Alzheimer disease ␤-amyloid (A␤) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and ␥-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 A␤ 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 A␤. 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.

Neuronal network dysfunction and cognitive decline constitute the most prominent features of Alzheimer’s disease (AD), although mechanisms causing such impairments are yet to be determined. Here we report that virus-mediated delivery of the active spliced transcription factor X-Box binding protein 1s (XBP1s) in the hippocampus rescued spine density, synaptic plasticity and memory function in a mouse model of AD. XBP1s transcriptionally activated Kalirin-7 (Kal7), a protein that controls synaptic plasticity. In addition, we found reduced levels of Kal7 in primary neurons exposed to Aβ oligomers, transgenic mouse models and human AD brains. Short hairpin RNA-mediated knockdown of Kal7 altered synaptic plasticity and memory formation in naive mice. Further, reduction of endogenous Kal7 compromised the beneficial effects of XBP1s in Alzheimer’s model. Hence, our findings reveal that XBP1s is neuroprotective through a mechanism that engages Kal7 pathway with therapeutic implications in AD pathology.

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.

␥-Secretase complexes achieve the production of amyloid peptides playing a key role in Alzheimer disease. These proteases have many substrates involved in important physiological functions. They are composed of two constant subunits, nicastrin and PEN2, and two variable ones, presenilin (PS1 or PS2) and APH1 (APH1aL, APH1aS, or APH1b). Whether the composition of a given ␥-secretase complex determines a specific cellular targeting remains unsolved. Here we combined a bidirectional inducible promoter and 2A peptide technology to generate constructs for the temporary, stoichiometric co-expression of six different combinations of the four ␥-secretase subunits including EGFP-tagged nicastrin. These plasmids allow for the formation of functional ␥-secretase complexes displaying specific activities and maturations. We show that PS1-containing ␥-secretase complexes were targeted to the plasma membrane, whereas PS2-containing ones were addressed to the trans-Golgi network, to recycling endosomes, and, depending on the APH1-variant, to late endocytic compartments. Overall, these novel constructs unravel a presenilin-dependent subcellular targeting of ␥-secretase complexes. These tools should prove useful to determine whether the cellular distribution of ␥-secretase complexes contributes to substrate selectivity and to delineate regulations of their trafficking.