested the hypothesis that several brain proteins involved in pathways such as energy and metabolism, antioxidant activity, proteasome degradation, and pH regulation are altered in J20Tg Vs M631LTg AD mice. http://www.ncbi.nlm.nih.gov/pubmed/22500616 Sat, 31 Dec 2011 00:00:00 -0800 Among the pathological factors known to be associated with Alzheimer disease (AD), oxidative stress induced by the amyloid peptide (A) has been demonstrated to play a key role in human brain and animal models of AD. Recently, we reported elevated levels of oxidative damage in the brain of a transgenic (Tg) AD mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) PDAPP mice, line J20, as evidenced by increased levels of protein carbonyls, 3nitrotyrosine, and proteinbound 4hydroxy2nonenal. This oxidative damage was dependent on the methionine 35 residue within the A peptide. Further insight into the molecular pathways affected in this Tg model of AD may be gained with discoverybased proteomics studies therefore, twodimensional gelbased expression proteomics was performed to compare differences in brain protein levels of J20 Tg mice with nontransgenic (NTg) littermate controls. Based on our studies, we identified six proteins that had significantly increased levels in J20 Tg relative to NTg mice: calcineurin subunit B type 1, GDPdissociation inhibitor 1, Tcomplex protein 1 subunit A, enolase, peptidylprolyl cistrans isomerase (Pin1), and ATP synthase subunit mitochondrial. Several of these proteins have previously been implicated in in vitro and in vivo models and subjects with AD. Additionally, using redox proteomics analyses we identified two oxidativelymodified proteins: phosphatidylethanolaminebinding protein 1 and Pin1 with decreased levels of protein 3nitrotyrosine in J20 Tg mice relative to NTg. Western blotting and immunoprecipitation analyses were used to validate proteomics results. Overall, these studies provide information about changes in the brain proteome as a result of A deposition and clues with which to further direct studies on elucidating AD pathogenesis. http://www.ncbi.nlm.nih.gov/pubmed/21223993 Fri, 31 Dec 2010 00:00:00 -0800 Previously, we identified valosincontaining protein (VCP) as a mediator of ER stressinduced cell death. Mutations in the VCP gene including R93, R155, and R191 have been described that manifest clinically as hereditary inclusion body myopathy with Paget's disease of bone and frontotemporal dementia. In addition, other studies have demonstrated that as a consequence of a mutation generated in the second ATP binding domain of VCP (K524A), cells accumulated large cytoplasmic vacuoles and underwent programmed cell death. In order to better understand the biochemical and molecular consequences of the clinically relevant VCP mutations as well as the genetically engineered ATPaseinactive mutant K524A and any relationship these may have to ER stressinduced cell death, we introduced analogous mutations separately and together into the human VCP gene and evaluated their effect on proteasome activity, Huntingtin protein aggregation and ER stressinduced cell death. Our results indicate that the VCP K524A mutant and the triple mutant VCP R93CR155CK524A block protein degradation, trigger Huntingtin aggregate formation, and render cells highly susceptible to ER stressinduced cell death as compared to VCPWT or other VCP mutants. http://www.ncbi.nlm.nih.gov/pubmed/21249466 Fri, 31 Dec 2010 00:00:00 -0800 The fourth meeting on dependence receptors featured descriptions of previously unknown dependence receptors. New mechanistic data were presented on the switch between the trophic, antiapoptotic response with the proapoptotic response that occurs with loss of trophic support. The possibility that the loss of trophic support may also involve the binding of an active antitrophin was also discussed. New in vivo data were presented on the roles of dependence receptors in development, angiogenesis, oncogenesis, and neurodegeneration, as well as new therapeutic approaches based on dependence receptor function. The next meeting on dependence receptors is scheduled for 2012. http://www.ncbi.nlm.nih.gov/pubmed/21266712 Fri, 31 Dec 2010 00:00:00 -0800 Statins are drugs commonly used to inhibit cholesterol synthesis, with the goal of reducing vascular diseases such as myocardial infarction and stroke. Statins have also been suggested as a therapeutic option for Alzheimer's disease (AD), although their benefit in AD remains controversial. We have previously shown that the intracellular Cterminal cleavage of the amyloid protein precursor (APP) is a major contributor to the neuronal toxicity seen in AD, and that this cleavage can be induced by amyloid. We now report that certain brain permeable statins are also able to induce the Cterminal cleavage of APP and associated cell death, whereas other statins do not. This statin effect on APP exceeded the effects of all other FDAapproved drugs in a library composed of these compounds, suggesting that this effect on APP cleavage is unique to a subset of the statins. Furthermore, the greatest effect occurred with cerivastatin, which has previously been shown to be the statin associated with the greatest risk of rhabdomyolysis. These results may have implications for the choice of which statins to evaluate in AD therapeutic trials furthermore, the results may inform statin choice in individuals who are at high risk for the development of AD, such as those with an apolipoprotein E 4 allele. http://www.ncbi.nlm.nih.gov/pubmed/21422530 Fri, 31 Dec 2010 00:00:00 -0800 Alzheimer's diseaseassociated amyloid (A) peptide is neurotoxic as an oligomer, but not as a monomer, by an unknown mechanism. We showed previously that A interacts with the amyloid protein precursor (APP), leading to caspase cleavage and cell death induction. To characterize this structure and interaction further, we purified the extracellular domain of APP695 (eAPP) and its complex with A oligomers (AOs) of varying sizes, and then performed small angle Xray scattering (SAXS). In the absence of any As, eAPP was a compact homodimer with a tight association between the E1 and E2 domains. Dimeric A oligomers induced monomerization of eAPP while larger oligomers also bound eAPP but preserved the homodimer. Efficient binding of the larger oligomers correlated with the presence of prefibrillar oligomers, suggesting that the eAPP binding is limited to a conformational subset of A oligomers. Both forms of A bound to eAPP at the Acognate region and induced dissociation of the E1 and E2 domains. Our data provide the first structural evidence for AAPP binding and suggest a mechanism for differential modulation of APP processing and cell death signaling by A dimers versus conformationallyspecific larger oligomers. http://www.ncbi.nlm.nih.gov/pubmed/21471643 Fri, 31 Dec 2010 00:00:00 -0800 The presence of misfolded proteins elicits cellular responses including an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Accumulation of these proteins in excessive amounts, however, overwhelms the "cellular quality control" system and impairs the protective mechanisms designed to promote correct folding and degrade misfolded proteins, ultimately leading to organelle dysfunction and cell death. Studies from multiple laboratories have identified the roles of several ER stressinduced cell death modulators and effectors. Earlier, we reported the role of the small cochaperone protein p23 in preventing ER stressinduced cell death. p23 undergoes caspasedependent cleavage to yield a 19kD product (p19), and mutation of this caspase cleavage site not only blocks the formation of the 19kD product but also attenuates the ER stressinduced cell death process triggered by various stressors. Thus, a critical question is whether p23 and/or p19 could serve as an in vivo marker for neurodegenerative diseases featuring misfolded proteins and cellular stress. In the present study, we used an antibody that recognizes both p23 and p19 as well as a specific neoepitope antibody that detects only the p19 fragment. These antibodies were used to detect the presence of both these proteins in cells, primary neurons, brain samples from a mouse model of Alzheimer's disease (AD), and fixed human AD brain samples. While patients with severe AD did display a consistent reduction in p23 levels, our inability to observe p19 in mouse or human AD brain samples suggests that the usefulness of the p23 neoepitope antibody is restricted to cells and primary neurons undergoing cellular stress. http://www.ncbi.nlm.nih.gov/pubmed/21691801 Fri, 31 Dec 2010 00:00:00 -0800 Transplantation of embryonic stem cell (ESC)derived precursors holds great promise for treating various disease conditions. Tracing of precursors derived from ESC after transplantation is important to determine their migration and fate. Chemical labeling, as well as transfection or viralmediated transduction of tracer genes in ESC or in ESCderived precursors, which are the methods that have been used in the generation of the vast majority of labeled ESCs, have serious drawbacks such as varying efficacy. To circumvent this problem we generated endogenously traceable mouse (m)ESC clones by direct derivation from blastocysts of transgenic mice expressing enhanced green fluorescent protein (EGFP) under control of the housekeeping actin promoter The only previous report of endogenously EGFPlabeled mESC derived directly from transgenic EGFP embryos is that of Ahn and colleagues (Ahn et al, 2008. Cytotherapy 10:759769), who used embryos from a different transgenic line and used a significantly different protocol for derivation. Cells from a highexpressing EGFPmESC clone, G11, retain high levels of EGFP expression after differentiation into derivatives of all three primary germ layers both in vitro and in vivo, and contribution to all tissues in chimeric progeny. To determine whether progenitor cells derived from G11 could be used in transplantation experiments, we differentiated them to early neuronal precursors and injected them into syngeneic mouse brains. Transplanted EGFPexpressing cells at different stages of differentiation along the neuronal lineage could be identified in brains by expression of EGFP twelve weeks after transplantation. Our results suggest that the EGFPmESC(G11) line may constitute a useful tool in ESCbased cell and tissue replacement studies. http://www.ncbi.nlm.nih.gov/pubmed/21874159 Fri, 31 Dec 2010 00:00:00 -0800 Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratomaforming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratomaforming cells. We focus here on the BCL2 family of proteins, which regulate mitochondrialdependent apoptosis. We used quantitative PCR to compare the steadystate expression profile of all human BCL2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the proapoptotic BH3only BCL2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of prosurvival BCL2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESCderived neoplasms. http://www.ncbi.nlm.nih.gov/pubmed/22174832 Fri, 31 Dec 2010 00:00:00 -0800 The amyloid protein precursor (APP) has been implicated in Alzheimer's disease (AD) not only as a precursor of the amyloid peptide but also as a mediator of signal transduction. We recently identified novel mediators of APP signaling via interactions with Mint/X11 family proteins Mint1 and Mint3. These mediators include transcriptional coactivators Taz and Yap. Here we show that Taz and Yap also mediate signaling via the APP paralogues APLP1 and APLP2 through interactions with Mint1 and Mint3. APLP1 and APLP2 formed transcriptionally active triple protein complexes with the adaptor protein Mint3 and each of the transcriptional regulators Taz and Yap, and complex formation was regulated by the secretase cleavage of APLP1 and APLP2. The presence of Mint1 instead of Mint3 in the complex prevented its translocation to the nucleus. APLP1 displayed much lower transactivation levels compared to APP and APLP2. These results indicate that all three APP family members are capable of activating gene transcription via Mint3Taz and Mint3Yap. http://www.ncbi.nlm.nih.gov/pubmed/21178287 Tue, 30 Nov 2010 00:00:00 -0800 Reports from multiple laboratories have now been published analyzing the critical nature of the caspase cleavage site of amyloid protein precursor (APP) for cell death induction, synaptic loss, hippocampal atrophy, longterm potentiation, memory loss, neophobia, and other aspects of the Alzheimer's phenotype. Here we review the results and implications of these studies for the understanding of Alzheimer's disease pathophysiology and the potential development of therapeutics that target this site in APP. http://www.ncbi.nlm.nih.gov/pubmed/20847422 Tue, 31 Aug 2010 00:00:00 -0700 This study describes the first proteomic analysis of paraptosis a nonapoptotic form of programmed cell death. As with apoptosis, the first description of paraptosis was based on morphological criteria. Since there are no known markers for paraptosis, the purpose of this study was to dissect changes in the proteome profile occurring during paraptosis. Using one and twodimensional SDSPAGE, Western analysis, and mass spectrometry, we show that during paraptosis, alterations occur mainly in cytoskeletal proteins, signal transduction proteins, mitochondrial proteins, and some metabolic proteins. We also report the identification of: 1) a paraptosis inhibitor, phosphatidylethanolamine binding protein (PEBP1), and 2) a candidate mediator of paraptosis, prohibitin. Identification of specific paraptotic changes will ultimately lead to tools to detect this type of programmed cell death in in vivo systems and allow for its further characterization. 