Scott A. Shaffer

The apolipoprotein E ε4 ( APOE4 ) allele is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD), yet the mechanistic underpinnings by which it alters human microglial function remain poorly understood. Here, we utilize CRISPR/Cas9-engineered isogenic human induced pluripotent stem cell-derived microglia-like cells (iMGs) expressing APOE3 or APOE4 to systematically interrogate genotype-dependent effects on microglial proteomes and their secreted extracellular vesicles (EVs) under basal and fibrillar amyloid-β (fAβ)-challenged conditions. Quantitative proteomics revealed that APOE4 iMGs exhibit downregulated RNA processing, impaired phagocytosis, and exacerbated pro-inflammatory signaling, particularly upon fAβ exposure. Functionally, APOE4 iMGs demonstrated increased NF-κB activation, enhanced neuronal apoptosis and tau pathology in 2D and 3D co-culture systems. Interestingly, APOE4 iMGs released greater amount of EVs enriched in proteasomal components over APOE3 iMG. Integrated proteomic analyses further highlighted APOE4 -specific selective packaging of EV cargo linked to neuron-glia communication, inflammatory signaling, and proteostasis dysregulation, suggesting a potent, non-cell-autonomous mechanism driving neuronal damage. Consistently, these APOE4 iMGEVs were preferentially internalized by neurons, leading to disrupted neuronal proteostasis, impaired neuronal firing, and increased tau phosphorylation, particularly under pathological stress. Notably, pharmacologic inhibition of NF-κB in APOE4 iMGs reduced EV cargo-loading of proteasomal molecules and mitigated iMGEV-driven neuronal dysfunction and tau pathology. These findings establish a mechanistic link between APOE4 , dysregulated microglial homeostasis, and microglial EV-mediated propagation of proteostatic dysfunction and neurodegeneration, advancing our understanding of how APOE4 contributes to AD pathogenesis.

The inner membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis . The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis . Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here, we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis . We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis . Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as arabinogalactan and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the subpolar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, α1,3-fucosyltransferase and fucosyl 4- O -methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis , and M. tuberculosis utilizes this membrane location to enrich biosynthetic enzymes for its surface-exposed lipid virulence factors. IMPORTANCE: M. tuberculosis remains an important public health threat, with more than one million deaths every year. The pathogen’s ability to survive in the human host for decades highlights the importance of understanding how this bacterium regulates and coordinates its metabolism, cell envelope elongation, and growth. The IMD is a membrane structure that associates with the subpolar growth zone of actively growing mycobacterial cell, but its existence is only known in a non-pathogenic model, M. smegmatis . Here, we demonstrated the presence of the IMD in M. tuberculosis , making the IMD an evolutionarily conserved plasma membrane compartment in mycobacteria. Furthermore, our study revealed that the IMD may function as the factory for synthesizing phenolic glycolipids, virulence factors produced by slow-growing pathogenic species.

P2x purinoreceptor 7 (P2RX7), an ATP-gated ion channel, is known to play pivotal roles in the progression of Alzheimer’s disease (AD), although its cell type-specific pathological mechanisms have yet to be elucidated. Here, we show that genetic deletion of P2rx7 mitigates brain atrophy, tau accumulation and cognitive impairment in PS19 tauopathy mice. Specific deletion of P2rx7 in microglia, but not astrocytes, significantly suppresses tau propagation from the entorhinal cortex to CA1 in the hippocampus, an early event in AD pathology. Single-cell (sc)-RNA sequencing of mouse brains revealed specific P2rx7 expression in microglia, inducing inflammatory changes accompanied by elevated extracellular vesicles (EVs) secretion in PS19 mice. Brain-derived EVs (BDEVs) proteome demonstrated that P2RX7 increases EV cargo loading of tau and mitochondrial molecules in BDEVs from PS19 mice, which was further validated by single-molecule super-resolution. Notably, following the injection of BDEVs isolated from PS19 mice with or without P2rx7 deficiency, the microglial transcriptome of recipient mice revealed enriched DNA-sensing and type II interferon signaling in response to BDEVs from PS19 mice, which was diminished in the group injected with P2rx7-deficient BDEVs. Thus, our results indicate that P2RX7 regulates EV-mediated tau and mitochondrial transfer and inflammatory activation in microglia with increased EV secretion, thereby contributing to tauopathy and neurodegeneration, highlighting the therapeutic potential of targeting the P2RX7-EV axis in AD.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc). Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression loss to a PD patient cohort. However, it is still unknown whether Rit2 loss itself impacts DA neuron function and/or viability. Here we report that conditional Rit2 silencing in mouse DA neurons drove motor dysfunction that occurred earlier in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreased DA release, striatal DA content, phenotypic DAergic markers, DA neurons, and DAergic terminals, with increased pSer129-alpha synuclein and pSer935-LRRK2 expression. These results provide clear evidence that Rit2 loss is causal for SNc cell death and motor dysfunction, and reveal key sex-specific differences in the response to Rit2 loss.

