Kulandaivelu S. Vetrivel

Amyloidogenic processing of β-amyloid precursor protein in intracellular compartments

Trafficking and proteolytic processing of amyloid precursor protein (APP) have been the focus of numerous investigations in the past two decades, since the identification of Aβ as the principal component of brain senile plaques and the cloning of APP cDNA. Tremendous progress has been made in the recent past toward the characterization of β- and γ-secretases. Here, we review the salient features of Alzheimer disease amyloidogenesis, and discuss the current knowledge on APP trafficking and amyloidogenic processing of APP in intracellular membrane compartments and microdomains.

Pathological and physiological functions of presenilins

Mutations in PSEN1 and PSEN2 genes account for the majority of cases of early-onset familial Alzheimer disease. Since the first prediction of a genetic link between PSEN1 and PSEN2 with Alzheimers disease, many research groups from both academia and pharmaceutical industry have sought to unravel how pathogenic mutations in PSEN cause presenile dementia. PSEN genes encode polytopic membrane proteins termed presenilins (PS1 and PS2), which function as the catalytic subunit of γ-secretase, an intramembrane protease that has a wide spectrum of type I membrane protein substrates. Sequential cleavage of amyloid precursor protein by BACE and γ-secretase releases highly fibrillogenic β-amyloid peptides, which accumulate in the brains of aged individuals and patients with Alzheimers disease. Familial Alzheimers disease-associated presenilin variants are thought to exert their pathogenic function by selectively elevating the levels of highly amyloidogenic Aβ42 peptides. In addition to Alzheimers disease, several recent studies have linked PSEN1 to familiar frontotemporal dementia. Here, we review the biology of PS1, its role in γ-secretase activity, and discuss recent developments in the cell biology of PS1 with respect to Alzheimers disease pathogenesis.

Alzheimer Disease Aβ Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts

Alzheimer disease β-amyloid (Aβ) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and γ-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Aβ production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Aβ. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

Nicastrin Is Critical for Stability and Trafficking but Not Association of Other Presenilin/γ-Secretase Components

γ-Secretase, which is responsible for the intramembranous cleavage of Alzheimer β-amyloid precursor protein and the signaling receptor Notch, is a multiprotein complex consisting of at least four components: presenilin (PS); nicastrin (Nct); APH-1 (anterior pharynx-defective-1); and presenilin enhancer-2 (PEN-2). Presenilin 1 (PS1) is known to be essential for the stability, interaction, and trafficking of the other PS1/γ-secretase components. However, the precise functions of the other components remain elusive. Here, we investigated the functions of Nct within the PS1/γ-secretase complex. We demonstrated that the loss of Nct expression in the embryonic fibroblast cells (Nct KO cells) results in dramatically decreased levels of APH-1, PEN-2, and PS1 fragments accompanied by a significant accumulation of full-length PS1. In the absence of Nct, PEN-2 and full-length PS1 are subjected to proteasome-mediated degradation, whereas the degradation of APH-1 is mediated by both proteasomal and lysosomal pathways. Unlike the case of wild type cells in which the γ-secretase complex mainly locates in the trans-Golgi network, the majority of residual PEN-2, APH-1, and the uncleaved full-length PS1 in Nct KO cells reside in the endoplasmic reticulum, which remain associated with each other in the absence of Nct. Interestingly, significant amounts of full-length PS1 and PEN-2, but not APH-1, are detected on the plasma membrane in Nct KO cells, suggesting the Nct-independent cell surface delivery of the PEN-2·PS1. Finally, the diminished PEN-2 protein level in Nct-deficient cells can be partially restored by overexpression of exogenous PS1, APH-1, or PEN-2 individually or collectively, indicating a dispensable role for Nct in controlling PEN-2 level. Taken together, our study demonstrates a critical role of Nct in the stability and proper intracellular trafficking of other components of the PS1/ γ-secretase complex but not in maintaining the association of PEN-2, APH-1, and full-length PS1.

Mechanisms of Disease: new therapeutic strategies for Alzheimer's disease—targeting APP processing in lipid rafts

Alzheimers disease (AD) is the most common cause of age-related dementia. Pathologically, AD is characterized by the deposition in the brain of amyloid-β peptides derived from proteolysis of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. A growing body of evidence implicates cholesterol and cholesterol-rich membrane microdomains in amyloidogenic processing of APP. Here, we review recent findings regarding the association of BACE1, γ-secretase and APP in lipid rafts, and discuss potential therapeutic strategies for AD that are based on knowledge gleaned from the membrane environment that fosters APP processing.

Dual roles of the transmembrane protein p23/TMP21 in the modulation of amyloid precursor protein metabolism

BackgroundAlzheimers disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides. Aβ is released from ectodomain cleaved amyloid precursor protein (APP) via intramembranous proteolysis by γ-secretase, a complex consisting of presenilin and a few other proteins. p23/TMP21, a member of the p24 family type I transmembrane proteins, was recently identified as a presenilin complex component capable of modulating γ-secretase cleavage. The p24 family proteins form oligomeric complexes and regulate vesicular trafficking in the early secretory pathway, but their role in APP trafficking has not been investigated.ResultsHere, we report that siRNA-mediated depletion of p23 in N2a neuroblastoma and HeLa cells produces concomitant knockdown of additional p24 family proteins and increases secretion of sAPP. Furthermore, intact cell and cell-free Aβ production increases following p23 knockdown, similar to data reported earlier using HEK293 cells. However, we find that p23 is not present in mature γ-secretase complexes isolated using an active-site γ-secretase inhibitor. Depletion of p23 and expression of a familial AD-linked PS1 mutant have additive effects on Aβ42 production. Knockdown of p23 expression confers biosynthetic stability to nascent APP, allowing its efficient maturation and surface accumulation. Moreover, immunoisolation analyses show decrease in co-residence of APP and the APP adaptor Mint3. Thus, multiple lines of evidence indicate that p23 function influences APP trafficking and sAPP release independent of its reported role in γ-secretase modulation.ConclusionThese data assign significance to p24 family proteins in regulating APP trafficking in the continuum of bidirectional transport between the ER and Golgi, and ascribe new relevance to the regulation of early trafficking in AD pathogenesis.