2010 WileyLiss, Inc. http://www.ncbi.nlm.nih.gov/pubmed/20830744 Tue, 31 Aug 2010 00:00:00 -0700 BACKGROUND: Reduced TOR signaling has been shown to significantly increase lifespan in a variety of organisms 1, 2, 3, 4. It was recently demonstrated that longterm treatment with rapamycin, an inhibitor of the mTOR pathway5, or ablation of the mTOR target p70S6K6 extends lifespan in mice, possibly by delaying aging. Whether inhibition of the mTOR pathway would delay or prevent ageassociated disease such as AD remained to be determined. METHODOLOGY/PRINCIPAL FINDINGS: We used rapamycin administration and behavioral tools in a mouse model of AD as well as standard biochemical and immunohistochemical measures in brain tissue to provide answers for this question. Here we show that longterm inhibition of mTOR by rapamycin prevented ADlike cognitive deficits and lowered levels of Abeta(42), a major toxic species in AD7, in the PDAPP transgenic mouse model. These data indicate that inhibition of the mTOR pathway can reduce Abeta(42) levels in vivo and block or delay AD in mice. As expected from the inhibition of mTOR, autophagy was increased in neurons of rapamycintreated transgenic, but not in nontransgenic, PDAPP mice, suggesting that the reduction in Abeta and the improvement in cognitive function are due in part to increased autophagy, possibly as a response to high levels of Abeta. CONCLUSIONS/SIGNIFICANCE: Our data suggest that inhibition of mTOR by rapamycin, an intervention that extends lifespan in mice, can slow or block AD progression in a transgenic mouse model of the disease. Rapamycin, already used in clinical settings, may be a potentially effective therapeutic agent for the treatment of AD. http://www.ncbi.nlm.nih.gov/pubmed/20376313 Wed, 31 Mar 2010 00:00:00 -0700 Numerous studies have demonstrated oxidative damage in the central nervous system in subjects with Alzheimer disease and in animal models of this dementing disorder. In this study, we show that transgenic mice modeling Alzheimer diseasePDAPP mice with Swedish and Indiana mutations in the human amyloid precursor protein (APP)develop oxidative damage in brain, including elevated levels of protein oxidation (indexed by protein carbonyls and 3nitrotyrosine) and lipid peroxidation (indexed by proteinbound 4hydroxy2nonenal). This oxidative damage requires the presence of a single methionine residue at position 35 of the amyloid betapeptide (Abeta), because all indices of oxidative damage in brain were completely prevented in genetically and agematched PDAPP mice with an M631L mutation in APP. No significant differences in the levels of APP, Abeta(142), and Abeta(140) or in the ratio Abeta(142)/Abeta(140) were found, suggesting that the loss of oxidative stress in vivo in the brain of PDAPP(M631L) mice results solely from the mutation of the Met35 residue to Leu in the Abeta peptide. However, a marked reduction in Abetaimmunoreactive plaques was observed in the M631L mice, which instead displayed small punctate areas of nonplaque immunoreactivity and a microglial response. In contrast to the requirement for Met at residue 35 of the Abeta sequence (M631 of APP) for oxidative damage, indices of spatial learning and memory were not significantly improved by the M631L substitution. Furthermore, a genetically matched line with a different mutationPDAPP(D664A)showed the reverse: no reduction in oxidative damage but marked improvement in memory. This is the first in vivo study to demonstrate the requirement for Abeta residue Met35 for oxidative stress in the brain of a mammalian model of Alzheimer disease. However, in this specific transgenic mouse model of AD, oxidative stress is neither required nor sufficient for memory abnormalities. http://www.ncbi.nlm.nih.gov/pubmed/19854267 Sun, 31 Jan 2010 00:00:00 -0800 Previous studies suggested that cleavage of the amyloid precursor protein (APP) at aspartate residue 664 by caspases may play a key role in the pathogenesis of Alzheimer's disease. Mutation of this site (D664A) prevents caspase cleavage and the generation of the Cterminal APP fragments C31 and Jcasp, which have been proposed to mediate amyloidbeta (Abeta) neurotoxicity. Here we compared human APP transgenic mice with (B254) and without (J20) the D664A mutation in a battery of tests. Before Abeta deposition, hAPPB254 and hAPPJ20 mice had comparable hippocampal levels of Abeta(142). At 23 or 57 months of age, hAPPB254 and hAPPJ20 mice had similar abnormalities relative to nontransgenic mice in spatial and nonspatial learning and memory, elevated plus maze performance, electrophysiological measures of synaptic transmission and plasticity, and levels of synaptic activityrelated proteins. Thus, caspase cleavage of APP at position D664 and generation of C31 do not play a critical role in the development of these abnormalities. http://www.ncbi.nlm.nih.gov/pubmed/20053918 Thu, 31 Dec 2009 00:00:00 -0800 Multiple recent reports implicate amyloid precursor protein (APP) signaling in the pathogenesis of Alzheimer's disease, but the APPdependent signaling network involved has not been defined. Here, we report a novel consensus sequence for interaction with the PDZ1 and PDZ2 domains of the APPinteracting proteins Mint1, Mint2, and Mint3 (X11alpha, X11beta, and X11gamma), and multiple novel interactors for these proteins, with the finding that transcriptional coactivators are highly represented among these interactors. Furthermore, we show that Mint3 interaction with a set of the transcriptional coactivators leads to nuclear localization and transactivation, whereas interaction of the same set with Mint1 or Mint2 prevents nuclear localization and transactivation. These results define new mediators of the signal transduction network mediated by APP. http://www.ncbi.nlm.nih.gov/pubmed/20016085 Mon, 30 Nov 2009 00:00:00 -0800 In addition to the cleavages that generate amyloidbeta (Abeta), the Abetaprecursor protein (APP) is processed at Asp664, releasing a second toxic peptide (APPC31). Transgenic mice otherwise identical to a wellcharacterized model of AD, PDAPP mice, but carrying a mutation that obliterates Asp664 show a reversal of ADlike deficits in memory and in noncognitive components of behaviour in spite of accumulating high levels of Abeta. These results suggest that cleavage of APP at Asp664 plays a role in the generation of ADlike deficits, and that a major pathway of Abeta toxicity in vivo, or a pathway that crucially impinges on it, may depend on cleavage of APP at Asp664. Since young PDAPP(D664A) mice showed an akinetic phenotype when first required to swim, we trained a 3monthold (mo) cohort to criterion (normal swimming), and briefly exposed it to the Morris water maze (MWM) environment prior to training at 7 mo, to avoid potentially confounding effects of the akinetic phenotype in MWM studies. Prior experience decreased floating in PDAPP(D664A) mice but not in PDAPP nor in nonTg groups. While learning was restored in experienced PDAPP(D664A) mice, it was indistinguishable from both nonTg as well as from PDAPP mice in nave PDAPP(D664A) animals. Floating did not correlate with worse performance in nave PDAPP(D664A) mice, suggesting that the contribution of prior experience to improved performance is related to its cognitive effects but not to noncognitive components of behaviour. Our results suggest that early experience reduces the contribution of noncognitive components of behaviour to performance, and may contribute to the restoration of learning at later ages in PDAPP(D664A) mice. http://www.ncbi.nlm.nih.gov/pubmed/19751769 Sat, 31 Oct 2009 00:00:00 -0700 Netrin1 was recently proposed to control tumorigenesis by inhibiting apoptosis induced by the dependence receptors DCC (Deleted in colorectal cancer) and UNC5H. Although the loss of these dependence receptors' expression has been described as a selective advantage for tumor growth and progression in numerous cancers, recent observations have shown that some tumors may use an alternative strategy to block dependence receptorinduced programmed cell death: the autocrine expression of netrin1. This alternative strategy has been observed in a large fraction of aggressive breast cancers, neuroblastoma, pancreatic adenocarcinoma, and lung cancer. This observation is of potential interest regarding future targeted therapy, as in such cases interfering with the ability of netrin1 to inhibit DCC or UNC5Hinduced cell death is associated with apoptosis of netrin1expressing tumor cells in vitro, and with inhibition of tumor growth or metastasis in different animal tumor models. The understanding of the mechanism by which netrin1 inhibits cell death is therefore of interest. Here, we show that netrin1 triggers the multimerization of both DCC and UNC5H2 receptors, and that multimerization of the intracellular domain of DCC and UNC5H2 is the critical step to inhibit the proapoptotic effects of both of these receptors. Taking advantage of this property, we utilized a recombinant specific domain of DCC that (i) interacts with netrin1 and (ii) inhibits netrin1induced multimerization, to trigger apoptosis in netrindependent tumor cells. http://www.ncbi.nlm.nih.gov/pubmed/19543238 Wed, 30 Sep 2009 00:00:00 -0700 Alterations in the processing of the amyloid precursor protein (APP) lead to familial Alzheimer's disease (AD). AD patients exhibit increased seizure susceptibility and alterations in their EEGs, which suggests that APP and its metabolites may modulate neuronal networks. Here we demonstrate that transgenic mice overexpressing APP intracellular domain (AICD) and its binding partner Fe65 exhibit abnormal spiking events and a susceptibility to induced seizures. These abnormalities are not observed in PDAPP(D664A) mice, which express high Abeta levels but harbor a mutation in the APP intracellular domain. These data suggest that alterations in the levels of AICD contribute to network dysfunction in AD. http://www.ncbi.nlm.nih.gov/pubmed/19828212 Wed, 30 Sep 2009 00:00:00 -0700 Resveratrol, a naturally occurring polyphenol, exhibits antioxidant, antiaging, and anticancer activity. Resveratrol has also been shown to inhibit tumor initiation, promotion, and progression in a variety of cell culture systems. Earlier, we showed that paraquat, a bipyridyl herbicide, triggers endoplasmic reticulum stress, cell dysfunction, and dopaminergic cell death. Due to its antioxidant activity, we assessed the ability of resveratrol to rescue cells from the toxic effects of paraquat. While resveratrol did not have any protective effect at low concentrations, it triggered endoplasmic reticulum (ER) stressinduced cell death at higher concentrations (50250 microM). The present study was carried out to determine the mechanism by which resveratrol triggers ER stress and cell death in dopaminergic N27 cells. Our studies demonstrate that resveratrol triggers ER stress and cell dysfunction, caspase activation, p23 cleavage and inhibition of proteasomal activity in dopaminergic N27 cells. While over expression of uncleavable p23 was associated with decreased cell death, downregulation of p23 protein expression by siRNA resulted in enhancement of ER stressinduced cell death triggered by resveratrol indicating a protective role for the small cochaperone p23 in dopaminergic cell death. http://www.ncbi.nlm.nih.gov/pubmed/19145491 Mon, 31 Aug 2009 00:00:00 -0700 The cytoplasmic tail of the amyloid precursor protein (APP) contains two putatively cytotoxic peptides, Jcasp and C31, derived by caspase cleavage of APP. Jcasp is a fragment starting from the epsilonsecretase site to position 664, while C31 is a fragment from position 665 to the Cterminus. Our studies now showed that compared to C31, Jcasp appeared to play a minor role in cytotoxicity. In particular, inhibition of Jcasp generation by treatment of gammasecretase inhibitor did not lead to any attenuation of C31induced toxicity. Secondly, because C31 toxicity is largely absent in cells lacking endogenous APP, we determined, using a split betagalactosidase complementary assay to monitor proteinprotein interactions, the presence of APP associated complexes. Our results demonstrated that both APP homomeric and C31/APP heteromeric complexes were correlated with cell death, indicating that C31 complexes with APP to recruit the interacting partners that initiate the signals related to cellular toxicity. http://www.ncbi.nlm.nih.gov/pubmed/19679105 Fri, 31 Jul 2009 00:00:00 -0700 Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the indepth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell deathrelated parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the pointofnoreturn of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells. http://www.ncbi.nlm.nih.gov/pubmed/19373242 Fri, 31 Jul 2009 00:00:00 -0700 ABSTRACT: Extensive genetic, biochemical, and histological evidence has implicated the amyloidbeta peptide (Abeta) in Alzheimer's disease pathogenesis, and several mechanisms have been suggested, such as metal binding, reactive oxygen species production, and membrane pore formation. However, recent evidence argues for an additional role for signaling mediated by the amyloid precursor protein, APP, in part via the caspase cleavage of APP at aspartate 664. Here we review the effects and implications of this cleavage event, and propose a model of Alzheimer's disease that focuses on the critical nature of this cleavage and its downstream effects. http://www.ncbi.nlm.nih.gov/pubmed/19558683 Tue, 30 Jun 2009 00:00:00 -0700 The betaamyloid precursor protein (APP) is an orphan transmembrane receptor whose physiological role is largely unknown. APP is cleaved by proteases generating amyloidbeta (Abeta) peptide, the main component of the amyloid plaques that are associated with Alzheimer's disease. Here, we show that APP binds netrin1, a multifunctional guidance and trophic factor. Netrin1 binding modulates APP signaling triggering APP intracellular domain (AICD)dependent gene transcription. Furthermore, netrin1 binding suppresses Abeta peptide production in brain slices from Alzheimer model transgenic mice. In this mouse model, decreased netrin1 expression is associated with increased Abeta concentration, thus supporting netrin1 as a key regulator of Abeta production. Finally, we show that netrin1 brain administration in Alzheimer model transgenic mice may be associated with an amelioration of the Alzheimer's phenotype. http://www.ncbi.nlm.nih.gov/pubmed/19148186 Thu, 30 Apr 2009 00:00:00 -0700 OBJECTIVE: Selective neuronal vulnerability in neurodegenerative diseases is poorly understood. In Alzheimer's disease, the basal forebrain cholinergic neurons are selectively vulnerable, putatively because of their expression of the cell death mediator p75(NTR) (the common neurotrophin receptor), and its interaction with proapoptotic ligands pronerve growth factor and amyloidbeta peptide. However, the relation between amyloid precursor protein (APP) and p75(NTR) has not been described previously. METHODS: APP and p75(NTR) were assayed for interaction by coimmunoprecipitation in vitro and in vivo, yeast twohybrid assay, bioluminescence resonance energy transfer, and confocal microscopy. Effects on APP processing and signaling were studied using immunoblotting, enzymelinked immunosorbent assays, and luciferase reporter assays. RESULTS: The results of this study are as follows: (1) p75(NTR) and APP interact directly (2) this interaction is modified by ligands nerve growth factor and betaamyloid (3) APP and p75(NTR) colocalization in vivo is modified in Alzheimer's model transgenic mice (4) APP processing is altered by p75(NTR), and to a lesser extent, p75(NTR) processing is altered by the presence of APP (5) APPdependent transcription mediated by Fe65 is blocked by p75(NTR) and (6) coexpression of APP and p75(NTR) triggers cell death. INTERPRETATION: These results provide new insight into the emerging signaling network that mediates the Alzheimer's phenotype and into the mechanism of basal forebrain cholinergic neuronal selective vulnerability. In addition, the results argue that the interaction between APP and p75(NTR) may represent a therapeutic target in Alzheimer's disease. http://www.ncbi.nlm.nih.gov/pubmed/19334058 Tue, 31 Mar 2009 00:00:00 -0700 Parkinson's disease (PD) features oxidative stress and accumulation of misfolded (unfolded, alternatively folded, or mutant) proteins with associated loss of dopaminergic neurons. Oxidative stress and the accumulated misfolded proteins elicit cellular responses that include an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Chronic ER stress and accumulation of misfolded proteins in excessive amounts, however, overwhelm the cellular 'quality control' system and impair the protective mechanisms designed to promote correct folding and degrade faulty proteins, ultimately leading to organelle dysfunction and neuronal cell death. Paraquat belongs to a class of bipyridyl herbicides and triggers oxidative stress and dopaminergic cell death. Epidemiological studies suggest an increased risk for developing PD following chronic exposure to paraquat. The present study was carried out to determine the role of paraquat in triggering cellular stress particularly ER stress and to elucidate the pathways that couple ER stress to dopaminergic cell death. We demonstrate that paraquat triggers ER stress, cell dysfunction, and dopaminergic cell death. p23, a small cochaperone protein, is cleaved during ER stressinduced cell death triggered by paraquat and blockage of the caspase cleavage site of p23 was associated with decreased cell death. Paraquat also inhibits proteasomal activity that may further trigger accumulation of misfolded proteins resulting in ER stress. Our results indicate a protective role for p23 in PDrelated programmed cell death. The data also underscore the involvement of ER, caspases, and the proteasomal system in ER stressinduced cell death process. http://www.ncbi.nlm.nih.gov/pubmed/18773310 Sat, 31 Jan 2009 00:00:00 -0800 Astrocytes emerge as key players in motor neuron degeneration in Amyotrophic Lateral Sclerosis (ALS). Whether astrocytes cause direct damage by releasing toxic factors or contribute indirectly through the loss of physiological functions is unclear. Here we identify in the hSOD1(G93A) transgenic mouse model of ALS a degenerative process of the astrocytes, restricted to those directly surrounding spinal motor neurons. This phenomenon manifests with an early onset