Neuron-derived extracellular vesicles (NDEVs) are potential biomarkers of neurological diseases although their reliable molecular target is not well established. Here, we demonstrate that ATPase Na ⁺ /K ⁺ transporting subunit alpha 3 (ATP1A3) is abundantly expressed in extracellular vesicles (EVs) isolated from induced human neuron, brain, cerebrospinal fluid, and plasma in comparison with the presumed NDEV markers NCAM1 and L1CAM by using super-resolution microscopy and biochemical assessments. Proteomic analysis of immunoprecipitated ATP1A3 ⁺ brain-derived EVs shows higher enrichment of synaptic markers and cargo proteins relevant to Alzheimer’s disease (AD) compared to NCAM1 ⁺ or LICAM ⁺ EVs. Single particle analysis shows the elevated amyloid-β positivity in ATP1A3 ⁺ EVs from AD plasma, providing better diagnostic prediction of AD over other plasma biomarkers. Thus, ATP1A3 is a reliable target to isolate NDEV from biofluids for diagnostic research.

Stimulator of interferon genes (STING) is an essential adaptor protein required for the inflammatory response to cytosolic DNA. dsDNA activates cGAS to generate cGAMP, which binds and activates STING triggering a conformational change, oligomerization, and the IRF3- and NFκB-dependent transcription of type I Interferons (IFNs) and inflammatory cytokines, as well as the activation of autophagy. Aberrant activation of STING is now linked to a growing number of both rare as well as common chronic inflammatory diseases. Here, we identify and characterize a potent small-molecule inhibitor of STING. This compound, BB-Cl-amidine inhibits STING signaling and production of type I IFNs, IFN-stimulated genes (ISGs) and NFκB-dependent cytokines, but not other pattern recognition receptors. In vivo, BB-Cl-amidine alleviated pathology resulting from accrual of cytosolic DNA in Trex-1 mutant mice. Mechanistically BB-Cl-amidine inhibited STING oligomerization through modification of Cys ¹⁴⁸ . Collectively, our work uncovers an approach to inhibit STING activation and highlights the potential of this strategy for the treatment of STING-driven inflammatory diseases.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc) . Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression anomalies to a PD patient cohort. However, it is still unknown whether Rit2 loss itself is causative for PD or PD-like symptoms. Here we report that conditional Rit2 silencing in mouse DA neurons drove a progressive motor dysfunction that was more rapid in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreases in DA release, striatal DA content, phenotypic DAergic markers, and a loss of DA neurons, with increased pSer129-alpha synuclein expression. These results provide the first evidence that Rit2 loss is causal for SNc cell death and a PD-like phenotype, and reveal key sex-specific differences in the response to Rit2 loss.

Proteins destined for the secretory compartment of the cell are cotranslationally translocated into the endoplasmic reticulum. The majority of these proteins are N-glycosylated, a co- and posttranslational modification that ensures proper protein folding, stability, solubility, and cellular localization. Here, we show that the β subunit of the signal recognition particle receptor (SR) is required for assembly of the N-glycosylation–competent translocon. We report that guanine analog chemical probes identified by high-throughput screening or mutation of the SR- β guanosine triphosphate binding site cause an N-glycosylation–deficient phenotype. Neither method alters the association of SR- α with SR- β , but both approaches reduce the association of SR- β with the oligosaccharyltransferase complex. These experiments demonstrate that SR- β has a previously unrecognized function coordinating endoplasmic reticulum translation with N-glycosylation.

Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (M^Pro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, M^Pro is a novel therapeutic target. We identified two novel M^Pro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the M^Pro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PL^Pro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC₅₀ values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of M^Pro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.

Significance: Efforts to improve tuberculosis therapy include optimizing multidrug regimens to take advantage of drug–drug synergies. However, the complex host environment has a profound effect on bacterial metabolic state and drug activity, making predictions of optimal drug combinations difficult. In this study, we leverage a newly developed library of conditional knockdown Mycobacterium tuberculosis mutants in which genetic depletion of essential genes mimics the effect of drug therapy. This tractable system allowed us to assess the effect of growth condition on predicted drug–drug interactions. We found that these interactions can be differentially sensitive to the metabolic state, and select in vitro–defined interactions can be leveraged to accelerate bacterial killing during infection. These findings suggest strategies for optimizing tuberculosis therapy.