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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1558-9) contains supplementary material, which is available to authorized users.

BackgroundBACE1 is one of the two enzymes that cleave amyloid precursor protein to generate Alzheimer's 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.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0124-1) contains supplementary material, which is available to authorized users.

Summary Alzheimer’s 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.

SUMMARY Abnormal accumulation of β-secretase (BACE1) in dystrophic neurites and presynaptic β-amyloid (Aβ) production contribute to Alzheimer's disease pathogenesis. Little, however, is known about BACE1 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, 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 axonal sorting and dynamic axonal transport of BACE1. 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 represent the first demonstration of unidirectional endocytic transport of any cargo in dendrites. Moreover, they reveal a novel role for EHD proteins in neuronal BACE1 transcytosis and Aβ production, processes that are highly relevant for Alzheimer's disease.

Background: NF-B regulates BACE1 but there is little data suggesting ␤APP and ␥-secretase involvement. Results: NF-B differentially regulates A␤ production at physiological and supraphysiological A␤ concentrations by modulating transactivation of ␤APP and ␥-secretase promoters, thereby controlling ␥-secretase activity. Conclusion: Under physiological conditions, NF-B regulates A␤ homeostasis while it contributes in increasing A␤ production in the pathological context. Significance: NF-B may be seen as a potential therapeutic target.

␤-Amyloid (A␤) peptides that accumulate in Alzheimer disease are generated from the ␤-amyloid precursor protein (␤APP) by cleavages by ␤-secretase BACE1 and by presenilindependent ␥-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 ␥-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 ␥-secretase. The overexpression of APP intracellular domain (AICD), the ␥/⑀-secretase-derived C-terminal product of ␤-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 APP⑀ (the N-terminal product of ␤APP generated by ⑀-secretase cleavage harboring the A␤ domain but lacking the AICD sequence), suggesting that the production of A␤ could account for the modulation of BACE1. Accordingly, we show that HEK293 cells overexpressing wild-type ␤APP exhibit a DFK167-sensitive increase in BACE1 promoter transactivation that is increased by the A␤-potentiating Swedish mutation. This effect was mimicked by exogenous application of A␤42 but not A␤40 or by transient transfection of cDNA encoding A␤42 sequence. The I B kinase inhibitor BMS345541 prevents A␤-induced BACE1 promoter transactivation suggesting that NF B could mediate this A␤-associated phenotype. Accordingly, the overexpression of wild-type or Swedish mutated ␤APP does not modify the transactivation of BACE1 promoter constructs lacking NF B-responsive element. Furthermore, APP/␤-amyloid precursor protein-like protein deficiency does not affect BACE1 activity and expression. Overall, these data suggest that physiological levels of endogenous A␤ are not sufficient per se to modulate BACE1 promoter transactivation but that exacerbated A␤ production linked to wild-type or Swedish mutated ␤APP overexpression modulates BACE1 promoter transactivation and activity via an NF B-dependent pathway.Alzheimer disease (AD) 3 is characterized by abnormal deposition of a set of hydrophobic peptides called amyloid ␤ (A␤) peptides. The increase of cerebral A␤ levels is one of the common denominators characterizing both sporadic and familial forms of AD and therefore, if not demonstrated as the etiological cause of AD pathology, is often considered as a key factor contributing to the degenerative process (1).The mechanisms by which A␤ peptides are generated are a matter of intense research in the AD field. A␤ peptides are released from a transmembrane protein, ␤-amyloid precursor protein (␤APP), by the sequential attacks by ␤-and ␥-secretase that liberate the N-and C-terminal moieties of A␤ peptides, respectively (2). All ␤-secretase-like activi...

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by pathological brain lesions and a decline in cognitive function. β-Amyloid peptides (Aβ), derived from proteolytic processing of amyloid precursor protein (APP), play a central role in AD pathogenesis. β-Site APP cleaving enzyme 1 (BACE1), the transmembrane aspartyl protease which initiates Aβ production, is axonally transported in neurons and accumulates in dystrophic neurites near cerebral amyloid deposits in AD. BACE1 is modified by S-palmitoylation at four juxtamembrane cysteine residues. S-palmitoylation is a dynamic posttranslational modification that is important for trafficking and function of several synaptic proteins. Here, we investigated the in vivo significance of BACE1 S-palmitoylation through the analysis of knock-in mice with cysteine-to-alanine substitution at the palmitoylated residues (4CA mice). BACE1 expression, as well as processing of APP and other neuronal substrates, was unaltered in 4CA mice despite the lack of BACE1 S-palmitoylation and reduced lipid raft association. Whereas steady-state Aβ levels were similar, synaptic activity-induced endogenous Aβ production was not observed in 4CA mice. Furthermore, we report a significant reduction of cerebral amyloid burden and BACE1 accumulation in dystrophic neurites in the absence of BACE1 S-palmitoylation in mouse models of AD amyloidosis. Studies in cultured neurons suggest that S-palmitoylation is required for dendritic spine localization and axonal targeting of BACE1. Finally, the lack of BACE1 S-palmitoylation mitigates cognitive deficits in 5XFAD mice. Using transgenic mouse models, these results demonstrate that intrinsic posttranslational S-palmitoylation of BACE1 has a significant impact on amyloid pathogenesis and the consequent cognitive decline.