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Weiming Xia

Publications and source records attributed to Weiming Xia.

At least 19 recordsLinked to original sources
PubMed2006

RNA interference silencing of the adaptor molecules ShcC and Fe65 differentially affect amyloid precursor protein processing and Abeta generation.

The amyloid precursor protein (APP) and its pathogenic by-product amyloid-beta protein (Abeta) play central roles in Alzheimer disease (AD) neuropathogenesis. APP can be cleaved by beta-secretase (BACE) and alpha-secretase to produce APP-C99 and APP-C83. These C-terminal fragments can then be cleaved by gamma-secretase to produce Abeta and p3, respectively. p3 has been reported to promote apoptosis, and Abeta is the key component of senile plaques in AD brain. APP adaptor proteins with phosphotyrosine-binding domains, including ShcA (SHC1), ShcC (SHC3), and Fe65 (APBB1), can bind to and interact with the conserved YENPTY motif in the APP-C terminus. Here we have described for the first time the effects of RNA interference (RNAi) silencing of ShcA, ShcC, and Fe65 expression on APP processing and Abeta production. RNAi silencing of ShcC led to reductions in the levels of APP-C-terminal fragments (APP-CTFs) and Abeta in H4 human neuroglioma cells stably overexpressing full-length APP (H4-FL-APP cells) but not in those expressing APP-C99 (H4-APP-C99 cells). RNAi silencing of ShcC also led to reductions in BACE levels in H4-FL-APP cells. In contrast, RNAi silencing of the homologue ShcA had no effect on APP processing or Abeta levels. RNAi silencing of Fe65 increased APP-CTF levels, although also decreasing Abeta levels in H4-FL-APP cells. These findings suggest that pharmacologically blocking interaction of APP with ShcC and Fe65 may provide novel therapeutic strategies against AD.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Increased App expression in a mouse model of Down's syndrome disrupts NGF transport and causes cholinergic neuron degeneration.

Degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive dysfunction in Alzheimer's disease (AD) and Down's syndrome (DS). We used Ts65Dn and Ts1Cje mouse models of DS to show that the increased dose of the amyloid precursor protein gene, App, acts to markedly decrease NGF retrograde transport and cause degeneration of BFCNs. NGF transport was also decreased in mice expressing wild-type human APP or a familial AD-linked mutant APP; while significant, the decreases were less marked and there was no evident degeneration of BFCNs. Because of evidence suggesting that the NGF transport defect was intra-axonal, we explored within cholinergic axons the status of early endosomes (EEs). NGF-containing EEs were enlarged in Ts65Dn mice and their App content was increased. Our study thus provides evidence for a pathogenic mechanism for DS in which increased expression of App, in the context of trisomy, causes abnormal transport of NGF and cholinergic neurodegeneration.

Alzheimer Disease↗
PubMed2006

The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production.

Neuropathological hallmarks of Alzheimer's disease are neurofibrillary tangles composed of tau and neuritic plaques comprising amyloid-beta peptides (Abeta) derived from amyloid precursor protein (APP), but their exact relationship remains elusive. Phosphorylation of tau and APP on certain serine or threonine residues preceding proline affects tangle formation and Abeta production in vitro. Phosphorylated Ser/Thr-Pro motifs in peptides can exist in cis or trans conformations, the conversion of which is catalysed by the Pin1 prolyl isomerase. Pin1 has been proposed to regulate protein function by accelerating conformational changes, but such activity has never been visualized and the biological and pathological significance of Pin1 substrate conformations is unknown. Notably, Pin1 is downregulated and/or inhibited by oxidation in Alzheimer's disease neurons, Pin1 knockout causes tauopathy and neurodegeneration, and Pin1 promoter polymorphisms appear to associate with reduced Pin1 levels and increased risk for late-onset Alzheimer's disease. However, the role of Pin1 in APP processing and Abeta production is unknown. Here we show that Pin1 has profound effects on APP processing and Abeta production. We find that Pin1 binds to the phosphorylated Thr 668-Pro motif in APP and accelerates its isomerization by over 1,000-fold, regulating the APP intracellular domain between two conformations, as visualized by NMR. Whereas Pin1 overexpression reduces Abeta secretion from cell cultures, knockout of Pin1 increases its secretion. Pin1 knockout alone or in combination with overexpression of mutant APP in mice increases amyloidogenic APP processing and selectively elevates insoluble Abeta42 (a major toxic species) in brains in an age-dependent manner, with Abeta42 being prominently localized to multivesicular bodies of neurons, as shown in Alzheimer's disease before plaque pathology. Thus, Pin1-catalysed prolyl isomerization is a novel mechanism to regulate APP processing and Abeta production, and its deregulation may link both tangle and plaque pathologies. These findings provide new insight into the pathogenesis and treatment of Alzheimer's disease.