and becomes significant concomitant with the loss of motor cells and the appearance of clinical symptoms. Contrary to wildtype astrocytes, mutant hSOD1expressing astrocytes are highly vulnerable to glutamate and undergo cell death mediated by the metabotropic type5 receptor (mGluR5). Blocking mGluR5 in vivo slows down astrocytic degeneration, delays the onset of the disease and slightly extends survival in hSOD1(G93A) transgenic mice. We propose that excitotoxicity in ALS affects both motor neurons and astrocytes, favouring their local interactive degeneration. This new mechanistic hypothesis has implications for therapeutic interventions. http://www.ncbi.nlm.nih.gov/pubmed/18617894 Fri, 31 Oct 2008 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/18828573 Tue, 30 Sep 2008 00:00:00 -0700 The microenvironment of cancerous cells includes endoplasmic reticulum (ER) stress the resistance to which is required for the survival and growth of tumors. Acute ER stress triggers the induction of a family of ER stress proteins that promotes survival and/or growth of the cancer cells, and also confers resistance to radiation and chemotherapy. Prolonged or severe ER stress, however, may ultimately overwhelm the cellular protective mechanisms, triggering cell death through specific programmed cell death (pcd) pathways. Thus, downregulation of the protective stress proteins may offer a new therapeutic approach to cancer treatment. In this regard, recent reports have demonstrated the roles of the phytochemical curcumin in the inhibition of proteasomal activity and triggering the accumulation of cytosolic Ca(2) by inhibiting the Ca(2)ATPase pump, both of which enhance ER stress. Using a mouse melanoma cell line, we investigated the possibility that curcumin may trigger ER stress leading to programmed cell death. Our studies demonstrate that curcumin triggers ER stress and the activation of specific cell death pathways that feature caspase cleavage and activation, p23 cleavage, and downregulation of the antiapoptotic Mcl1 protein. http://www.ncbi.nlm.nih.gov/pubmed/18493855 Sat, 31 May 2008 00:00:00 -0700 The deficits of Alzheimer's disease (AD) are believed to result, at least in part, from neurotoxicity of betaamyloid (Abeta), a set of 3843 amino acid fragments derived from the betaamyloid precursor protein (APP). In addition, APP generates the APPC31 and Jcasp toxic fragments intracellularly by cleavage at Asp664. We reported that mutation of Asp664 to Ala in a FADhuman APP transgene prevented ADlike deficits but did not affect Abeta production or deposition in PDAPP mice, arguing that D664A plays a crucial role in the generation of ADlike deficits. Whether D664A simply delays or completely prevents ADlike deficits, however, remained undefined. To address this question, we performed behavioral studies longitudinally on a pretrained mouse cohort at 9 and 13 months (mo) of age. While behavioral deficits were present in PDAPP mice, performance of Tg PDAPP(D664A) mice was not significantly different from nonTg littermates' across all ages tested. Moreover, aberrant patterns in noncognitive components of behavior in PDAPP mice were ameliorated in PDAPP(D664A) animals as well. A trend towards poorer retention at 9 mo and poorer learning at 13 mo that did not reach statistical significance was observed in PDAPP(D664A) mice. These results support and extend recent studies showing that cleavage of APP at Asp664 (or proteinprotein interactions dependent on Asp664) is a crucial event in the generation of ADlike deficits in PDAPP mice. Our results thus further demonstrate that the D664A mutation either completely precludes, or markedly delays (beyond 13 mo) the appearance of ADlike deficits in this mouse model of AD. http://www.ncbi.nlm.nih.gov/pubmed/18485495 Wed, 30 Apr 2008 00:00:00 -0700 Programmed cell death (pcd) is a form of cell death in which the cell plays an active role in its own demise. Pcd plays a critical role in the development of the nervous system, as well as in its response to insult. Both antipcd and propcd modulators play prominent roles in development and disease, including neurodegeneration, cancer, and ischemic vascular disease, among others. Over 100,000 published studies on one form of programmed cell deathapoptosishave appeared, but recent studies from multiple laboratories suggest the existence of nonapoptotic forms of programmed cell death, such as autophagic programmed cell death. In addition, there appear to be programmatic cell deaths that do not fit the criteria for either apoptosis or autophagic cell death, arguing that additional programs may also be available to cells. Constructing a mechanistic taxonomy of all forms of pcdbased on inhibitors, activators, and identified biochemical pathways involved in each form of pcdshould offer new insight into cell deaths associated with various disease states, and ultimately offer new therapeutic approaches. http://www.ncbi.nlm.nih.gov/pubmed/18473818 Wed, 30 Apr 2008 00:00:00 -0700 In addition to the proteolytic cleavages that give rise to amyloidbeta (Abeta), the amyloidbeta protein precursor (AbetaPP) is cleaved at Asp664 intracytoplasmically. This cleavage releases a cytotoxic peptide, APPC31, removes AbetaPPinteraction motifs required for signaling and internalization, and is required for the generation of ADlike deficits in a mouse model of the disease. Although we and others had previously shown that Asp664 cleavage of AbetaPP is increased in AD brains, the distribution of the Asp664cleaved forms of AbetaPP in nondiseased and AD brains at different ages had not been determined. Confirming previous reports, we found that Asp664cleaved forms of AbetaPP were increased in neuronal cytoplasm and nuclei in earlystage AD brains but were absent in agematched, nondiseased control brains and in latestage AD brains. Remarkably, however, Asp664cleaved AbetaPP was prominent in neuronal somata and in processes in entorhinal cortex and hippocampus of nondiseased human brains at ages 45 years. Our observations suggest that Asp664 cleavage of AbetaPP may be part of the normal proteolytic processing of AbetaPP in young (45 years) human brain and that this cleavage is downregulated with normal aging, but is aberrantly increased and altered in location in early AD. http://www.ncbi.nlm.nih.gov/pubmed/18334752 Fri, 29 Feb 2008 00:00:00 -0800 Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response. http://www.ncbi.nlm.nih.gov/pubmed/18188003 Thu, 31 Jan 2008 00:00:00 -0800 While much of the focus on Alzheimer's disease therapeutics has been directed at betaamyloid peptide or at cholinergic synaptic transmission, recent data suggest that targeting signal transduction by the amyloid precursor protein (APP) itself may be an alternative approach with significant potential 1. Here we discuss the possibility that APPmediated signal transduction, downstream from amyloidbeta peptide production itself, may be an appropriate therapeutic target in Alzheimer's disease. http://www.ncbi.nlm.nih.gov/pubmed/18220517 Mon, 31 Dec 2007 00:00:00 -0800 The deficits in Alzheimer disease (AD) stem at least partly from neurotoxic betaamyloid peptides generated from the amyloid precursor protein (APP). APP may also be cleaved intracellularly at Asp664 to yield a second neurotoxic peptide, C31. Previously, we showed that cleavage of APP at the Cterminus is required for the impairments seen in APP transgenic mice, by comparing elements of the disease in animals modeling AD, with (plateletderived growth factor Bchain promoterdriven APP transgenic mice PDAPP) versus without (PDAPP D664A) a functional Asp664 caspase cleavage site. However, the signaling mechanism(s) by which Asp664 contributes to these deficits remains to be elucidated. In this study, we identify a kinase protein, recently shown to bind APP at the Cterminus and to contribute to AD, whose activity is modified in PDAPP mice, but normalized in PDAPP D664A mice. Specifically, we observed a significant increase in nuclear p21activated kinase (isoforms 1, 2, and or 3 PAK1/2/3) activation in hippocampus of 3 month old PDAPP mice compared with nontransgenic littermates, an effect completely prevented in PDAPP D664A mice. In contrast, 13 month old PDAPP mice displayed a significant decrease in PAK1/2/3 activity, which was once again absent in PDAPP D664A mice. Similarly, in hippocampus of early and severe AD subjects, there was a progressive and subcellularspecific reduction in active PAK1/2/3 compared with normal controls. Interestingly, total PAK1/2/3 protein was increased in early AD subjects, but declined in moderate AD and declined further, to significantly below that of control levels, in severe AD. These findings are compatible with previous suggestions that PAK may be involved in the pathophysiology of AD, and demonstrate that both early activation and late inactivation in the murine AD model require the cleavage of APP at Asp664. http://www.ncbi.nlm.nih.gov/pubmed/17986220 Mon, 31 Dec 2007 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/18077970 Fri, 30 Nov 2007 00:00:00 -0800 The function of neurogenesis in the adult brain is still unknown. Interventions such as environmental enrichment and exercise impinge on neurogenesis, suggesting that the process is regulated by experience. Conversely, a role for neurogenesis in learning has been proposed through 'cellular plasticity', a process akin to synaptic plasticity but operating at the network level. Although neurogenesis is stimulated by acute injury, and possibly by neurodegenerative processes such as Alzheimer's disease (AD), it does not suffice to restore function. While the role and direction of change in the neurogenic response at different stages of AD is still a matter of debate, it is possible that a deficit in neurogenesis may contribute to AD pathogenesis since at least one of the two regions ostensibly neurogenic in the adult human brain (the subgranular zone of the dentage gyrus and the ventriculoolfactory neurogenic system) support highlevel functions affected in early AD (associative memory and olfaction respectively). The age of onset and the rate of progression of sporadic forms of AD are highly variable. Sporadic AD may have a component of insufficient neurogenic replacement or insufficient neurogenic stimulation that is correlated with traits of personal history the rate of neurogenesis and the survival of replicating progenitors is strongly modified by behavioral interventions known to impinge on the rate of neurogenesis and the probability of survival of newly born neuronsexercise, enriched experience, and learning. This view is consistent with epidemiological data suggesting that higher education and increased participation in intellectual, social and physical aspects of daily life are associated with slower cognitive decline in healthy elderly ("cognitive reserve") and may reduce the risk of AD. Although neurogenesis can be modulated exogenously by growth factors, stimulation of neurogenesis as a mean to treat neurodegeneration is still for the most part speculative. Moreover, it is possible that different roles of neurogenesis during the course of AD are dictated by the degree of permissibility of the environment in which the process is taking place. A unique opportunity may exist in which the therapeutic stimulation of neurogenesis might contribute to functional 'repair' of the adult diseased brain, before damage to whole neuronal networks has ensued. In spite of the considerable gaps in our knowledge of neurogenesis, and of the considerable limitations that will need to be overcome before we can intervene in the process, that new neurons are added continuously to the adult mammalian brain is a discovery that has already changed the way we think about neurobiology, and may soon change the way we understand and approach neurodegenerative diseases such as AD. http://www.ncbi.nlm.nih.gov/pubmed/18045158 Wed, 31 Oct 2007 00:00:00 -0700 The heat shock protein (Hsp) system is a cell defense mechanism constitutively expressed at the basal state and essential for cell survival in response to damaging stimuli. Apoptosis is a physiological cell death program that preserves tissue homeostasis. We investigated the intrinsic pathway of apoptosis at various stages of brain maturation in CD1 mice, triggered by two mitochondrial proapoptotic proteins, cytochrome c and Smac/DIABLO, and the pathway's regulation by Hsp70. Smac/DIABLO and Hsp70 proteins were upregulated 2fold and 1.53fold, respectively, after birth. In contrast, in the presence of cytochrome c/2'deoxyadenosine 5'triphosphate (dATP), caspase activity in mouse brain cellfree extracts increased 90fold and 61fold, at fetal and neonatal stages, whereas no activation was detected 15 days postnatally or at any subsequent times. These results indicate that the activation pattern of the intrinsic pathway of apoptosis undergoes a marked shift during postnatal maturation. http://www.ncbi.nlm.nih.gov/pubmed/17914183 Sun, 30 Sep 2007 00:00:00 -0700 Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin7 is cleaved by caspase7, and we map two putative caspase7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin7 protein. Sitedirected mutagenesis of these two caspase7 cleavage sites in the polyQexpanded form of ataxin7 produces an ataxin7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ lengthdependent manner, expression of the noncleavable D266N/D344N form of polyQexpanded ataxin7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase7 truncation product of ataxin769Q or 92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin7, showed increased cellular toxicity. We also detected Nterminal polyQexpanded ataxin7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase7 into the nucleus by polyQexpanded ataxin7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin7 by caspase7 may contribute to SCA7 disease pathogenesis. http://www.ncbi.nlm.nih.gov/pubmed/17646170 Sun, 30 Sep 2007 00:00:00 -0700 Specific protein associations define the wiring of protein interaction networks and thus control the organization and functioning of the cell as a whole. Peptide recognition by PDZ and other protein interaction domains represents one of the beststudied classes of specific protein associations. However, a mechanistic understanding of the relationship between selectivity and promiscuity commonly observed in the interactions mediated by peptide recognition modules as well as its functional meaning remain elusive. To address these questions in a comprehensive manner, two large populations of artificial and natural peptide ligands of six archetypal PDZ domains from the synaptic proteins PSD95 and SAP97 were generated by targetassisted iterative screening (TAIS) of combinatorial peptide libraries and by synthesis of proteomic fragments, correspondingly. A comparative statistical analysis of affinityranked artificial and natural ligands yielded a comprehensive picture of known and novel PDZ ligand specificity determinants, revealing a hitherto unappreciated combination of specificity and adaptive plasticity inherent to PDZ domain recognition. We propose a reconceptualization of the PDZ domain in terms of a complex adaptive system representing a flexible compromise between the rigid order of exquisite specificity and the chaos of unselective promiscuity, which has evolved to mediate two mutually contradictory properties required of such higher order subcellular organizations as synapses, cell junctions, and othersorganizational structure and organizational plasticity/adaptability. The generalization of this reconceptualization in regard to other protein interaction modules and specific protein associations is consistent with the image of the cell as a complex adaptive macromolecular system as opposed to clockwork. http://www.ncbi.nlm.nih.gov/pubmed/17895993 Fri, 31 Aug 2007 00:00:00 -0700 The amyloid precursor protein (APP) is a type I transmembrane protein translocated to neuronal terminals, whose function is still unknown. The Cterminus of APP mediates its interaction with cellular adaptor and signaling