Proximity labeling proteomics reveals Kv1.3 potassium channel immune interactors in microglia.

Microglia are resident immune cells of the brain and regulate its inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease associated microglia (DAM). DAM express higher levels of proinflammatory signaling molecules, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to identify the potential proteins interacting with Kv1.3 during transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia following TLR4-mediated activation. Electrophysiology, western blotting, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during a TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C-terminus. We determined that the N-terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g. NUDC, TIMM50). Whereas, the C-terminus interacts with immune signaling proteins in an LPS-induced inflammatory response (e.g. STAT1, TLR2, and C3). There are 70 proteins that rely on the C-terminal PDZ-binding domain to interact with Kv1.3 (e.g. ND3, Snx3, and Sun1). Furthermore, we used Kv1.3 blockade to verify functional coupling between Kv1.3 and interferon-mediated STAT1 activation. Overall, we highlight that the Kv1.3 potassium channel functions beyond conducting the outward flux of potassium ions in an inflammatory context and that Kv1.3 modulates the activity of key immune signaling proteins, such as STAT1 and C3.

Profiling the neuroimmune cascade in 3xTg-AD mice exposed to successive mild traumatic brain injuries.

Repetitive mild traumatic brain injuries (rmTBI) sustained within a window of vulnerability can result in long term cognitive deficits, depression, and eventual neurodegeneration associated with tau pathology, amyloid beta (Aß) plaques, gliosis, and neuronal and functional loss. However, a comprehensive study relating acute changes in immune signaling and glial reactivity to neuronal changes and pathological markers after single and repetitive mTBIs is currently lacking. In the current study, we addressed the question of how repeated injuries affect the brain neuroimmune response in the acute phase of injury (< 24 h) by exposing the 3xTg-AD mouse model of tau and Aß pathology to successive (1x-5x) once-daily weight drop closed-head injuries and quantifying immune markers, pathological markers, and transcriptional profiles at 30 min, 4 h, and 24 h after each injury. We used young adult 2-4 month old 3xTg-AD mice to model the effects of rmTBI in the absence of significant tau and Aß pathology. We identified pronounced sexual dimorphism in this model, with females eliciting more diverse changes after injury compared to males. Specifically, females showed: (1) a single injury caused a decrease in neuron-enriched genes inversely correlated with inflammatory protein expression and an increase in AD-related genes within 24 h, (2) each injury significantly increased a group of cortical cytokines (IL-1α, IL-1ß, IL-2, IL-9, IL-13, IL-17, KC) and MAPK phospho-proteins (phospho-Atf2, phospho-Mek1), several of which co-labeled with neurons and correlated with phospho-tau, and (3) repetitive injury caused increased expression of genes associated with astrocyte reactivity and macrophage-associated immune function. Collectively our data suggest that neurons respond to a single injury within 24 h, while other cell types, including astrocytes, transition to inflammatory phenotypes within days of repetitive injury.

Proteomic changes in the human cerebrovasculature in Alzheimer's disease and related tauopathies linked to peripheral biomarkers in plasma and cerebrospinal fluid.

INTRODUCTION: Cerebrovascular dysfunction is a pathological hallmark of Alzheimer's disease (AD). Nevertheless, detecting cerebrovascular changes within bulk tissues has limited our ability to characterize proteomic alterations from less abundant cell types. METHODS: We conducted quantitative proteomics on bulk brain tissues and isolated cerebrovasculature from the same individuals, encompassing control (N = 28), progressive supranuclear palsy (PSP) (N = 18), and AD (N = 21) cases. RESULTS: Protein co-expression network analysis identified unique cerebrovascular modules significantly correlated with amyloid plaques, cerebrovascular amyloid angiopathy (CAA), and/or tau pathology. The protein products within AD genetic risk loci were concentrated within cerebrovascular modules. The overlap between differentially abundant proteins in AD cerebrospinal fluid (CSF) and plasma with cerebrovascular network highlighted a significant increase of matrisome proteins, SMOC1 and SMOC2, in CSF, plasma, and brain. DISCUSSION: These findings enhance our understanding of cerebrovascular deficits in AD, shedding light on potential biomarkers associated with CAA and vascular dysfunction in neurodegenerative diseases.

Development and validation of a SCORing systEm for pre-thrombectomy diagnosis of IntraCranial Atherosclerotic Disease (Score-ICAD).

