BackgroundExosomes, small extracellular vesicles of endosomal origin, have been suggested to be involved in both the metabolism and aggregation of Alzheimer’s disease (AD)-associated amyloid β-protein (Aβ). Despite their ubiquitous presence and the inclusion of components which can potentially interact with Aβ, the role of exosomes in regulating synaptic dysfunction induced by Aβ has not been explored.ResultsWe here provide in vivo evidence that exosomes derived from N2a cells or human cerebrospinal fluid can abrogate the synaptic-plasticity-disrupting activity of both synthetic and AD brain-derived Aβ. Mechanistically, this effect involves sequestration of synaptotoxic Aβ assemblies by exosomal surface proteins such as PrPC rather than Aβ proteolysis.ConclusionsThese data suggest that exosomes can counteract the inhibitory action of Aβ, which contributes to perpetual capability for synaptic plasticity.

BACE1 (␤-site ␤-amyloid precursor protein (APP)-cleaving enzyme 1) mediates the first proteolytic cleavage of APP, leading to amyloid ␤-peptide (A␤) production. It has been reported that BACE1 intracellular trafficking, in particular endosometo-TGN sorting, is mediated by adaptor complexes, such as retromer and Golgi-localized ␥-ear-containing ARF-binding proteins (GGAs). Here we investigated whether sortilin, a Vps10p domain-sorting receptor believed to participate in retromer-mediated transport of select membrane cargoes, contributes to the subcellular trafficking and activity of BACE1. Our initial studies revealed increased levels of sortilin in post-mortem brain tissue of AD patients and that overexpression of sortilin leads to increased BACE1-mediated cleavage of APP in cultured cells. In contrast, RNAi suppression of sortilin results in decreased BACE1-mediated cleavage of APP. We also found that sortilin interacts with BACE1 and that a sortilin construct lacking its cytoplasmic domain, which contains putative retromer sorting motifs, remains bound to BACE1. However, expression of this truncated sortilin redistributes BACE1 from the trans-Golgi network to the endosomes and substantially reduces the retrograde trafficking of BACE1. Site-directed mutagenesis and chimera experiments reveal that the cytoplasmic tail of sortilin, but not those from other VPS10p domain receptors (e.g. SorCs1b and SorLA), plays a unique role in BACE1 trafficking. Our studies suggest a new function for sortilin as a modulator of BACE1 retrograde trafficking and subsequent generation of A␤.

Pharmacological screening in physiologically relevant brain cells is crucial for identifying neuroactive compounds that better translate into in vivo biology and efficacious therapeutics. Pharmacological enhancement of apolipoprotein E (apoE), a cholesterol-transporting apolipoprotein, has been proposed as a promising therapeutic approach for Alzheimer's disease. Several nuclear receptor agonists were initially shown to increase brain apoE levels together with ATP-binding cassette transporter 1 (ABCA1), but their underlying mechanisms remain unclear. To gain an insight on brain apoE regulation, we performed an unbiased high-throughput screening of known drugs and bioactive compounds in cultured human primary astrocytes, the major apoE-producing cell type in the brain. We have identified several small molecules that increase apoE secretion via previously unknown mechanisms, including those not co-inducing ABCA1. These newly identified compounds are active preferentially in human astrocytes but not in an astrocytoma cell line, furnishing new tools for investigating biological pathways underlying brain apoE production.

Disrupted cross-cellular communication signaling (cellular crosstalk) has been implicated in neurodegenerative diseases, including Alzheimer's disease (AD). However, there is currently no systematic characterization of brain crosstalk networks in health and disease. We systematically characterized brain cellular crosstalk networks using single-nucleus transcriptomics data from a large cohort of control and AD brain donors (n=67). We found that crosstalk interactions between microglia and neurons were highly enriched to directly involve reported AD risk genes as ligands or receptors. Computational reconstruction of the co-expression networks associated with neuron-microglia crosstalk revealed they perturb additional known AD risk genes in microglia. We identified the interaction of neuronal SEMA6D (a PLXNA1 ligand) with a highly connected microglial regulatory sub-network involving TREM2, APOE, and HLA genes, which we predict is disrupted in late AD stages. Using CRISPR-modified human induced pluripotent stem cell (iPSC)-derived microglia and treatment with recombinant SEMA6D, we experimentally demonstrated that SEMA6D promotes microglial phagocytosis and cytokine (TNFα and IL-6) release in a TREM2-dependent manner. The novel discovery that the SEMA6D-PLXNA1/TREM2 signaling axis is involved in the regulation of microglia function demonstrates that our systematic characterization of cellular crosstalk networks is an important strategy for discovering specific mediators of significant cross-cellular interactions important to AD pathogenesis, gaining wider insights into the biology of this disease, and uncovering novel therapeutics.

SUMMARY Given the complex nature of Alzheimer’s disease (AD), a cell-based model that recapitulates physiological properties of the target neuronal population carries significant value in discovering improved drug candidates and chemical probes for uncovering disease mechanisms. We established phenotypic neuronal assays for biogenesis and synaptic action of amyloid β-peptide (Aβ) based on embryonic stem (ES) cell-derived neurons (ESNs). ESNs enriched with pyramidal neurons were robust, scalable and amenable to a small molecule screening assay, overcoming apparent limitations of neuronal models derived from human pluripotent cells. Small molecule screening of clinical compounds identified four compounds capable of reducing Aβ levels in ESNs derived from the Tg2576 mouse model of AD. Our approach is therefore highly suitable for phenotypic screening in AD drug discovery and has the potential to identify therapeutic candidates with improved efficacy and safety potential.