proteins, some of which signal to the stressactivated protein kinase (SAPK) pathway. Here we show that ASK1, a MAPKKK that activates two SAPKs, cJun Nterminalkinase (JNK) and p38, is present in a complex containing APP, phosphoMKK6, JIP1 and JNK1. In primary neurons deprived of growth factors, as well as in brains of (FAD)APPtransgenic mice, ASK1 was upregulated in neuronal projections, where it interacted with APP. In nontransgenic brains, ASK1 and APP associated mainly in the ER. Our results indicate that recruitment of ASK1 to stresssignaling complexes assembled with APP may be triggered and enhanced by cellular stress. Thus, ASK1 may be the apical MAPKKK in a signaling complex assembled with APP as a response to stress. http://www.ncbi.nlm.nih.gov/pubmed/17719230 Fri, 31 Aug 2007 00:00:00 -0700 Programmed cell death (PCD) plays critical roles during development and in disease states. One form of programmed cell death utilizes autophagya cellular mechanism of degrading bulk cytosolic componentsto destroy cells. Previously, the broadspectrum caspase inhibitor zValAlaAsp(OMe)fluoromethylketone (zVAD) was shown to induce autophagic cell death. The mechanism of Zvadinduced cell death was proposed to require caspase8 inhibition. In our report, we extend these findings to show thatas is the case for apoptosisinduction of autophagic cell death in response to zVAD results in phosphatidylserine exposure prior to loss of membrane integrity. Additionally, we show that caspase8 inhibition is insufficient to cause autophagic cell death. Rather, the activity of a calpainlike protease must also be blocked. These results reveal the existence of an autophagic PCDinhibiting calpainlike cysteine protease. http://www.ncbi.nlm.nih.gov/pubmed/17404499 Tue, 31 Jul 2007 00:00:00 -0700 Programmed cell death signaling is a critical feature of development, cellular turnover, oncogenesis, and neurodegeneration, among other processes. Such signaling may be transduced via specific receptors, either following ligand bindingto death receptorsor following the withdrawal of trophic ligandsfrom dependence receptors. Although dependence receptors display functional similarities, no common structural domains have been identified. Therefore, we employed the Multiple Expectation Maximization for Motif Elicitation and the Motif Alignment and Search Tool software programs to identify a novel transmembrane motif, dubbed dependenceassociated receptor transmembrane (DART) motif, that is common to all described dependence receptors. Of 3,465 human transmembrane proteins, 25 (0.7) display the DART motif. The predicted secondary structure features an alpha helical structure, with an unusually high percentage of valine residues. At least four of the proteins undergo regulated intramembrane proteolysis. To date, we have not identified a function for this putative domain. We speculate that the DART motif may be involved in protein processing, interaction with other proteins or lipids, or homomultimerization. http://www.ncbi.nlm.nih.gov/pubmed/17520022 Mon, 30 Apr 2007 00:00:00 -0700 Despite the increasing number of published protein structures, and the fact that each protein's function relies on its threedimensional structure, there is limited access to automatic programs used for the identification of critical residues from the protein structure, compared with those based on protein sequence. Here we present a new algorithm based on network analysis applied exclusively on protein structures to identify critical residues. Our results show that this method identifies critical residues for protein function with high reliability and improves automatic sequencebased approaches and previous networkbased approaches. The reliability of the method depends on the conformational diversity screened for the protein of interest. We have designed a web site to give access to this software at http://bis.ifc.unam.mx/jamming/. In summary, a new method is presented that relates critical residues for protein function with the most traversed residues in networks derived from protein structures. A unique feature of the method is the inclusion of the conformational diversity of proteins in the prediction, thus reproducing a basic feature of the structure/function relationship of proteins. http://www.ncbi.nlm.nih.gov/pubmed/17502913 Mon, 30 Apr 2007 00:00:00 -0700 Cells exposed to sustained endoplasmic reticulum (ER) stress undergo programmed cell death and display features typical of apoptosis, such as cysteine aspartyl protease (caspase) activation, cytochrome c release, and DNA fragmentation. Here, we show that the execution of cell death induced by ER stress is mediated via the proteasome. Inhibition of the proteasome by lactacystin prevented ER stressinduced degradation of Bcl2, release of cytochrome c, processing of effector caspase3, and exposure of phosphatidylserine. Owing to the ability of lactacystin to inhibit cytochrome c release, we propose that the proapoptotic activity of the proteasome lies upstream of mitochondrial activation. Thus, the proteasome serves as a principal mediator of ER stressinduced cell death in this system. http://www.ncbi.nlm.nih.gov/pubmed/17396132 Mon, 30 Apr 2007 00:00:00 -0700 BACKGROUND AND PURPOSE: Programmed cell death (pcd) plays a critical role in the development of the nervous system, as well as in its response to insult. Both antipcd and propcd modulators play prominent roles in development and disease, including ischemic cerebrovascular disease. The purpose of this article is therefore to review the basics of programmed cell death. METHODS: There have been over 100 000 scientific and clinical publications on the topic of programmed cell death and its most well known form, apoptosis. The principles emerging from these studies are reviewed here. RESULTS: Programmed cell death is a form of cell death in which the cell plays an active role in its own demise. Apoptosis is the most welldefined form of pcd, but recent studies have begun to characterize an alternative program, autophagic cell death. In addition, there appear to be programmatic cell deaths that do not fit the criteria for either apoptosis or autophagic cell death, arguing that additional programs may also be available to cells. CONCLUSIONS: Constructing a mechanistic taxonomy of all forms of pcdbased on inhibitors, activators, and identified biochemical pathways involved in each form of pcdshould offer new insight into cell deaths associated with cerebrovascular disease and other diseases, and ultimately offer new therapeutic approaches. http://www.ncbi.nlm.nih.gov/pubmed/17261709 Sun, 31 Dec 2006 00:00:00 -0800 Targetassisted iterative screening (TAIS) has been applied to a random phagedisplayed peptide library in a search for novel ligands of the third baculovirus IAP ('inhibitors of apoptosis') repeat (BIR) domain of cIAP1. The peptides selected in the screen fall into two distinct specificity groups, one that conforms to a known IAPbinding motif (IBM) and another one that reveals a novel BIR domain interacting motif, NH(2)SR(V/P)W. The biochemical profiling of selected sequences with synthetic peptides, which included alanine scanning and N and Cterminal truncations as well as competition with the Smac peptide, suggests a major energetic contribution of tryptophan at the 4 position of peptide ligands to binding and identifies the latter together with the respective pocket on the BIR domain surface as a 'hot spot' of the interaction. A peptide featuring the novel motif selectively binds the fulllength cIAP1 protein in cell lysates. A 'twopocket' model of BIR domain recognition mechanism is proposed as the basis of differential BIR domain interactions with different IBMs. http://www.ncbi.nlm.nih.gov/pubmed/17094177 Sun, 31 Dec 2006 00:00:00 -0800 Synaptic dysfunction has been shown to be one of the earliest correlates of disease progression in animal models of Alzheimer's disease. Amyloidbeta protein (Abeta) is thought to play an important role in diseaserelated synaptic dysfunction, but the mechanism by which Abeta leads to synaptic dysfunction is not understood. Here we describe evidence that cleavage of APP in the C terminus may be necessary for the deficits present in APP transgenic mice. In APP transgenic mice with a mutated cleavage site at amino acid 664, normal synaptic transmission, synaptic plasticity, and learning were maintained despite the presence of elevated levels of APP, Abeta42, and even plaque accumulation. These results indicate that cleavage of APP may play a critical role in the development of synaptic and behavioral dysfunction in APP transgenic mice. http://www.ncbi.nlm.nih.gov/pubmed/17192425 Thu, 30 Nov 2006 00:00:00 -0800 Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease trigger neuronal cell death through endogenous suicide pathways. Surprisingly, although the cell death itself may occur relatively late in the course of the degenerative process, the mediators of the underlying celldeath pathways have shown promise as potential therapeutic targets. http://www.ncbi.nlm.nih.gov/pubmed/17051206 Sat, 30 Sep 2006 00:00:00 -0700 The intrinsic (mitochondriadependent) pathway of apoptosis is one of the major pathways leading to cell death. We evaluated cytochrome c/apoptotic protease activation factor1 (Apaf1)dependent activation of caspase3 in brain and liver of different strains of rodents at different stages of development. In cellfree extracts from brain and liver of SpragueDawley rats, caspase was activated by cytochrome c/2'deoxyadenosine 5'triphosphate at both neonatal and adult stages. In adult brain extracts from Wistar rats, no activation of caspase was observed while extracts from neonatal brain and liver and from adult liver were activated. In CD1 mouse, only neonatal extracts were activated. Alteration in levels of endogenous inhibitors of apoptosis were not responsible for the lack of activation observed. Instead, decrease in the content of Apaf1 and caspase3 and some degradation of caspase9 during brain ageing were observed. These results suggest that a decrease in apoptosis activation during ageing is not tissuespecific, but rather displays a complex dependence on species and strains of animals. http://www.ncbi.nlm.nih.gov/pubmed/16777102 Wed, 31 May 2006 00:00:00 -0700 Amyloid betapeptide (Abeta) is postulated to play a central role in the pathogenesis of Alzheimer's disease. We recently proposed a pathway of Abetainduced toxicity that is APP dependent and involves the facilitation of APP complex formation by Abeta. The APPdependent component requires cleavage of APP at position 664 in the cytoplasmic domain, presumably by caspases or caspaselike proteases, with release of a potentially cytotoxic C31 peptide. In this study we show that Abeta interacted directly and specifically with membranebound APP to facilitate APP homooligomerization. Using chimeric APP molecules, this interaction was shown to take place between Abeta and its homologous sequence on APP. Consistent with this finding, we demonstrated that Abeta also facilitated the oligomerization of betasecretase cleaved APP Cterminal fragment (C99). We found that the YENPTY domain in the APP cytoplasmic tail and contained within C31 is critical for this cell death pathway. Deletion or alanine scanning mutagenesis through this domain significantly attenuated cell death apparently without affecting either APP dimerization or cleavage at position 664. This indicated that sequences within C31 are required after its release from APP. As the YENPTY domain has been shown to interact with a number of cytosolic adaptor molecules, it is possible that the interaction of APP, especially dimeric forms of APP, with these molecules contribute to cell death. http://www.ncbi.nlm.nih.gov/pubmed/16636103 Wed, 31 May 2006 00:00:00 -0700 The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of betaamyloid peptides, a set of 3943 amino acid fragments derived proteolytically from betaamyloid precursor protein (APP). APP also is cleaved intracytoplasmically at Asp664 to generate a second cytotoxic peptide, APPC31, but whether this Cterminal processing of APP plays a role in the pathogenesis of AD is unknown. Therefore, we compared elements of the Alzheimer's phenotype in transgenic mice modeling AD with vs. without a functional Asp664 caspase cleavage site. Surprisingly, whereas betaamyloid production and plaque formation were unaltered, synaptic loss, astrogliosis, dentate gyral atrophy, increased neuronal precursor proliferation, and behavioral abnormalities were completely prevented by a mutation at Asp664. These results suggest that Asp664 plays a critical role in the generation of Alzheimerrelated pathophysiological and behavioral changes in human APP transgenic mice, possibly as a cleavage site or via proteinprotein interactions. http://www.ncbi.nlm.nih.gov/pubmed/16641106 Sun, 30 Apr 2006 00:00:00 -0700 The endoplasmic reticulum (ER) is the principal organelle for the biosynthesis of proteins, steroids and many lipids, and is highly sensitive to alterations in its environment. Perturbation of Ca(2) homeostasis, elevated secretory protein synthesis, deprivation of glucose or other sugars, altered glycosylation and/or the accumulation of misfolded proteins may all result in ER stress, and prolonged ER stress triggers cell death. Studies from multiple laboratories have identified the roles of several ER stressinduced celldeath modulators and effectors through the use of biochemical, pharmacological and genetic tools. In the present work, we describe the role of p23, a small chaperone protein, in preventing ER stressinduced cell death. p23 is a highly conserved chaperone protein that modulates HSP90 activity and is also a component of the steroid receptors. p23 is cleaved during ER stressinduced cell death this cleavage, which occurs close to the carboxyterminus, requires caspase3 and/or caspase7, but not caspase8. Blockage of the caspase cleavage site of p23 was associated with decreased cell death induced by ER stress. Immunodepletion of p23 or inhibition of p23 expression by siRNA resulted in enhancement of ER stressinduced cell death. While p23 coimmunoprecipitated with the BH3only protein PUMA (p53upregulated modulator of apoptosis) in untreated cells, prolonged ER stress disrupted this interaction. The results define a protective role for p23, and provide further support for a model in which ER stress is coupled to the mitochondrial intrinsic apoptotic pathway through the activities of BH3 family proteins. http://www.ncbi.nlm.nih.gov/pubmed/16195741 Tue, 31 Jan 2006 00:00:00 -0800 The intrinsic pathway of apoptosis was investigated in cellfree extracts of neurones and astrocytes at various stages of maturation. Neuronal extracts were activated 65fold after 3 days, 9fold after 7 days, and were not activated after 10 days in culture. In contrast, astrocyte extracts were activated to a similar extent at all stages, up to 60 days in culture. The coincubation of neuronal and astrocyte extracts followed by addition of cytochrome c/2'deoxyadenosine 5'triphosphate led to a 40fold activation, suggesting that the developmentassociated neuronal shift does not involve the appearance of a dominant inhibitor, but rather downregulation of some key component(s) involved in caspase activation. http://www.ncbi.nlm.nih.gov/pubmed/16242128 Mon, 31 Oct 2005 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/16264422 Mon, 31 Oct 2005 00:00:00 -0800 BACKGROUND: Phylogenetic approaches are commonly used to predict which amino acid residues are critical to the function of a given protein. However, such approaches display inherent limitations, such as the requirement for identification of multiple homologues of the protein under consideration. Therefore, complementary or alternative approaches for the prediction of critical residues would be desirable. Network analyses have been used in the modelling of many complex biological systems, but only very recently have they been used to predict critical residues from a protein's threedimensional structure. Here we compare a couple of phylogenetic