Alzheimer Disease↗
PubMed2006

The cytosolic loop of the gamma-secretase component presenilin enhancer 2 protects zebrafish embryos from apoptosis.

The gamma-secretase complex, composed of presenilin, presenilin enhancer 2 (Pen-2), nicastrin, and Aph-1, catalyzes the final cleavage of amyloid precursor protein to generate the toxic amyloid beta protein, the major component of plaques in the brains of Alzheimer disease patients. To understand the in vivo function of Pen-2, we used morphant technology available in zebrafish and transiently knocked down the expression of endogenous Pen-2 by injecting the morpholino (MO) against Pen-2. Two truncated Pen-2 proteins lacking either the cytosolic or the C-terminal domain were expressed in MO-injected embryos. This deletion analysis demonstrated that the Pen-2 cytosolic loop is essential for protecting developing embryos from caspase-dependent apoptosis caused by the reduction of Pen-2. Twelve amino acids in the C terminus of Pen-2 were dispensable and could not rescue the Pen-2 knockdown-induced apoptotic phenotype. Surprisingly, double knockdown of Pen-2 and nuclear factor kappaB component p65 abrogated the single Pen-2 MO-induced caspase activation, indicating that a previously reported pro-apoptotic role of NF-kappaB in some cell types could be manifested in a whole animal and that knockdown of Pen-2 may trigger pro-apoptotic activation of NF-kappaB.

Amyloid Precursor Protein Secretases↗
PubMed2006

Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2) demonstrate excessive p53-dependent apoptosis and neuronal loss.

Gamma-secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen-2), nicastrin, and Aph-1, is the final proteolytic step in generating amyloid beta protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen-2 in neuronal survival. We found that (i) knockdown of Pen-2 using antisense morpholino led to a reduction of islet-1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen-2 or other gamma-secretase components, (iii) neuronal loss in Pen-2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen-2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet-1 or acetylated tubulin positive neurons in Pen-2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen-2 directly induces a p53-dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen-2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo.

Alzheimer Disease↗
PubMed2006

Presenilin-1-mediated retention of APP derivatives in early biosynthetic compartments.

Processing of the amyloid precursor protein (APP) leads to the production of amyloid-beta (Abeta), the major component of extracellular plaques in the brains of Alzheimer's disease (AD) patients. Presenilin-1 (PS-1) plays a key role in the final step of Abeta formation, the gamma-secretase cleavage. Previously, we showed that PS-1 is retained in pre-Golgi compartments by incorporation into COPI-coated membranes of the vesicular tubular clusters (VTCs) between endoplasmic reticulum (ER) and Golgi complex. Here, we show that PS-1 also mediates the retention of the beta-cleavage-derived APP-C-terminal fragment (CTFbeta) and/or Abeta in pre-Golgi membranes. Overexpression of PS-1 increased the percentage of CTFbeta and/or Abeta in VTCs as well as their distribution to COPI-coated VTC membranes. By contrast, overexpression of the dominant-negative aspartate mutant PS-1(D257A) or PS-knockout decreased incorporation of these APP derivatives into COPI-coated membranes. Sorting of APP derivatives to COPI-coated VTC membranes was not depending on the APP cytosolic tail. In post-Golgi compartments, PS-1 expression enhanced the association of full-length APP/APPs with endosomal compartments at the expense of plasma membrane-bound APP. We conclude that PS-1, in addition to its role in gamma-secretase cleavage, is also required for the subcellular routing of APP and its derivatives. Malfunctioning of PS-1 in this role may have important consequences for the progress of AD.

Alzheimer Disease↗
PubMed2005

Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

Animals↗
PubMed2004

Dimerization of Parkinson's disease-causing DJ-1 and formation of high molecular weight complexes in human brain.

Mutations in the DJ-1 gene have been implicated in the PARK7-linked autosomal recessive form of Parkinson's disease (PD). The molecular properties of DJ-1WT, DJ-1L166P, and a newly identified disease-causing mutant DJ-1M26I were explored after they were transiently expressed in mammalian cells. Treatment of intact, living cells with the chemical crosslinker disuccinimidyl suberate (DSS) revealed that DJ-1WT and mutant DJ-1M26I were present as stable homodimers; DJ-1L166P in particular tended to form high-order complexes as well. In contrast to DJ-1L166P that is quickly degraded by the proteasome, DJ-1M26I was found to be an efficiently expressed and stable variant of DJ-1, suggesting that these mutations have distinct biochemical effects on DJ-1. We further provide evidence that in human brain, under nondenaturing conditions, DJ-1 is present in high molecular weight (HMW) complexes of approximately 250-700 kDa containing parkin, another PD-associated protein.

Animals↗
PubMed2003

APP processing is regulated by cytoplasmic phosphorylation.

