Virginie Buggia-Prévot

A paired RNAi and RabGAP overexpression screen identifies Rab11 as a regulator of β-Amyloid production

Alzheimers disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides, which are generated from amyloid precursor protein (APP) by β- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aβ levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Aβ production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled β-secretase endosomal recycling to the plasma membrane and thus affected Aβ production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Aβ levels and show how systems biology approaches can unravel the molecular complexity underlying AD.

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.

NFκB-dependent Control of BACE1 Promoter Transactivation by Aβ42

β-Amyloid (Aβ) peptides that accumulate in Alzheimer disease are generated from the β-amyloid precursor protein (βAPP) by cleavages by β-secretase BACE1 and by presenilin-dependent γ-secretase activities. Very few data document a putative cross-talk between these proteases and the regulatory mechanisms underlying such interaction. We show that presenilin deficiency lowers BACE1 maturation and affects both BACE1 activity and promoter transactivation. The specific γ-secretase inhibitor DFK167 triggers the decrease of BACE1 activity in wild-type but not in presenilin-deficient fibroblasts. This decrease is also elicited by catalytically inactive γ-secretase. The overexpression of APP intracellular domain (AICD), the γ/ϵ-secretase-derived C-terminal product of β-amyloid precursor protein, does not modulate BACE1 activity or promoter transactivation in fibroblasts and does not alter BACE1 expression in AICD transgenic brains of mice. A DFK167-sensitive increase of BACE1 activity is observed in cells overexpressing APPϵ (the N-terminal product of βAPP generated by ϵ-secretase cleavage harboring the Aβ domain but lacking the AICD sequence), suggesting that the production of Aβ could account for the modulation of BACE1. Accordingly, we show that HEK293 cells overexpressing wild-type βAPP exhibit a DFK167-sensitive increase in BACE1 promoter transactivation that is increased by the Aβ-potentiating Swedish mutation. This effect was mimicked by exogenous application of Aβ42 but not Aβ40 or by transient transfection of cDNA encoding Aβ42 sequence. The IκB kinase inhibitor BMS345541 prevents Aβ-induced BACE1 promoter transactivation suggesting that NFκB could mediate this Aβ-associated phenotype. Accordingly, the overexpression of wild-type or Swedish mutated βAPP does not modify the transactivation of BACE1 promoter constructs lacking NFκB-responsive element. Furthermore, APP/β-amyloid precursor protein-like protein deficiency does not affect BACE1 activity and expression. Overall, these data suggest that physiological levels of endogenous Aβ are not sufficient per se to modulate BACE1 promoter transactivation but that exacerbated Aβ production linked to wild-type or Swedish mutated βAPP overexpression modulates BACE1 promoter transactivation and activity via an NFκB-dependent pathway.

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.

Presynaptic dystrophic neurites surrounding amyloid plaques are sites of microtubule disruption, BACE1 elevation, and increased Aβ generation in Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by amyloid plaques composed of the β-amyloid (Aβ) peptide surrounded by swollen presynaptic dystrophic neurites consisting of dysfunctional axons and terminals that accumulate the β-site amyloid precursor protein (APP) cleaving enzyme (BACE1) required for Aβ generation. The cellular and molecular mechanisms that govern presynaptic dystrophic neurite formation are unclear, and elucidating these processes may lead to novel AD therapeutic strategies. Previous studies suggest Aβ may disrupt microtubules, which we hypothesize have a critical role in the development of presynaptic dystrophies. To investigate this further, here we have assessed the effects of Aβ, particularly neurotoxic Aβ42, on microtubules during the formation of presynaptic dystrophic neurites in vitro and in vivo. Live-cell imaging of primary neurons revealed that exposure to Aβ42 oligomers caused varicose and beaded neurites with extensive microtubule disruption, and inhibited anterograde and retrograde trafficking. In brain sections from AD patients and the 5XFAD transgenic mouse model of amyloid pathology, dystrophic neurite halos with BACE1 elevation around amyloid plaques exhibited aberrant tubulin accumulations or voids. At the ultrastructural level, peri-plaque dystrophies were strikingly devoid of microtubules and replete with multi-lamellar vesicles resembling autophagic intermediates. Proteins of the microtubule motors, kinesin and dynein, and other neuronal proteins were aberrantly localized in peri-plaque dystrophies. Inactive pro-cathepsin D also accumulated in peri-plaque dystrophies, indicating reduced lysosomal function. Most importantly, BACE1 accumulation in peri-plaque dystrophies caused increased BACE1 cleavage of APP and Aβ generation. Our study supports the hypothesis that Aβ induces microtubule disruption in presynaptic dystrophic neurites that surround plaques, thus impairing axonal transport and leading to accumulation of BACE1 and exacerbation of amyloid pathology in AD.

Nuclear Factor-κB Regulates βAPP and β- and γ-Secretases Differently at Physiological and Supraphysiological Aβ Concentrations

Nuclear factorB regulates APP and and -secretases differently at physiological and supraphysiological A concentrations. Linda Chami, Virginie Buggia-Prévot, Eric Duplan, Dolores Del Prete, Mounia Chami, Jean-François Peyron, and Frédéric Checler Dr. Del Prete’s name was misspelled. The correct spelling is Dolores Del Prete, as shown in the author line. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 50, p. 29758, December 11, 2015

Ca2+ Influx through Store-operated Ca2+ Channels Reduces Alzheimer Disease β-Amyloid Peptide Secretion

Background: Dysregulation of Ca2+ homeostasis has been implicated in Alzheimer disease pathogenesis, but the effects of Ca2+ on amyloid precursor protein processing are not well understood. Results: Constitutive activation of the store-operated calcium entry pathway reduces β-amyloid generation. Conclusion: Elevation of Ca2+ influx affects amyloid precursor protein processing. Significance: Alteration of Ca2+ homeostasis in Alzheimer disease may influence pathogenesis directly through modulation of β-amyloid production. Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of β-amyloid peptides (Aβ) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca2+ homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca2+ homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aβ peptides. Whereas these studies support the hypothesis that disruption of Ca2+ homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aβ production from their effects on Ca2+ homeostasis. Here, we developed a system in which cellular Ca2+ homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aβ production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca2+ entry pathway, to generate cells with constitutive and store depletion-induced Ca2+ entry. We found striking effects of Ca2+ entry induced by overexpression of the constitutively active STIM1D76A mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca2+ entry by expression of STIM1D76A significantly reduced Aβ secretion. Our results suggest that disruptions in Ca2+ homeostasis may influence AD pathogenesis directly through the modulation of Aβ production.