approaches to several different networkbased methods for the prediction of critical residues, and show that a combination of one phylogenetic method and one networkbased method is superior to other methods previously employed. RESULTS: We associate a network with each member of a set of proteins for which the threedimensional structure is known and the critical residues have been previously determined experimentally. We show that several networkbased centrality measurements (connectivity, 2connectivity, closeness centrality, betweenness and cluster coefficient) accurately detect residues critical for the protein's function. Phylogenetic approaches render predictions as reliable as the networkbased measurements, although, interestingly, the two general approaches tend to predict different sets of critical residues. Hence we propose a hybrid method that is composed of one networkbased calculationthe closeness centralityand one phylogenetic approachthe Conseq server. This hybrid approach predicts critical residues more accurately than the other methods tested here. CONCLUSION: We show that network analysis can be used to improve the prediction of amino acids critical for protein function, when utilized in combination with phylogenetic approaches. It is proposed that such improvement is due to the complementary nature of these approaches: networkbased methods tend to predict as critical those residues that are highly connected and internal (i.e., nonsurface), although some surface residues are indeed identified as critical by network analyses whereas residues chosen by phylogenetic approaches display a lower overall probability of being surface inaccessible. http://www.ncbi.nlm.nih.gov/pubmed/16124876 Wed, 31 Aug 2005 00:00:00 -0700 Several approaches have been used in an effort to identify proteins that interact with betaamyloid precursor protein (APP). However, few studies have addressed the identification of proteins associated with APP in brain tissue from patients with Alzheimer's disease. We report the results of a pilot proteomic study performed on complexes immunoprecipitated with APP in brain samples of patients with Alzheimer's disease and normal control subjects. The 21 proteins identified could be grouped into five functional classes: molecular chaperones, cytoskeletal and structural proteins, proteins involved in trafficking, adaptors, and enzymes. Among the proteins identified, six had been reported previously as direct, indirect, or genetically inferred APP interactors. The other 15 proteins immunoprecipitated with APP were novel potential partners. We confirmed the APP interaction by Western blotting and coimmunolocalization in brain tissues, for 5 of the 21 interactors. In agreement with previous studies, our results are compatible with an involvement of APP in axonal transport and vesicular trafficking, and with a potential association of APP with cellular protein folding/protein degradation systems. http://www.ncbi.nlm.nih.gov/pubmed/16049941 Sun, 31 Jul 2005 00:00:00 -0700 Cells depend for their survival on stimulation by trophic factors and other prosurvival signals, the withdrawal of which induces apoptosis, both via the loss of antiapoptotic signaling and the activation of proapoptotic signaling via specific receptors. These receptors, dubbed dependence receptors, activate apoptotic pathways following the withdrawal of trophic factors and other supportive stimuli. Such receptors may feature in developmental cell death, carcinogenesis (including metastasis), neurodegeneration, and possibly subapoptotic events such as neurite retraction and somal atrophy. Mechanistic studies of dependence receptors suggest that these receptors form liganddependent complexes that include specific caspases. Complex formation in the absence of ligand leads to caspase activation by a mechanism that is typically dependent on caspase cleavage of the receptor itself, releasing proapoptotic peptides. Cellular dependence receptors, considered in the aggregate, may thus form a system of molecular integration, analogous to the electrical integration system provided by dendritic arbors in the nervous system. http://www.ncbi.nlm.nih.gov/pubmed/16015380 Thu, 30 Jun 2005 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/15665411 Fri, 31 Dec 2004 00:00:00 -0800 The two predominant pathological concomitants of Alzheimer's disease (AD) are senile plaques and neurofibrillary tangles. Although many biochemical studies have addressed the composition and formation of these AD hallmarks, very little is known about the interrelationship between the two. Here we present evidence that the tau phosphorylation characteristic of neurofibrillary tangles may be mediated by a physical association of MKK6 (mitogenassociated protein kinase kinase 6) with tau and subsequent phosphorylation of tau by the MKK6 substrate, p38 MAPK and that APP (betaamyloid precursor protein) may be coimmunoprecipitated both with MKK6 and its upstream MAPKKK, ASK1. Taken together with recent data demonstrating APP dimerization by betaamyloid peptide (Abeta) (Lu et al., 2003), and the possible activation of ASK1 via APP dimerization (Hashimoto et al., 2003), these results suggest a model of AD in which Abeta peptide dimerizes APP directly, leading to the activation of ASK1, MKK6, and p38, with subsequent phosphorylation of tau at sites characteristic of AD. http://www.ncbi.nlm.nih.gov/pubmed/15626821 Tue, 30 Nov 2004 00:00:00 -0800 The accumulation of misfolded proteins (e.g. mutant or damaged proteins) triggers cellular stress responses that protect cells against the toxic buildup of such proteins. However, prolonged stress due to the buildup of these toxic proteins induces specific death pathways. Dissecting these pathways should be valuable in understanding the pathogenesis of, and ultimately in designing therapy for, neurodegenerative diseases that feature misfolded proteins. http://www.ncbi.nlm.nih.gov/pubmed/15530777 Sun, 31 Oct 2004 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/15379849 Tue, 31 Aug 2004 00:00:00 -0700 Summary Aging and lifespan determination have been viewed, in the most wellaccepted theories, as nonprogrammatic, and are thought to result from the evolutionary selection for early fitness at the expense of late survival. Here, recent data implicating potentially programmatic aspects of aging and lifespan determination are discussed, and analogies between programmed cell death and programmed organismal death are offered. It is hoped that the recognition of at least the possibility of a programmatic aspect, or aspects, to the determination of longevity and the process of aging will help to optimize our chances to identify appropriate therapeutic targets both for longevity enhancement and disease prevention. http://www.ncbi.nlm.nih.gov/pubmed/15379848 Tue, 31 Aug 2004 00:00:00 -0700 The expression of the protein DCC (deleted in colorectal cancer) is lost or markedly reduced in numerous cancers and in the majority of colorectal cancers due to loss of heterozygosity in chromosome 18q, and has therefore been proposed to be a tumour suppressor. However, the rarity of mutations found in DCC, the lack of cancer predisposition of DCC mutant mice, and the presence of other tumour suppressor genes in 18q have raised doubts about the function of DCC as a tumour suppressor. Unlike classical tumour suppressors, DCC has been shown to induce apoptosis conditionally: by functioning as a dependence receptor, DCC induces apoptosis unless DCC is engaged by its ligand, netrin1 (ref. 3). Here we show that inhibition of cell death by enforced expression of netrin1 in mouse gastrointestinal tract leads to the spontaneous formation of hyperplastic and neoplastic lesions. Moreover, in the adenomatous polyposis coli mutant background associated with adenoma formation, enforced expression of netrin1 engenders aggressive adenocarcinomatous malignancies. These data demonstrate that netrin1 can promote intestinal tumour development, probably by regulating cell survival. Thus, a netrin1 receptor or receptors function as conditional tumour suppressors. http://www.ncbi.nlm.nih.gov/pubmed/15343335 Tue, 31 Aug 2004 00:00:00 -0700 Neurogenesis continues in the adult brain and is increased in certain pathological states. We reported recently that neurogenesis is enhanced in hippocampus of patients with Alzheimer's disease (AD). We now report that the effect of AD on neurogenesis can be reproduced in a transgenic mouse model. PDGFAPP(Sw,Ind) mice, which express the Swedish and Indiana amyloid precursor protein mutations, show increased incorporation of BrdUrd and expression of immature neuronal markers in two neuroproliferative regions: the dentate gyrus and subventricular zone. These changes, consisting of approximately 2fold increases in the number of BrdUrdlabeled cells, were observed at age 3 months, when neuronal loss and amyloid deposition are not detected. Because enhanced neurogenesis occurs in both AD and an animal model of AD, it seems to be caused by the disease itself and not by confounding clinical factors. As neurogenesis is increased in PDGFAPP(Sw,Ind) mice in the absence of neuronal loss, it must be triggered by more subtle disease manifestations, such as impaired neurotransmission. Enhanced neurogenesis in AD and animal models of AD suggests that neurogenesis may be a compensatory response and that measures to enhance neurogenesis further could have therapeutic potential. http://www.ncbi.nlm.nih.gov/pubmed/15340159 Tue, 31 Aug 2004 00:00:00 -0700 Programmed cell death (pcd) may take the form of apoptotic or nonapoptotic pcd. Whereas cysteine aspartylspecific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here, we report that paraptosis, an alternative, nonapoptotic cell death program that may be induced by the insulinlike growth factor I receptor (among other inducers), is mediated by mitogenactivated protein kinases (MAPKs) and inhibited by AIP1/Alix. The inhibition by AIP1/Alix is specific for paraptosis since apoptosis was not inhibited. Caspases were not activated in this paradigm, nor were caspase inhibitors effective in blocking cell death. However, insulinlike growth factor I receptor (IGFIR)induced paraptosis was inhibited by MEK2specific inhibitors and by antisense oligonucleotides directed against cjun Nterminal kinase1 (JNK1). These results suggest that IGFIRinduced paraptosis is mediated by MAPKs, and inhibited by AIP1/Alix. http://www.ncbi.nlm.nih.gov/pubmed/15195070 Tue, 31 Aug 2004 00:00:00 -0700 One of the most important pathological features of Alzheimer's disease (AD) is extracellular senile plaques, whose major component is amyloidbeta peptides (Abeta). Abeta binds to the extracellular domain of p75NTR (p75 neurotrophin receptor) and induces neuronal cell death. We investigated the molecular mechanism of Abetainduced neurotoxicity in detail from the standpoint of interaction between p75NTR and its recently identified relative, PLAIDD (p75like apoptosisinducing death domain). Using F11 neuronal hybrid cells, we demonstrate that there are two distinct pathways for Abetainduced toxicity mediated by p75NTR. One pathway that has been previously elucidated, is mediated by p75NTR, Go, JNK, NADPH oxidase and caspase3related caspases. We found that PLAIDD and Gi proteins, heterotrimeric G proteins, are involved in the alternative Abetainduced neurotoxicity mediated by p75NTR. The alternative pathway triggered by Abeta is thus mediated by p75NTR, PLAIDD, Gi, JNK, NADPH oxidase and caspase3related caspases. In addition, we found that HN, ADNF, IGFI, or bFGF inhibits both pathways of Abetainduced neurotoxicity mediated by p75NTR. http://www.ncbi.nlm.nih.gov/pubmed/15255932 Wed, 30 Jun 2004 00:00:00 -0700 Programmed cell death (pcd) may take the form of apoptosis or of nonapoptotic pcd. Whereas cysteine aspartylspecific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here we report that alternative, nonapoptotic pcd induced by the neurokinin1 receptor (NK(1)R) activated by its ligand Substance P, is mediated by a MAPK phosphorylation cascade recruited by the scaffold protein arrestin 2. The activation of the protein kinases Raf1, MEK2, and ERK2 is essential for this form of nonapoptotic pcd, leading to the phosphorylation of the orphan nuclear receptor Nur77. NK(1)Rmediated cell death was inhibited by a dominant negative form of arrestin 2, Raf1, or Nur77, by MEK1/2specific inhibitors, and by RNA interference directed against ERK2 or MEK2 but not ERK1 or MEK1 and against Nur77. The MAPK pathway is also activated in neurons in primary culture undergoing NK(1)Rmediated death, since the MEK inhibitor PD98059 inhibited Substance Pinduced death in primary striatal neurons. These results suggest that Nur77, which is regulated by a MAPK pathway activated via arrestin 2, modulates NK(1)Rmediated nonapoptotic pcd. http://www.ncbi.nlm.nih.gov/pubmed/14769794 Wed, 31 Mar 2004 00:00:00 -0800 Classical signal transduction is initiated by ligandreceptor interactions. We have described an alternative form of signal transduction that is initiated by the withdrawal of ligands from specific receptors referred to as dependence receptors. This process is widespread, featuring in developmental cell death, carcinogenesis (especially metastasis), neurodegeneration, and possibly subapoptotic events such as neurite retraction and somal atrophy. Initial mechanistic studies of dependence receptors suggest that these receptors form complexes that include specific caspases. Complex formation appears to be a function of ligandreceptor interaction, and dependence receptors appear to exist in at least two conformational states. Complex formation in the absence of ligand leads to caspase activation by a mechanism that in at least some cases is dependent on caspase cleavage of the receptor itself, releasing proapoptotic peptides. Thus these receptors may serve in caspase amplification, and in so doing create cellular states of dependence on their respective ligands. http://www.ncbi.nlm.nih.gov/pubmed/15044679 Sun, 29 Feb 2004 00:00:00 -0800 The novel screening format, targetassisted iterative screening (TAIS), comprises a simple and rapid twostep procedure for in vitro affinity selection of specific binders from enormous molecular diversities to the target molecule of interest. This detailed protocol describes the application of TAIS to a T7 phagedisplayed complementary DNA (cDNA) library with a protein domain as a target. Protocols for purification of the target as glutathioneStransferase (GST) fusion protein and modifications of the purified target that are required for the screening complement the TAIS protocol. The described application is a method of choice for the researchers interested in the identification and characterization of novel proteinprotein interactions mediated by peptide recognition domains. http://www.ncbi.nlm.nih.gov/pubmed/15020779 Sun, 29 Feb 2004 00:00:00 -0800 The endoplasmic reticulum (ER) regulates protein synthesis, protein folding and trafficking, cellular responses to stress and intracellular calcium (Ca(2)) levels. Alterations in Ca(2) homeostasis and accumulation of misfolded proteins in the ER cause ER stress that ultimately leads to apoptosis. Prolonged ER stress is linked to the pathogenesis of several different neurodegenerative disorders. Apoptosis is a form of cell death that involves the concerted action of a number of intracellular signaling pathways including members of the caspase family of cysteine proteases. The two main apoptotic pathways, the death receptor ('extrinsic') and mitochondrial ('intrinsic') pathways, are activated by caspase8 and 9, respectively, both of which are found in the cytoplasm. Recent studies point to the ER as a third subcellular compartment implicated in apoptotic execution. Here, we review evidence for the contribution of various cellular molecules that contribute to ER stress and subsequent cellular death. It is hoped that dissection of the molecular components and pathways that alter ER structure and function and ultimately promote cellular death will provide a framework for understanding