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.

Alzheimer Disease↗
PubMed2003

Intramembrane proteolysis by presenilin and presenilin-like proteases.

Regulated intramembrane proteolysis is a novel mechanism involving proteases that hydrolyze their substrates in a hydrophobic environment. Presenilin (PS) 1 and PS 2 are required for intramembrane cleavage of an increasing number of type I membrane proteins, including the amyloid precursor protein of Alzheimer's disease and the Notch receptor, which signals during differentiation and development. Mutagenesis, affinity labeling, biochemical isolation, and reconstitution in cells reveal that PS, in complex with co-factors nicastrin, APH-1 and PEN-2, apparently contains the active site of gamma-secretase, a novel membrane aspartyl protease. In addition, other related aspartyl proteases have been identified. These include members of the type-4 prepilin peptidase family in bacteria, which are known proteases and carry a GD motif conserved in PS. A group of multi-pass membrane proteins found in eukaryotes also contain YD and LGXGD motifs in two transmembrane domains that are conserved in PS and postulated to constitute an aspartyl protease active site. Among these is signal peptide peptidase (SPP), which cleaves remnant signal peptides derived from signal-peptidase-mediated ectodomain shedding. SPP cuts type II membrane proteins, illustrating that PS-like proteases play a key role in intramembrane proteolysis of single-pass membrane proteins oriented in either direction.

Alzheimer Disease↗
PubMed2003

Functional gamma-secretase complex assembly in Golgi/trans-Golgi network: interactions among presenilin, nicastrin, Aph1, Pen-2, and gamma-secretase substrates.

Gamma-secretase is a proteolytic complex whose substrates include Notch, beta-amyloid precursor protein (APP), and several other type I transmembrane proteins. Presenilin (PS) and nicastrin are known components of this high-molecular-weight complex, and recent genetic screens in invertebrates have identified two additional gene products, Aph1 and Pen-2, as key factors in gamma-secretase activity. Here, we examined the interaction of the components of the gamma-secretase complex in Chinese hamster ovary cells stably expressing human forms of APP, PS1, Aph1, and Pen-2. Subcellular fractionation of membrane vesicles and subsequent coimmunoprecipitation of individual gamma-secretase components revealed that interactions among all proteins occurred in the Golgi/trans-Golgi network (TGN) compartments. Furthermore, incubation of the Golgi/TGN-enriched vesicles resulted in de novo generation of amyloid beta-protein and APP intracellular domain. Immunofluorescent staining of the individual gamma-secretase components supported our biochemical evidence that the gamma-secretase components assemble into the proteolytically active gamma-secretase complex in the Golgi/TGN compartment.

Amyloid Precursor Protein Secretases↗
PubMed2003

Presenilin-1 exists in both pre- and post-Golgi compartments and recycles via COPI-coated membranes.

Presenilin-1 is involved in intramembrane proteolysis of various proteins, but its intracellular site of action has remained elusive. Here, we determined by quantitative immunogold-electron microscopy that presenilin-1 in Chinese hamster ovary cells is present in pre-Golgi compartments as well as at the plasma membrane and endosomes. Notably, a high percentage of presenilin-1 resides in COPI-coated membranes between the endoplasmic reticulum and the Golgi complex, indicating significant recycling to the endoplasmic reticulum. By contrast, the inactive aspartate mutant presenilin-1D257A is relatively excluded from COPI-coated membranes, concomitant with increased post-Golgi levels. These data provide critical evidence for the scenario that the complex containing presenilin-1 can serve as gamma-secretase at the plasma membrane or endosomes and suggest a role for COPI-mediated retrograde transport in regulating post-Golgi levels of presenilin-1.

Amino Acid Substitution↗
PubMed2003

Presenilin endoproteolysis mediated by an aspartyl protease activity pharmacologically distinct from gamma-secretase.

Presenilin (PS)-dependent gamma-secretase cleavage is the final proteolytic step in generating amyloid beta protein (A beta), a key peptide involved in the pathogenesis of Alzheimer's disease. PS undergoes endoproteolysis by an unidentified 'presenilinase' to generate the functional N-terminal and C-terminal fragment heterodimers (NTF/CTF) that may harbor the gamma-secretase active site. To better understand the relationship between presenilinase and gamma-secretase, we characterized the biochemical properties of presenilinase and compared them with those of gamma-secretase. Similar to gamma-secretase, presenilinase was most active at acidic pH 6.3. Aspartyl protease inhibitor pepstatin A blocked presenilinase activity with an IC50 of approximately 1 microM. Difluoroketone aspartyl protease transition state analogue MW167 was relatively selective for presenilinase (IC50 < 1 microM) over gamma-secretase (IC50-16 microM). Importantly, removing the transition state mimicking moiety simultaneously abolished both presenilinase and gamma-secretase inhibition, suggesting that presenilinase, like gamma-secretase, is an aspartyl protease. Interestingly, several of the most potent gamma-secretase inhibitors (IC50 = 0.3 or 20 nM) failed to block presenilinase activity. Although de novo generation of PS1 fragments coincided with production of A beta in vitro, blocking presenilinase activity without reducing pre-existing fragment levels permitted normal de novo generation of A beta and amyloid intracellular domain. Therefore, presenilinase has characteristics of an aspartyl protease, but this activity is distinct from gamma-secretase.