Predominant Expression of Alzheimer’s Disease-Associated BIN1 in Mature Oligodendrocytes and Localization to White Matter Tracts

BackgroundGenome-wide association studies have identified BIN1 within the second most significant susceptibility locus in late-onset Alzheimer’s disease (AD). BIN1 undergoes complex alternative splicing to generate multiple isoforms with diverse functions in multiple cellular processes including endocytosis and membrane remodeling. An increase in BIN1 expression in AD and an interaction between BIN1 and Tau have been reported. However, disparate descriptions of BIN1 expression and localization in the brain previously reported in the literature and the lack of clarity on brain BIN1 isoforms present formidable challenges to our understanding of how genetic variants in BIN1 increase the risk for AD.MethodsIn this study, we analyzed BIN1 mRNA and protein levels in human brain samples from individuals with or without AD. In addition, we characterized the BIN1 expression and isoform diversity in human and rodent tissue by immunohistochemistry and immunoblotting using a panel of BIN1 antibodies.ResultsHere, we report on BIN1 isoform diversity in the human brain and document alterations in the levels of select BIN1 isoforms in individuals with AD. In addition, we report striking BIN1 localization to white matter tracts in rodent and the human brain, and document that the large majority of BIN1 is expressed in mature oligodendrocytes whereas neuronal BIN1 represents a minor fraction. This predominant non-neuronal BIN1 localization contrasts with the strict neuronal expression and presynaptic localization of the BIN1 paralog, Amphiphysin 1. We also observe upregulation of BIN1 at the onset of postnatal myelination in the brain and during differentiation of cultured oligodendrocytes. Finally, we document that the loss of BIN1 significantly correlates with the extent of demyelination in multiple sclerosis lesions.ConclusionOur study provides new insights into the brain distribution and cellular expression of an important risk factor associated with late-onset AD. We propose that efforts to define how genetic variants in BIN1 elevate the risk for AD would behoove to consider BIN1 function in the context of its main expression in mature oligodendrocytes and the potential for a role of BIN1 in the membrane remodeling that accompanies the process of myelination.

Overexpression of the Insulin-Like Growth Factor II Receptor Increases β-Amyloid Production and Affects Cell Viability

ABSTRACT Amyloid β (Aβ) peptides originating from amyloid precursor protein (APP) in the endosomal-lysosomal compartments play a critical role in the development of Alzheimers disease (AD), the most common type of senile dementia affecting the elderly. Since insulin-like growth factor II (IGF-II) receptors facilitate the delivery of nascent lysosomal enzymes from the trans-Golgi network to endosomes, we evaluated their role in APP metabolism and cell viability using mouse fibroblast MS cells deficient in the murine IGF-II receptor and corresponding MS9II cells overexpressing the human IGF-II receptors. Our results show that IGF-II receptor overexpression increases the protein levels of APP. This is accompanied by an increase of β-site APP-cleaving enzyme 1 levels and an increase of β- and γ-secretase enzyme activities, leading to enhanced Aβ production. At the cellular level, IGF-II receptor overexpression causes localization of APP in perinuclear tubular structures, an increase of lipid raft components, and increased lipid raft partitioning of APP. Finally, MS9II cells are more susceptible to staurosporine-induced cytotoxicity, which can be attenuated by β-secretase inhibitor. Together, these results highlight the potential contribution of IGF-II receptor to AD pathology not only by regulating expression/processing of APP but also by its role in cellular vulnerability.

Regulation of dynamic BACE1 trafficking in neurons

Proteolytic processing of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase generates Aβ peptides. APP is constitutively trafficked through the secretory and endocytic pathways in cultured cells and neurons. The identities of cellular organelles and sorting pathways involved in amyloidogenic processing of APP have been extensively investigated. Although a consensus has not yet emerged, there is a general agreement from biochemical and genetic studies on the importance of endocytic APP trafficking for Aβ production. In neurons, APP is trafficked anterogradely along peripheral and central axons, and proteolytically processed during transit. Recent in vivo studies estimated that ~70% of Aβ released in the brain requires ongoing endocytosis, and synaptic activity regulates the vast majority of this endocytosis-dependent Aβ. BACE1 cleavage is thought to be the rate-limiting step in amyloidogenic processing of APP. Little, however, is known about endocytic BACE1 sorting and dynamic transport in neurons. We investigated BACE1 trafficking in cultured hippocampal neurons using live-cell imaging and selective labeling. This approach revealed dynamic neuronal transport of internalized BACE1 in dendrites and axons. BACE1 was transported in vesicles that were positive for markers of recycling endosomes. Dominant-negative expression and siRNA knock-down of proteins involved in endocytic transit revealed that BACE1 is dynamically transported in recycling endosomes and this process significantly contributes to amyloidogenic APP processing. Our results suggest that BACE1 trafficking in neuronal recycling endosomes is likely relevant for presynaptic Aβ production and contributes to Alzheimer’s disease pathogenesis.