degenerative disorders that feature misfolded proteins. http://www.ncbi.nlm.nih.gov/pubmed/14765132 Sun, 29 Feb 2004 00:00:00 -0800 Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of fulllength mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase2 into an apoptosomelike complex. Binding of caspase2 to Htt is polyglutamine repeatlength dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing fulllength Htt (YAC72). Expression of catalytically inactive (dominantnegative) forms of caspase2, caspase7, and to some extent caspase6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase3, 8, and 9 did not. Histological analysis of postmortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase2 correlates directly with decreased levels of brainderived neurotrophic factor in the cortex and striatum of 3month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase2 in the selective neuronal cell death associated with HD in the striatum and cortex. http://www.ncbi.nlm.nih.gov/pubmed/14713958 Sun, 29 Feb 2004 00:00:00 -0800 We report here that the checkpoint kinase Chk1 and the inhibitor of apoptosis protein (IAP) family member XIAP can be found in a complex in association with condensed chromosomes aligned at the metaphase plate during mitosis. The interaction between Chk1 and XIAP was transient and followed the breakdown of the nuclear envelope. Chk1 and XIAP also formed a complex in vitro and in coimmunoprecipitation experiments. The interaction between Chk1 and the BIR3 domain of XIAP in vitro required an Nterminal sequence in Chk1 that is identical to the BIRbinding motif at the Nterminus of HID. An interaction of Chk1 and XIAP may imply a mechanism of coupling between the regulatory networks that control cell cycle progression and apoptosis during mitosis. http://www.ncbi.nlm.nih.gov/pubmed/14759516 Sat, 31 Jan 2004 00:00:00 -0800 In many of autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with SOD1 mutation, a missense point mutation may induce the disease by its gain of adverse property. Reduction of such a mutant protein expression is expected to improve the disease phenotype. Duplex of 21nt RNA, known as siRNA, has recently emerged as a powerful tool to silence gene, but the sequence specificity and efficacies have not been fully studied in comparison with ribozyme and DNA enzyme. We could make the siRNA which recognized even a single nucleotide alternation and selectively suppress G93A SOD1 expression leaving wildtype SOD1 intact. In mammalian cells, the siRNA much more efficiently suppressed the expression of mutant SOD1 than ribozyme or DNA enzyme. Furthermore, these siRNAs could suppress cell death of Neuro2a induced by overexpression of mutant SOD1s with stress of proteasome inhibition. Our results support the feasibility of utilizing siRNAbased gene therapy of familial ALS with mutant SOD1. http://www.ncbi.nlm.nih.gov/pubmed/14715277 Wed, 31 Dec 2003 00:00:00 -0800 A new family of functionallyrelated receptors has recently been proposed, dubbed dependence receptors. These proteins, only some of which share sequence similarities, display the common property that they transduce two different intracellular signals: in the presence of ligand, these receptors transduce a positive signal leading to survival, differentiation or migration conversely, in the absence of ligand, the receptors initiate or amplify a signal for programmed cell death. Thus cells that express these proteins at sufficient concentrations manifest a state of dependence on their respective ligands. The signaling that mediates cell death induction upon ligand withdrawal is in large part uncharacterized, but typically includes a required interaction with, and cleavage by, specific caspases. Here, we review the current knowledge concerning dependence receptors, including the shared mechanisms for cell death induction and their potential relevance in nervous system development and regulation of tumorigenesis. http://www.ncbi.nlm.nih.gov/pubmed/14739597 Wed, 31 Dec 2003 00:00:00 -0800 The amyloidbeta protein precursor, a type 1 transmembrane protein, gives rise to the amyloid betaprotein, a neurotoxic peptide postulated to be involved in the pathogenesis of Alzheimer's disease. Here, we show that soluble amyloid beta protein accelerates amyloid precursor protein complex formation, a process that contributes to neuronal cell death. The mechanism of cell death involves the recruitment of caspase8 to the complex, followed by intracytoplasmic caspase cleavage of amyloid precursor protein. In vivo, the levels of soluble amyloid beta protein correlated with caspasecleaved fragments of the amyloid precursor protein in brains of Alzheimer's disease subjects. These findings suggest that soluble amyloid beta proteininduced multimerization of the amyloid precursor protein may be another mechanism by which amyloid beta protein contributes to synapse loss and neuronal cell death seen in Alzheimer's disease. http://www.ncbi.nlm.nih.gov/pubmed/14681887 Sun, 30 Nov 2003 00:00:00 -0800 Programmed cell death occurs in response to both the presence of various extracellular factors and the lack of specific factors. Receptors that can mediate cell death in the absence of ligand binding are called dependence receptors, and they were the topic of the meeting held during the summer of 2003 in Fondation des Treilles, France. Not only is progress being made in the identification of new dependence receptors, but the partners that carry out this "negative" signal are also coming to light. With several of the receptors implicated in various human developmental disorders or disease states, gaining an understanding of the molecular mechanisms controlling dependence receptormediated cell death has clear clinical relevance. http://www.ncbi.nlm.nih.gov/pubmed/14679288 Sun, 30 Nov 2003 00:00:00 -0800 Alterations in Ca2 homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) cause ER stress that ultimately leads to programmed cell death. Recent studies have shown that ER stress triggers programmed cell death via an alternative intrinsic pathway of apoptosis that, unlike the intrinsic pathway described previously, is independent of Apaf1 and cytochrome c. In the present work, we have used a set of complementary approaches, including twodimensional gel electrophoresis coupled with matrixassisted laser desorption ionizationtimeofflight mass spectrometry and nanoliquid chromatographyelectrospray ionization mass spectrometry with tandem mass spectrometry, RNA interference, coimmunoprecipitation, immunodepletion of candidate proteins, and reconstitution studies, to identify mediators of the ER stressinduced cell death pathway. Our data identify two molecules, valosincontaining protein and apoptosislinked gene2 (ALG2), that appear to play a role in mediating ER stressinduced cell death. http://www.ncbi.nlm.nih.gov/pubmed/14561754 Sun, 30 Nov 2003 00:00:00 -0800 The amyloid betaprotein precursor (APP) is proteolytically cleaved to generate the amyloid betaprotein (Abeta), the principal constituent of senile plaques found in Alzheimer's disease (AD). In addition, Abeta in its oligomeric and fibrillar forms have been hypothesized to induce neuronal toxicity. We and others have previously shown that APP can be cleaved by caspases at the Cterminus to generate a potentially cytotoxic peptide termed C31. Furthermore, this cleavage event and caspase activation were increased in the brains of AD, but not control, cases. In this study, we show that in cultured cells, Abeta induces caspase cleavage of APP in the Cterminus and that the subsequent generation of C31 contributes to the apoptotic cell death associated with Abeta. Interestingly, both Abeta toxicity and C31 pathway are dependent on the presence of APP. Both APPdependent Abeta toxicity and C31induced apoptotic cell death involve apical or initiator caspases8 and 9. Our results suggest that Abetamediated toxicity initiates a cascade of events that includes caspase activation and APP cleavage. These findings link C31 generation and its potential cell death activity to Abeta cytotoxicity, the leading mechanism proposed for neuronal death in AD. http://www.ncbi.nlm.nih.gov/pubmed/14535955 Tue, 30 Sep 2003 00:00:00 -0700 Accumulation of amyloid beta peptides (Abeta) in the brain, which is a hallmark of Alzheimer's disease (AD), is associated with progressive damage to neuronal processes resulting in extensive neuritic dystrophy. This process may contribute to cognitive decline, but it is not known how Abeta elicits neuritic injury. Our analysis of AD brains and related transgenic mouse models suggests an involvement of the interferoninduced serinethreonine protein kinase, PKR, which is best known for its activation upon binding to doublestranded RNA. PKR activation is a component of stressactivated pathways that mobilize somatic cell death programs, but its roles in neurological disease largely remain to be defined. An antibody specific to the activated form of PKR (phosphorylated at T451) was used to determine the pattern of PKR activation in postmortem brain tissues from humans or from transgenic mice that express high levels of familial ADmutant human amyloid precursor protein (hAPP) and hAPPderived Abeta in neurons. In contrast to nondemented controls, AD cases showed prominent granular phosphoPKR immunoreactivity in association with neuritic plaques and pyramidal neurons in the hippocampus and neocortex. The distribution of phosphoPKR matched the distributions of abnormally phosphorylated tau and active p38 MAP kinase in adjacent sections. Compared with nontransgenic controls, hAPP transgenic mice also showed strong increases in phosphoPKR in the brain, primarily in association with plaques and dystrophic neurites. These findings support a role for PKR activation in the pathogenesis of AD. http://www.ncbi.nlm.nih.gov/pubmed/13678666 Sun, 31 Aug 2003 00:00:00 -0700 Targetassisted iterative screening applied to random peptide libraries unveiled a novel and atypical recognition consensus shared by CIN85/SETA/Ruk SH3 domains, PX(P/A)XXR. Confirmed by mutagenesis and in vitro binding experiments, the novel consensus allowed for the accurate mapping of CIN85 SH3 binding sites within known CIN85 interactors, cCbl, BLNK, Cblb, AIP1/Alix, SB1, and CD2 proteins, as well as the prediction of CIN85 novelinteracting partners in protein databases. Synaptojanin 1, PAK2, ZO2, and TAFII70, which contain CIN85 SH3 recognition consensus sites, were selectively precipitated from mouse brain lysates by CIN85 SH3 domains in glutathione Stransferase pulldown experiments. A direct interaction of synaptojanin 1 and PAK2 with CIN85 SH3 domains was confirmed by Far Western blotting. http://www.ncbi.nlm.nih.gov/pubmed/12829691 Sun, 31 Aug 2003 00:00:00 -0700 Protein engineering is an emerging area that has expanded our understanding of protein folding and laid the groundwork for the creation of unprecedented structures with unique functions. We previously designed the first nativelike poreforming protein, small globular protein (SGP). We show here that this artificially engineered protein has membranedisrupting properties and antitumor activity in several cancer animal models. We propose and validate a mechanism for the selectivity of SGP toward cell membranes in tumors. SGP is the prototype for a new class of artificial proteins designed for therapeutic applications. http://www.ncbi.nlm.nih.gov/pubmed/12750379 Sun, 31 Aug 2003 00:00:00 -0700 The common neurotrophin receptor p75(NTR) remains one of the most enigmatic of the tumor necrosis factor receptor (TNFR) superfamily: on the one hand, it displays a death domain and has been shown to be capable of mediating programmed cell death (PCD) upon ligand binding on the other hand, its death domain is of type II (unlike that of Fas or TNFR I), and it has also been shown to be capable of mediating cell death in response to the withdrawal of ligand. Thus, p75(NTR) may function as a death receptorsimilar to Fas or TNFR Ior a dependence receptorsimilar to deleted in colorectal cancer (DCC) or uncoordinated gene5 homologues 13 (UNC5H13). Here, we review the data relating to the mediation of PCD by p75(NTR), and suggest that one reasonable model for the apparently paradoxical effects of p75(NTR) is that this receptor functions as a "quality control" in that it is capable of mediating PCD in at least four situations: (1). withdrawal of neurotrophins (2). exposure to mismatched neurotrophins (3). exposure to unprocessed neurotrophins and (4). exposure of inappropriately immature cells to neurotrophins. Results to date suggest that these functions are mediated through different underlying mechanisms, and that their respective signaling pathways are cell type and coreceptor dependent. http://www.ncbi.nlm.nih.gov/pubmed/12787561 Sat, 31 May 2003 00:00:00 -0700 The type 1 insulinlike growth factor receptor (IGFIR) is a receptortyrosine kinase that plays a critical role in signaling cell survival and proliferation. IGFIR binding to its ligand, insulinlike growth factor (IGFI) activates phosphoinositide 3kinase (PI3K), promotes cell proliferation by activating the mitogenactivated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins such as BAD. Apoptosis signalregulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that is required for cJun Nterminal kinase (JNK) and p38 activation in response to Fas and tumor necrosis factor (TNF) receptor stimulation, and for oxidative stress and TNFalphainduced apoptosis. The results presented here indicate that ASK1 forms a complex with the IGFIR and becomes phosphorylated on tyrosine residue(s) in a manner dependent on IGFIR activity. IGFIR signaling inhibited ASK1 irrespective of TNFalphainduced ASK1 activation and resulted in decreased ASK1dependent JNK1 stimulation. Signaling through IGFIR rescued cells from ASK1induced apoptotic cell death in a manner independent of PI3K activity. These results indicate that IGFIR signaling suppresses the ASK1mediated stimulation of JNK/p38 and the induction of programmed cell death. The simultaneous activation of MAP kinases and the inhibition of the stressactivated arm of the cascade by IGFIR may constitute a potent proliferative signaling system and is possibly a mechanism by which IGFI can stimulate growth and inhibit cell death in a wide variety of cell types and biological settings. http://www.ncbi.nlm.nih.gov/pubmed/12556535 Mon, 31 Mar 2003 00:00:00 -0800 Huntington's disease (HD) results from polyglutamine expansion in huntingtin (htt), a protein with several consensus caspase cleavage sites. Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo. Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration. Using antibodies that detect only caspasecleaved htt, we demonstrate that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552. We detect caspasecleaved htt in control human brain as well as in HD brains with early grade neuropathology, including one homozygote. Cleaved htt is also seen in wildtype and HD transgenic mouse brains before the onset of neurodegeneration. These results suggest that caspase cleavage of htt may be a normal physiological event. However, in HD, cleavage of mutant htt would release Nterminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract. Furthermore, htt fragments were detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD. http://www.ncbi.nlm.nih.gov/pubmed/12223539 Sat, 31 Aug 2002 00:00:00 -0700 Several receptors that mediate apoptosis have been identified, such as Fas and tumor necrosis factor receptor I. Studies of the signal transduction pathways utilized by these receptors have played an important role in the understanding of apoptosis. Here we report the first ligandreceptor pairthe neuropeptide substance P and its receptor, neurokinin1 receptor (NK(1)R)that mediates an alternative, nonapoptotic form of programmed cell death. This pair is widely distributed in the central and peripheral nervous systems, and has been implicated in pain mediation and depression, among other effects. Here we demonstrate that substance P induces a nonapoptotic form of programmed cell death in hippocampal, striatal, and cortical neurons. This cell death requires gene expression, displays a nonapoptotic morphology, and is independent of caspase activation. The same form of cell death is induced by substance P in NK(1)Rtransfected human embryonic kidney cells. These results argue that NK(1)R activates a death pathway different than apoptosis, and provide a signal transduction system by which to study an alternative, nonapoptotic cell death program. http://www.ncbi.nlm.nih.gov/pubmed/12107824 Wed, 31 Jul 2002 00:00:00 -0700 The synapse loss and neuronal cell death characteristic of Alzheimer's disease (AD) are believed to result in large part from the neurotoxic effects of betaamyloid peptide (Abeta), a 4042 amino acid peptide(s) derived proteolytically from betaamyloid precursor protein (APP). However, APP is also cleaved intracellularly to generate a second cytotoxic peptide, C31, and this cleavage event occurs in vivo as well as in vitro and preferentially in the brains of AD patients (Lu et al. 2000). Here we show that APPC31 is toxic to neurons in primary culture, and that like APP, the APP family members APLP1 and possibly APLP2 are cleaved by caspases at their Ctermini. The carboxyterminal peptide derived from caspase cleavage of APLP1 shows a degree of neurotoxicity comparable to APPC31. Our results suggest that even though APLP1 and APLP2 cannot generate Abeta, they may potentially contribute to the pathology of AD by generating peptide fragments whose toxicity is comparable to that of APPC31. http://www.ncbi.nlm.nih.gov/pubmed/12124429 Sun, 30 Jun 2002 00:00:00 -0700 We describe the cloning and characterization of a rat single transmembrane protein that is homologous to the common neurotrophin receptor p75NTR in its death domain and the transmembrane region but dissimilar outside these regions. We have dubbed this protein PLAIDD, for p75like apoptosisinducing death domain protein. PLAIDD messenger RNA, which is ubiquitously distributed, is highly expressed in the embryo, but downregulated in adult tissues. Alternative splicing within the extracellular region of PLAIDD generates four RNA species, but only two of them are translated, PLAIDDL and PLAIDDS (long and short isoforms, respectively). While the amino acid sequence of the intracellular region of PLAIDD displays 41 identity with the intracellular region of p75NTR, the extracellular region of PLAIDD does not reveal any homology with p75NTR. Overexpression of each isoform of PLAIDD led to cytotoxicity in superior cervical ganglion neurons and in human embryonic kidney 293T cells. Both isoforms of PLAIDD could be coimmunoprecipitated with p75NTR, suggesting an interaction between these molecules. http://www.ncbi.nlm.nih.gov/pubmed/12095158 Sun, 30 Jun 2002 00:00:00 -0700 Accumulation of misfolded proteins and alterations in Ca2 homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signaltransducing events that connect ER stress to cell death pathways are incompletely understood. To discern the pathway by which ER stressinduced cell death proceeds, we performed studies on Apaf1(/) (null) fibroblasts that are known to be relatively resistant to apoptotic insults that induce the intrinsic apoptotic pathway. While these cells were resistant to cell death initiated by proapoptotic stimuli such as tamoxifen, they were susceptible to apoptosis induced by thapsigargin and brefeldinA, both of which induce ER stress. This pathway was inhibited by catalytic mutants of caspase12 and caspase9 and by a peptide inhibitor of caspase9 but not by caspase8 inhibitors. Cleavage of caspases and poly(ADPribose) polymerase was observed in cellfree extracts lacking cytochrome c that were isolated from thapsigargin or brefeldintreated cells. To define the molecular requirements for this Apaf1 and cytochrome cindependent apoptosis pathway further, we developed a cellfree system of ER stressinduced apoptosis the addition of microsomes prepared from ER stressinduced cells to a normal cell extract lacking mitochondria or cytochrome c resulted in processing of caspases. Immunodepletion experiments suggested that caspase12 was one of the microsomal components required to activate downstream caspases. Thus, ER stressinduced programmed cell death defines a novel, mitochondrial and Apaf1independent, intrinsic apoptotic pathway. http://www.ncbi.nlm.nih.gov/pubmed/11919205 Fri, 31 May 2002 00:00:00 -0700 A new in vitro screening method has been developed and applied to a commercial phagedisplayed cDNA library to search for novel proteinprotein interactions. PDZ, WW and SH3 domains from PSD95, Nedd4, Src, Abl and Crk proteins were used as targets. 12 novel putative and 2 previously reported interactions were identified in test screens. The novel screening format, dubbed TAIS (targetassisted iterative screening), is discussed as an alternative platform to existing technologies for a pairwise characterization of proteinprotein interactions. http://www.ncbi.nlm.nih.gov/pubmed/12023804 Tue, 30 Apr 2002 00:00:00 -0700 Alterations in Ca(2) homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucoseregulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stressinduced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome cmediated caspase activation in a cellfree system, and expression of GRP78 blocks both caspase activation and caspasemediated cell death. GRP78 forms a complex with caspase7 and 12 and prevents release of caspase12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase12 into the cytoplasm to set in motion the cytosolic component of the ER stressinduced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stressinduced cell death. http://www.ncbi.nlm.nih.gov/pubmed/11943137 Sun, 31 Mar 2002 00:00:00 -0800 Organ specific drug targeting was explored in mice as a possible alternative to surgery to treat prostate diseases. Peptides that specifically recognize the vasculature in the prostate were identified from phagedisplayed peptide libraries by selecting for phage capable of homing into the prostate after an i.v. injection. One of the phage selected in this manner homed to the prostate 1015 times more than to other organs. Unselected phage did not show this preference. The phage bound also to vasculature in the human prostate. The peptide displayed by the prostatehoming phage, SMSIARL (single letter code), was synthesized and shown to inhibit the homing of the phage when coinjected into mice with the phage. Systemic treatment of mice with a chimeric peptide consisting of the SMSIARL homing peptide, linked to a proapoptotic peptide that disrupts mitochondrial membranes, caused tissue destruction in the prostate, but not in other organs. The chimeric peptide delayed the development of the cancers in prostate cancerprone transgenic mice (TRAMP mice). These results suggest that it may be possible to develop an alternative to surgical prostate resection and that such a treatment may also reduce future cancer risk. http://www.ncbi.nlm.nih.gov/pubmed/11830668 Thu, 31 Jan 2002 00:00:00 -0800 The recent demonstration that biochemical pathways from diverse organisms are arranged in scalefree, rather than random, systems Jeong et al., Nature 407 (2000) 651654, emphasizes the importance of developing methods for the identification of biochemical nexusesthe nodes within biochemical pathways that serve as the major input/output hubs, and therefore represent potentially important targets for modulation. Here we describe a bioinformatics approach that identifies candidate nexuses for biochemical pathways without requiring functional gene annotation we also provide proofofprinciple experiments to support this technique. This approach, called Nexxus, may lead to the identification of new signal transduction pathways and targets for drug design. http://www.ncbi.nlm.nih.gov/pubmed/11741594 Fri, 30 Nov 2001 00:00:00 -0800 Ligand binding to tumor necrosis factor receptorI (TNFRI) can promote cell survival or activate the apoptotic caspase cascade. Cytoplasmic interaction of TNFRI with TRAF2 and RIP allows for the activation of JNK and NFkappaB pathways. Alternatively, a carboxy terminal death domain protein interaction motif can recruit TRADD, which then recruits FADD/MORT1, and finally procaspase 8. Aggregation of these components form a death inducing signaling complex, leading to the cleavage and activation of caspase 8. We have found that during apoptosis human TNFRI protein is lost in a caspasedependent manner. The cytoplasmic tail of human TNFRI was found to be susceptible to caspase cleavage but not by caspase 8. Instead, the downstream executioner caspase 7 was the only caspase capable of cleaving TNFRI, in vitro. Identification and characterization of the cleavage site revealed a derivative of the classic EXD motif that incorporates a glutamate (E) in the P1 position. Using several criteria to establish that caspase activity was responsible for cleavage at this site, we confirmed that caspase 7 can cleave at a GELE motif. Mutation of the cleavage site prevented the apoptosisassociated cleavage of TNFRI. This ability of caspase 7 to cleave at a nonEXD or DXXD motif suggests that the specificity of caspases may be broader than is currently held. http://www.ncbi.nlm.nih.gov/pubmed/11755217 Fri, 30 Nov 2001 00:00:00 -0800 Bcl2 is a gene family involved in the suppression of apoptosis in response to a wide range of cellular insults. Multiple papers have suggested a link between Bcl2 and oxidative damage/antioxidant protection. We therefore examined parameters of antioxidant defense and oxidative damage in two different cell lines, NT2/D1 (NT2) and SKNMC, overexpressing Bcl2 as compared with vectoronly controls. Bcl2 transfectants of both cell lines were more resistant to H(2)O(2) and showed increases in GSH level and Cu/Znsuperoxide dismutase (SOD1) activity, but not in Mnsuperoxide dismutase, glutathione peroxidase, or glutathione reductase activities. Catalase activity was increased in SKNMC cells. Overexpression of Bcl2 did not significantly decrease levels of oxidative DNA damage (measured as 8hydroxyguanine) or lipid peroxidation, but it decreased levels of 3nitrotyrosine in both cell lines and protein carbonyls in SKNMC cells only. It also increased proteasome activity in both cell lines. We conclude that Bcl2 raises cellular antioxidant defense status, but this is not necessarily reflected in decreased levels of oxidative damage to DNA and lipids. The ability of Bcl2 overexpression to decrease 3nitrotyrosine levels suggests that it may decrease formation of peroxynitrite or other reactive nitrogen species this was confirmed as decreased production of NO(2)()/NO(3)() in the transfected cells and a fall in the level of nNOS protein. http://www.ncbi.nlm.nih.gov/pubmed/11744329 Fri, 30 Nov 2001 00:00:00 -0800 Cellular organelles, such as the Golgi apparatus and the endoplasmic reticulum, adopt characteristic structures depending on their function. While the tubular shapes of these structures result from complex proteinlipid interactions that are not fully understood, some fundamental machinery must be required. We show here that a de novodesigned 18mer amphipathic alphahelical peptide, Hel 135, transforms spherical liposomes made from a Golgispecific phospholipid mixture into nanotubules on the scale of and resembling the shape of the nanotubules that form the Golgi apparatus. Furthermore, we show that that the size and the shape of such nanotubules depend on lipid composition and peptide properties such as length and the ratio of hydrophobic to hydrophilic amino acids. Although the question of precisely how nature engineers organellar membranes remains unknown, our simple novel system provides a basic set of tools to begin addressing this question. http://www.ncbi.nlm.nih.gov/pubmed/11406635 Wed, 31 Oct 2001 00:00:00 -0800 The mechanisms underlying disruption of physiologic celldeath programs are slowly being revealed. Information gleaned from genetic profiles soon may allow physicians to delay onset of many silent, agerelated diseases. While development of actual cures could prove elusive, simply postponing clinical onset would have an impressive effect on the goal of healthy aging. http://www.ncbi.nlm.nih.gov/pubmed/11565741 Fri, 31 Aug 2001 00:00:00 -0700 The endoplasmic reticulum (ER) is the site of assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. It also regulates cellular responses to stress and intracellular Ca(2) levels. A variety of toxic insults can result in ER stress that ultimately leads to apoptosis. Apoptosis is initiated by the activation of members of the caspase family and serves as a central mechanism in the cell death process. The present study was carried out to determine the role of caspases in triggering ER stressinduced cell death. Treatment of cells with ER stress inducers such as brefeldinA or thapsigargin induces the expression of caspase12 protein and also leads to translocation of cytosolic caspase7 to the ER surface. Caspase12, like most other members of the caspase family, requires cleavage of the prodomain to activate its proapoptotic form. Caspase7 associates with caspase12 and cleaves the prodomain to generate active caspase12, resulting in increased cell death. We propose that any cellular insult that causes prolonged ER stress may induce apoptosis through caspase7mediated caspase12 activation. The data underscore the involvement of ER and caspases associated with it in the ER stressinduced apoptotic process. http://www.ncbi.nlm.nih.gov/pubmed/11448953 Fri, 31 Aug 2001 00:00:00 -0700 The cell surface molecules controlling apoptosis in cortical neurons are largely unknown. A monoclonal antibody was derived that induces cultured neocortical neurons to undergo apoptosis. A Fab fragment of the antibody, however, lacked the ability to induce cell death. The antigen was purified, and characterized by compositional analysis, fast atom bombardment (FAB) mass spectrometry, sequential exoglycosidase treatments, methylation analysis, and (1)Hnuclear magnetic resonance spectroscopy, proving to be isoglobotetraosylceramide (IsoGb4). IsoGb4 has been shown previously to be a metastasis marker, antibodies against which block metastases in a mammary adenocarcinoma model (S. A. Carlsen et al., Cancer Res., 53: 29062911, 1993). Addition of the purified antigen to cells lacking this glycolipid demonstrated that it is capable of functioning as a portable apoptosistransducing molecule. Intracellular ceramide levels were increased after the treatment with the apoptosisinducing antibody, but the membrane sphingomyelin level remained unchanged. Fumonisin B1 inhibited both the ceramide increase and the apoptosis induced via IsoGb4, which indicated that the ceramide synthase pathway is likely to be involved in apoptosis induction by IsoGb4. http://www.ncbi.nlm.nih.gov/pubmed/11479210 Tue, 31 Jul 2001 00:00:00 -0700 Fourteen neurological diseases have been associated with the expansion of trinucleotide repeat regions. These diseases have been categorized into those that give rise to the translation of toxic polyglutamine proteins and those that are untranslated. Thus far, compelling evidence has not surfaced for the inclusion of a model in which a common mechanism may participate in the pathobiology of both translated and untranslated trinucleotide diseases. In these studies we show that a doublestranded RNAbinding protein, PKR, which has previously been linked to virallyinduced and stressmediated apoptosis, preferentially binds mutant huntingtin RNA transcripts immobilized on streptavidin columns that have been incubated with human brain extracts. These studies also show, by immunodetection in tissue slices, that PKR is present in its activated form in both human Huntington autopsy material and brain tissue derived from Huntington yeast artificial chromosome transgenic mice. The increased immunolocalization of the activated kinase is more pronounced in areas most affected by the disease and, coupled with the RNA binding results, suggests a role for PKR activation in the disease process. http://www.ncbi.nlm.nih.gov/pubmed/11468270 