Proximity Proteome Analysis Reveals Novel TREM2 Interactors in the ER-Mitochondria Interface of Human Microglia.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a central role in microglial biology and the pathogenesis of Alzheimer's disease (AD). Besides DNAX-activating protein 12 (DAP12), a communal adaptor for TREM2 and many other receptors, other cellular interactors of TREM2 remain largely elusive. We employed a 'proximity labeling' approach using a biotin ligase, TurboID, for mapping protein-protein interactions in live mammalian cells. We discovered novel TREM2-proximal proteins with diverse functions, including those localized to the Mitochondria-ER contact sites (MERCs), a dynamic subcellular 'hub' implicated in a number of crucial cell physiology such as lipid metabolism. TREM2 deficiency alters the thickness (inter-organelle distance) of MERCs, a structural parameter of metabolic state, in microglia derived from human induced pluripotent stem cells. Our TurboID-based TREM2 interactome study suggest novel roles for TREM2 in the structural plasticity of the MERCs, raising the possibility that dysregulation of MERC-related TREM2 functions contribute to AD pathobiology.

Bioactive Compound Screen for Pharmacological Enhancers of Apolipoprotein E in Primary Human Astrocytes.

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.

Exosomes neutralize synaptic-plasticity-disrupting activity of Aß assemblies in vivo.

BACKGROUND: Exosomes, 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 ß-protein (Aß). Despite their ubiquitous presence and the inclusion of components which can potentially interact with Aß, the role of exosomes in regulating synaptic dysfunction induced by Aß has not been explored. RESULTS: We 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 Aß. Mechanistically, this effect involves sequestration of synaptotoxic Aß assemblies by exosomal surface proteins such as PrPC rather than Aß proteolysis. CONCLUSIONS: These data suggest that exosomes can counteract the inhibitory action of Aß, which contributes to perpetual capability for synaptic plasticity.

Phenotypic assays for ß-amyloid in mouse embryonic stem cell-derived neurons.

Given the complex nature of Alzheimer's disease (AD), a cell-based model that recapitulates the physiological properties of the target neuronal population would be extremely valuable for discovering improved drug candidates and chemical probes to uncover disease mechanisms. We established phenotypic neuronal assays for the biogenesis and synaptic action of amyloid ß peptide (Aß) based on embryonic stem cell-derived neurons (ESNs). ESNs enriched with pyramidal neurons were robust, scalable, and amenable to a small-molecule screening assay, overcoming the apparent limitations of neuronal models derived from human pluripotent cells. Small-molecule screening of clinical compounds identified four compounds capable of reducing Aß 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.

BACE1 retrograde trafficking is uniquely regulated by the cytoplasmic domain of sortilin.

BACE1 (ß-site ß-amyloid precursor protein (APP)-cleaving enzyme 1) mediates the first proteolytic cleavage of APP, leading to amyloid ß-peptide (Aß) production. It has been reported that BACE1 intracellular trafficking, in particular endosome-to-TGN sorting, is mediated by adaptor complexes, such as retromer and Golgi-localized γ-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 Aß.