Axonal BACE1 dynamics and targeting in hippocampal neurons: a role for Rab11 GTPase.

BackgroundBACE1 is one of the two enzymes that cleave amyloid precursor protein to generate Alzheimers disease (AD) beta amyloid peptides. It is widely believed that BACE1 initiates APP processing in endosomes, and in the brain this cleavage is known to occur during axonal transport of APP. In addition, BACE1 accumulates in dystrophic neurites surrounding brain senile plaques in individuals with AD, suggesting that abnormal accumulation of BACE1 at presynaptic terminals contributes to pathogenesis in AD. However, only limited information is available on BACE1 axonal transport and targeting.ResultsBy visualizing BACE1-YFP dynamics using live imaging, we demonstrate that BACE1 undergoes bi-directional transport in dynamic tubulo-vesicular carriers along axons in cultured hippocampal neurons and in acute hippocampal slices of transgenic mice. In addition, a subset of BACE1 is present in larger stationary structures, which are active presynaptic sites. In cultured neurons, BACE1-YFP is preferentially targeted to axons over time, consistent with predominant in vivo localization of BACE1 in presynaptic terminals. Confocal analysis and dual-color live imaging revealed a localization and dynamic transport of BACE1 along dendrites and axons in Rab11-positive recycling endosomes. Impairment of Rab11 function leads to a diminution of total and endocytosed BACE1 in axons, concomitant with an increase in the soma. Together, these results suggest that BACE1 is sorted to axons in endosomes in a Rab11-dependent manner.ConclusionOur results reveal novel information on dynamic BACE1 transport in neurons, and demonstrate that Rab11-GTPase function is critical for axonal sorting of BACE1. Thus, we suggest that BACE1 transcytosis in endosomes contributes to presynaptic BACE1 localization.

S-Palmitoylation of γ-Secretase Subunits Nicastrin and APH-1

Proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases generates β-amyloid (Aβ) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Aβ production. Previously, we and others reported that the four integral subunits of the γ-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of γ-secretase subunits. We report that in cultured cells and in brain the γ-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys689, and APH-1 is S-palmitoylated at Cys182 and Cys245. S-Palmitoylation-defective nicastrin and APH-1 form stable γ-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for γ-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Aβ40, Aβ42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate γ-secretase processing of APP and other substrates.

A Function for EHD Family Proteins in Unidirectional Retrograde Dendritic Transport of BACE1 and Alzheimer’s Disease Aβ Production

Abnormal accumulation of β-secretase (BACE1) in dystrophic neurites and presynaptic β-amyloid (Aβ) production contribute to Alzheimers disease pathogenesis. Little, however, is known about BACE1 sorting and dynamic transport in neurons. We investigated BACE1 trafficking in hippocampal neurons using live-cell imaging and selective labeling. We report that transport vesicles containing internalized BACE1 in dendrites undergo exclusive retrograde transport toward the soma, whereas they undergo bidirectional transport in axons. Unidirectional dendritic transport requires Eps15-homology-domain-containing (EHD) 1 and 3 protein function. Furthermore, loss of EHD function compromises dynamic axonal transport and overall BACE1 levels in axons. EHD1/3 colocalize with BACE1 and APP β-C-terminal fragments in hippocampal mossy fiber terminals, and their depletion in neurons significantly attenuates Aβ levels. These results demonstrate unidirectional endocytic transport of a dendritic cargo and reveal a role for EHD proteins in neuronal BACE1 transcytosis and Aβ production, processes that are highly relevant for Alzheimers disease.

Evidence That CD147 Modulation of β-Amyloid (Aβ) Levels Is Mediated by Extracellular Degradation of Secreted Aβ

Cerebral deposition of β-amyloid (Aβ) peptides is a pathological hallmark of Alzheimer disease. Intramembranous proteolysis of amyloid precursor protein by a multiprotein γ-secretase complex generates Aβ. Previously, it was reported that CD147, a glycoprotein that stimulates production of matrix metalloproteinases (MMPs), is a subunit of γ-secretase and that the levels of secreted Aβ inversely correlate with CD147 expression. Here, we show that the levels and localization of CD147 in fibroblasts, as well as postnatal expression and distribution in brain, are distinct from those of integral γ-secretase subunits. Notably, we show that although depletion of CD147 increased extracellular Aβ levels in intact cells, membranes isolated from CD147-depleted cells failed to elevate Aβ production in an in vitro γ-secretase assay. Consistent with an extracellular source that modulates Aβ metabolism, synthetic Aβ was degraded more rapidly in the conditioned medium of cells overexpressing CD147. Moreover, modulation of CD147 expression had no effect on ϵ-site cleavage of amyloid precursor protein and Notch1 receptor. Collectively, our results demonstrate that CD147 modulates Aβ levels not by regulating γ-secretase activity, but by stimulating extracellular degradation of Aβ. In view of the known function of CD147 in MMP production, we postulate that CD147 expression influences Aβ levels by an indirect mechanism involving MMPs that can degrade extracellular Aβ.