Sat, 30 Jun 2001 00:00:00 -0700 We have previously described a novel cancer chemotherapeutic approach based on the induction of apoptosis in targeted cells by homing proapoptotic peptides. In order to improve this approach we developed a computational method (approach for detecting potential apoptotic peptides, APAP) to detect short PAPs, based on the prediction of the helical content of peptides, the hydrophobic moment, and the isoelectric point. PAPs are toxic against bacteria and mitochondria, but not against mammalian cells when applied extracellularly. Among other peptides, substance P was identified as a PAP and subsequently demonstrated to be a proapoptotic peptide experimentally. APAP thus provides a method to detect and ultimately improve proapoptotic peptides for chemotherapy. http://www.ncbi.nlm.nih.gov/pubmed/11311243 Sat, 31 Mar 2001 00:00:00 -0800 The expression of DCC (deleted in colorectal cancer) is often markedly reduced in colorectal and other cancers. However, the rarity of point mutations identified in DCC coding sequences and the lack of a tumor predisposition phenotype in DCC hemizygous mice have raised questions about its role as a tumor suppressor. DCC also mediates axon guidance and functions as a dependence receptor such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand. We now show that DCC drives cell death independently of both the mitochondriadependent pathway and the death receptor/caspase8 pathway. Moreover, we demonstrate that DCC interacts with both caspase3 and caspase9 and drives the activation of caspase3 through caspase9 without a requirement for cytochrome c or Apaf1. Hence, DCC defines an additional pathway for the apoptosomeindependent caspase activation. http://www.ncbi.nlm.nih.gov/pubmed/11248093 Wed, 28 Feb 2001 00:00:00 -0800 Apoptosis is a form of cell death that is driven by an intrinsic cellular suicide program. The roles of apoptosis and other forms of programmed cell death in neural development, maintenance, and disease states are increasingly being recognized and defined. Therapies directed at the apoptotic program have seen at least some degree of success in animal models of neurodegenerative disease, vascular disease, and traumatic CNS injury. This article describes the signal transduction pathways that mediate apoptosis. Broadly speaking, intrinsic and extrinsic pathways for apoptosis activation may be distinguished, as can be crosstalk between these two. These pathways converge on a system of proteases referred to as "capases" (cysteinyl aspartic proteinases), and modulators exist that multimerize, activate, amplify, or inhibit caspases. Activated caspases are the executioners of the apoptotic program, and carry out this function by cleaving specific cellular substrates. Modulation of this process holds promise as a therapeutic approach in neurotrauma. http://www.ncbi.nlm.nih.gov/pubmed/11063049 Wed, 31 Jan 2001 00:00:00 -0800 We investigated the mechanism of lysosomemediated cell death using purified recombinant proapoptotic proteins, and cellfree extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin Lrelated protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis. http://www.ncbi.nlm.nih.gov/pubmed/11073962 Wed, 31 Jan 2001 00:00:00 -0800 The term apoptosis often has been used interchangeably with the term programmed cell death. Here we describe a form of programmed cell death that is distinct from apoptosis by the criteria of morphology, biochemistry, and response to apoptosis inhibitors. Morphologically, this alternative form of programmed cell death appears during development and in some cases of neurodegeneration. Despite its lack of response to caspase inhibitors and Bclx(L), we show that this form of cell death is driven by an alternative caspase9 activity that is Apaf1independent. Characterization of this alternative form of programmed cell death should lead to new insight into cell death programs and their roles in development and degeneration. http://www.ncbi.nlm.nih.gov/pubmed/11121041 Sun, 31 Dec 2000 00:00:00 -0800 Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collageninduced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD4C) was covalently linked to a proapoptotic heptapeptide dimer, D(KLAKLAK)2, and was systemically administered to mice with collageninduced arthritis. A phage displaying an RGDcontaining cyclic peptide (RGD4C) that binds selectively to the alpha(v)beta3 and alpha(v)beta5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD4C phage to inflamed synovium was inhibited by coadministration of soluble RGD4C. Intravenous injections of the RGD4CD(KLAKLAK)2 chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis. http://www.ncbi.nlm.nih.gov/pubmed/11714389 Sun, 31 Dec 2000 00:00:00 -0800 The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the proapoptotic effect. Within the 29residue domain required for apoptosis induction by p75NTR, a 14residue region is sufficient as a peptide inducer of apoptosis. This 14residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the fulllength p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains. http://www.ncbi.nlm.nih.gov/pubmed/11303785 Thu, 30 Nov 2000 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/10914037 Sat, 30 Sep 2000 00:00:00 -0700 Both transgenic mouse and cell culture models of familial amyotrophic lateral sclerosis (FALS) support a gainoffunction effect for the mutations in copperzinc superoxide dismutase (CuZnSOD) associated with FALS, but the nature of the function gained remains incompletely characterized. We previously reported an enhanced peroxidase activity for FALSassociated CuZnSOD mutants. Because one of the targets of such activity is CuZnSOD itself, we examined peroxidemediated inactivation of wildtype and mutant CuZnSODs, and found that the mutants are more readily inactivated. Inactivation of the mutants was associated with fragmentation, which did not occur in the wildtype enzyme under these conditions. Furthermore, the reduction of the FALSassociated mutants by ascorbate was enhanced markedly when compared to the wildtype enzyme. The visible spectra of the mutants showed a consistent blue shift of the peak at 680 nm in the wildtype enzyme, suggesting an alteration in coppersite geometry. These results extend previous studies demonstrating enhanced peroxidase activity in the mutants, and suggest that the toxic function that leads to motor neuron degeneration may result from a loss of specificity of the redox reactions catalyzed by CuZnSOD. http://www.ncbi.nlm.nih.gov/pubmed/11220787 Sat, 30 Sep 2000 00:00:00 -0700 The RET (rearranged during transfection) protooncogene encodes a tyrosine kinase receptor involved in both multiple endocrine neoplasia type 2 (MEN 2), an inherited cancer syndrome, and Hirschsprung disease (HSCR), a developmental defect of enteric neurons. We report here that the expression of RET receptor induces apoptosis. This proapoptotic effect of RET is inhibited in the presence of its ligand glial cell linederived neurotrophic factor (GDNF). Furthermore, we present evidence that RET induces apoptosis via its own cleavage by caspases, a phenomenon allowing the liberation/exposure of a proapoptotic domain of RET. In addition, we report that Hirschsprungassociated RET mutations impair GDNF control of RET proapoptotic activity. These results indicate that HSCR may result from apoptosis of RETexpressing enteric neuroblasts. http://www.ncbi.nlm.nih.gov/pubmed/10921886 Mon, 31 Jul 2000 00:00:00 -0700 The signaling pathways invoked by ligand binding to the common neurotrophin receptor p75NTR are incompletely understood. Using the yeast twohybrid system, we identified the mitogenactivated protein (MAP) kinase p38beta2 as a specific interactor with the 5th and 6th alpha helices of the p75NTR intracytoplasmic region. The consequences of this interaction were studied, using primary cultures of Schwann cells and the 293T cell line. Phosphorylation of p75NTR by p38beta2 was induced in vitro and in vivo by MAP kinase kinases (MKK) 6 activation. This pathway demonstrated feedback in that nerve growth factor (NGF) binding increased p38beta2 activity, causing an increase of nuclear factorkappaB (NFkappaB) activation and a decrease of AP1 activation. The mechanisms described explain at least in part why NGF binding to p75NTR increases cell survival in certain circumstances. http://www.ncbi.nlm.nih.gov/pubmed/11211234 Mon, 31 Jul 2000 00:00:00 -0700 The dependence receptor notion was based on the observation that the effects of a number of receptors that function in both nervous system development and tumorigenesis (especially metastasis) cannot be explained simply by a positive effect of signal transduction induced by ligand binding. Receptors such as the common neurotrophin receptor p75NTR, the androgen receptor (AR), DCC (deleted in colorectal cancer), and RET (rearranged during transfection) demonstrate effects that are more adequately explained when these are considered to be dependence receptors. These receptors show two distinct forms of signal transduction depending on their respective ligand availability: in the presence of their ligands, they transduce a signal for either proliferation or differentiation however, they are not inactive in the absence of their ligands, but rather induce an active signal for cell death. Such receptors thus create a cellular state of dependence on their ligands, the loss of ligand availability inducing cell suicide or enhancing the likelihood of cellular suicide. This new concept is reviewed here enlightening the molecular mechanisms of these receptors and their potential relevance in vivo in the development of the nervous system and in the control of tumorigenesis. http://www.ncbi.nlm.nih.gov/pubmed/10969210 Fri, 30 Jun 2000 00:00:00 -0700 We have studied neurotoxicity induced by pharmacological concentrations of 3hydroxykynurenine (3HK), an endogenous toxin implicated in certain neurodegenerative diseases, in cerebellar granule cells, PC12 pheochromocytoma cells, and GT17 hypothalamic neurosecretory cells. In all three cell types, the toxicity was induced in a dosedependent manner by 3HK at high micromolar concentrations and had features characteristic of apoptosis, including chromatin condensation and internucleosomal DNA cleavage. In cerebellar granule cells, the 3HK neurotoxicity was unaffected by xanthine oxidase inhibitors but markedly potentiated by superoxide dismutase and its hemelike mimetic, MnTBAP manganese(III) tetrakis(benzoic acid)porphyrin chloride. Catalase blocked 3HK neurotoxicity in the absence and presence of superoxide dismutase or MnTBAP. The formation of H(2)O(2) was demonstrated in PC12 and GT17 cells treated with 3HK, by measuring the increase in the fluorescent product, 2',7'dichlorofluorescein. In both PC12 and cerebellar granule cells, inhibitors of the neutral amino acid transporter that mediates the uptake of 3HK failed to block 3HK toxicity. However, their toxicity was slightly potentiated by the iron chelator, deferoxamine. Taken together, our results suggest that neurotoxicity induced by pharmacological concentrations of 3HK in these cell types is mediated primarily by H(2)O(2), which is formed most likely by autooxidation of 3HK in extracellular compartments. 3HKinduced death of PC12 and GT17 cells was protected by dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. The protection by dantrolene was associated with a marked increase in the protein level of Bcl2, a prominent antiapoptotic gene product. Moreover, overexpression of Bcl2 in GT17 cells elicited by gene transfection suppressed 3HK toxicity. Thus, dantrolene may elicit its neuroprotective effects by mechanisms involving upregulation of the level and function of Bcl2 protein. http://www.ncbi.nlm.nih.gov/pubmed/10854250 Fri, 30 Jun 2000 00:00:00 -0700 Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a sitedirected htt mutant that is resistant to caspase3 cleavage at positions 513 and 530 and to caspase6 cleavage at position 586. In contrast to cleavable htt, caspaseresistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease. http://www.ncbi.nlm.nih.gov/pubmed/10770929 Wed, 31 May 2000 00:00:00 -0700 The biochemical mechanism by which neurons become dependent on neurotrophins for survival is unknown. We found previously that the common neurotrophin receptor, p75(NTR), is a mediator of neurotrophin dependence and that this effect requires a novel type of domain dubbed a neurotrophin dependence domain. We report here that, in contrast to other proapoptotic receptors such as Fas, apoptosis induction by p75(NTR) requires monomerization, with dimerization inhibiting the effect. Blocking the proapoptotic effect of the monomer by dimerization requires a distinct domain that lies at the carboxyterminus of p75(NTR). These results define a novel type of domain required for inhibiting apoptosis induction by p75(NTR). http://www.ncbi.nlm.nih.gov/pubmed/10820429 Wed, 31 May 2000 00:00:00 -0700 Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso1 and iso2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a posttranslational modification of lysine 72, Nepsilontrimethylation. This modification was found to abrogate proapoptotic activity of metazoan cytochrome c expressed in yeast. However, iso1 cytochrome c lacking the trimethylation modification also was devoid of proapoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso1 sequence preclude proapoptotic activity. Competition studies suggest that the lack of proapoptotic activity was associated with a low affinity for Apaf1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities. http://www.ncbi.nlm.nih.gov/pubmed/10821864 Sun, 30 Apr 2000 00:00:00 -0700 The amyloid betaprotein precursor gives rise to the amyloid betaprotein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid betaprotein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspasecleaved amyloid betaprotein precursor and activated caspase9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid betaprotein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease. http://www.ncbi.nlm.nih.gov/pubmed/10742146 Fri, 31 Mar 2000 00:00:00 -0800 The common neurotrophin receptor p75(NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP1 (Fasassociated phosphatase1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fasmediated apoptosis. Here we have shown by coimmunoprecipitation that FAP1 also binds to the p75(NTR) cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP1 and Cterminal SerProVal residues of p75(NTR). Furthermore, cells expressing a FAP1/green fluorescent protein showed intracellular colocalization of FAP1 and p75(NTR) at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptorassociated factor 6)mediated NFkappaB activation and tamoxifeninduced apoptosis in 293T cells expressing p75(NTR). The results revealed that TRAF6mediated NFkappaB activation was suppressed by p75(NTR) and that the p75(NTR)mediated NFkappaB suppression was reduced by FAP1 expression. Interestingly, a mutant of the p75(NTR) intracellular domain with a single substitution of a Met for Val in its Cterminus, which cannot interact with FAP1, displayed enhanced proapoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP1 may be involved in suppressing p75(NTR)mediated proapoptotic signaling through its interaction with three Cterminal amino acids (tSPV). Thus, FAP1 may regulate p75(NTR)mediated signal transduction by physiological interaction through its third PDZ domain. http://www.ncbi.nlm.nih.gov/pubmed/10544233 Thu, 30 Sep 1999 00:00:00 -0700 We have designed short peptides composed of two functional domains, one a tumor blood vessel 'homing' motif and the other a programmed cell deathinducing sequence, and synthesized them by simple peptide chemistry. The 'homing' domain was designed to guide the peptide to targeted cells and allow its internalization. The proapoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anticancer activity in mice. This approach may yield new therapeutic agents. http://www.ncbi.nlm.nih.gov/pubmed/10470080 Tue, 31 Aug 1999 00:00:00 -0700