Amyloid Precursor Protein Secretases↗
PubMed2003

Relationship between presenilinase and gamma-secretase.

Genetic and neuropathological studies suggest that processing of amyloid precursor protein (APP) to yield amyloid beta-protein (Abeta) plays an important role in the pathogenesis of Alzheimer's disease (AD). One of the current therapeutic efforts for AD is directed towards blocking the gamma-secretase activity that produces Abeta. Compelling evidence for presenilin (PS) possessing gamma-secretase activity includes a lack of Abeta production in PS knockout neurons and in cultured cells carrying a dominant negative mutation at either of two critical aspartate residues in PS, which may constitute the active site of gamma-secretase. In vitro studies have shown a binding of transition-state analog gamma-secretase inhibitors to PS N-terminal fragment (NTF) and C-terminal fragment (CTF), the functional form of PS detected in the high-molecular-weight gamma-secretase complex that also contains nicastrin, Aph-1 and PEN-2. Conversion of full-length PS into functional NTF and CTF is mediated by an unknown protease activity named presenilinase. Endoproteolysis of PS into NTF/CTF by presenilinase also requires two critical aspartate residues, suggesting that full-length PS may undergo autoproteolysis and PS itself is presenilinase. Similar to gamma-secretase, presenilinase seems to be an aspartyl protease, as aspartyl protease inhibitor pepstatin A is the most potent inhibitor toward presenilinase among different classes of protease inhibitors. While several well-characterized gamma-secretase inhibitors can block presenilinase activity in vivo and in vitro, the potency of inhibitors blocking presenilinase and gamma-secretase are not correlated. Lack of presenilinase inhibition by several potent gamma-secretase inhibitors suggests that these two protease activities are pharmacologically distinct.

Alzheimer Disease↗
PubMed2003

Amyloid inhibitors and Alzheimer's disease.

Neuritic plaques composed of amyloid beta-protein (A beta) are an early and invariant neuropathological feature of Alzheimer's disease (AD). The current preclinical search for drugs is mainly focused on decreasing A beta production by inhibiting beta- or gamma-secretase, blocking the formation of these plaques by preventing A beta protofibril and fibril formation, and alleviating the toxic effects of neuritic plaque deposition. Increasing numbers of drugs currently used as therapies for other diseases are now entering clinical trials for AD, but the molecular targets of these drugs and their relevance to A beta toxicity needs to be thoroughly addressed. This knowledge will allow us to fully understand the A beta-related pathways in AD pathogenesis and explore novel therapeutic interventions.

Alzheimer Disease↗
PubMed2002

Endoproteolysis of presenilin in vitro: inhibition by gamma-secretase inhibitors.

The final proteolytic step to generate the amyloid beta-protein (Abeta) of Alzheimer's disease (AD) from beta-amyloid precursor protein (APP) is achieved by presenilin (PS)-dependent gamma-secretase cleavage. AD-causing mutations in PS1 and PS2 result in a selective and significant increase in production of the more amyloidogenic Abeta42 peptide. PS1 and PS2 undergo endoproteolysis by an unknown enzyme termed presenilinase to generate the functional complex of N- and C-terminal fragments (NTF/CTF). To investigate the endoproteolytic activity that generates active PS, we used a mammalian cell-free system that allows de novo human PS NTF and CTF generation. PS NTF and CTF generation in vitro was observed in endoplasmic reticulum (ER)-enriched fractions of membrane vesicles and to a lesser extent in Golgi/trans-Golgi-network (TGN)-enriched fractions. AD-causing mutations in PS1 and PS2 did not alter de novo generation of PS fragments. Removal of peripheral membrane-associated and cytosolic proteins did not prevent de novo generation of fragments, indicating that presenilinase activity corresponds to an integral membrane protein. Among several general inhibitors of different protease classes that blocked the presenilinase activity, pepstatin A was the most potent inhibitor. Screening available transition state analogue gamma-secretase inhibitors led to the identification of two compounds that were able to prevent the de novo generation of PS fragments, with an expected inhibition of Abeta generation. Our studies provide a biochemical approach to characterize and identify this elusive presenilinase.

Amyloid Precursor Protein Secretases↗