BACKGROUND: Early identification of intracranial atherosclerotic disease (ICAD) may impact the management of patients undergoing mechanical thrombectomy (MT). We sought to develop and validate a scoring system for pre-thrombectomy diagnosis of ICAD in anterior circulation large vessel/distal medium vessel occlusion strokes (LVOs/DMVOs). METHODS: Retrospective analysis of two prospectively maintained comprehensive stroke center databases including patients with anterior circulation occlusions spanning 2010-22 (development cohort) and 2018-22 (validation cohort). ICAD cases were matched for age and sex (1:1) to non-ICAD controls. RESULTS: Of 2870 MTs within the study period, 348 patients were included in the development cohort: 174 anterior circulation ICAD (6% of 2870 MTs) and 174 controls. Multivariable analysis ß coefficients led to a 20 point scale: absence of atrial fibrillation (5); vascular risk factor burden (1) for each of hypertension, diabetes, smoking, and hyperlipidemia; multifocal single artery stenoses on CT angiography (3); absence of territorial cortical infarct (3); presence of borderzone infarct (3); or ipsilateral carotid siphon calcification (2). The validation cohort comprised 56 ICAD patients (4.1% of 1359 MTs): 56 controls. Area under the receiver operating characteristic curve was 0.88 (0.84-0.91) and 0.82 (0.73-0.89) in the development and validation cohorts, respectively. Calibration slope and intercept showed a good fit for the development cohort although with overestimated risk for the validation cohort. After intercept adjustment, the overestimation was corrected (intercept 0, 95% CI -0.5 to -0.5; slope 0.8, 95% CI 0.5 to 1.1). In the full cohort (n=414), ≥11 points showed the best performance for distinguishing ICAD from non-ICAD, with 0.71 (95% CI 0.65 to 0.78) sensitivity and 0.82 (95% CI 0.77 to 0.87) specificity, and 3.92 (95% CI 2.92 to 5.28) positive and 0.35 (95% CI 0.28 to 0.44) negative likelihood ratio. Scores ≥12 showed 90% specificity and 63% sensitivity. CONCLUSION: The proposed scoring system for preprocedural diagnosis of ICAD LVOs and DMVOs presented satisfactory discrimination and calibration based on clinical and non-invasive radiological data.

Native-state proteomics of Parvalbumin interneurons identifies unique molecular signatures and vulnerabilities to early Alzheimer's pathology.

Dysfunction in fast-spiking parvalbumin interneurons (PV-INs) may represent an early pathophysiological perturbation in Alzheimer's Disease (AD). Defining early proteomic alterations in PV-INs can provide key biological and translationally-relevant insights. We used cell-type-specific in-vivo biotinylation of proteins (CIBOP) coupled with mass spectrometry to obtain native-state PV-IN proteomes. PV-IN proteomic signatures include high metabolic and translational activity, with over-representation of AD-risk and cognitive resilience-related proteins. In bulk proteomes, PV-IN proteins were associated with cognitive decline in humans, and with progressive neuropathology in humans and the 5xFAD mouse model of Aß pathology. PV-IN CIBOP in early stages of Aß pathology revealed signatures of increased mitochondria and metabolism, synaptic and cytoskeletal disruption and decreased mTOR signaling, not apparent in whole-brain proteomes. Furthermore, we demonstrated pre-synaptic defects in PV-to-excitatory neurotransmission, validating our proteomic findings. Overall, in this study we present native-state proteomes of PV-INs, revealing molecular insights into their unique roles in cognitive resiliency and AD pathogenesis.

Proteomic Changes in the Human Cerebrovasculature in Alzheimer's Disease and Related Tauopathies Linked to Peripheral Biomarkers in Plasma and Cerebrospinal Fluid.

Dysfunction of the neurovascular unit stands as a significant pathological hallmark of Alzheimer's disease (AD) and age-related neurodegenerative diseases. Nevertheless, detecting vascular changes in the brain within bulk tissues has proven challenging, limiting our ability to characterize proteomic alterations from less abundant cell types. To address this challenge, we conducted quantitative proteomic analyses on both bulk brain tissues and cerebrovascular-enriched fractions from the same individuals, encompassing cognitively unimpaired control, progressive supranuclear palsy (PSP), and AD cases. Protein co-expression network analysis identified modules unique to the cerebrovascular fractions, specifically enriched with pericytes, endothelial cells, and smooth muscle cells. Many of these modules also exhibited significant correlations with amyloid plaques, cerebral amyloid angiopathy (CAA), and/or tau pathology in the brain. Notably, the protein products within AD genetic risk loci were found concentrated within modules unique to the vascular fractions, consistent with a role of cerebrovascular deficits in the etiology of AD. To prioritize peripheral AD biomarkers associated with vascular dysfunction, we assessed the overlap between differentially abundant proteins in AD cerebrospinal fluid (CSF) and plasma with a vascular-enriched network modules in the brain. This analysis highlighted matrisome proteins, SMOC1 and SMOC2, as being increased in CSF, plasma, and brain. Immunohistochemical analysis revealed SMOC1 deposition in both parenchymal plaques and CAA in the AD brain, whereas SMOC2 was predominantly localized to CAA. Collectively, these findings significantly enhance our understanding of the involvement of cerebrovascular abnormalities in AD, shedding light on potential biomarkers and molecular pathways associated with CAA and vascular dysfunction in neurodegenerative diseases.

Proteomic Signaling of Dual-Specificity Phosphatase 4 (DUSP4) in Alzheimer's Disease.

DUSP4 is a member of the DUSP (dual-specificity phosphatase) subfamily that is selective to the mitogen-activated protein kinases (MAPK) and has been implicated in a range of biological processes and functions in Alzheimer's disease (AD). In this study, we utilized the stereotactic delivery of adeno-associated virus (AAV)-DUSP4 to overexpress DUSP4 in the dorsal hippocampus of 5xFAD and wildtype (WT) mice, then used mass spectrometry (MS)-based proteomics along with the label-free quantification to profile the proteome and phosphoproteome in the hippocampus. We identified protein expression and phosphorylation patterns modulated in 5xFAD mice and examined the sex-specific impact of DUSP4 overexpression on the 5xFAD proteome/phosphoproteome. In 5xFAD mice, a substantial number of proteins were up- or down-regulated in both male and female mice in comparison to age and sex-matched WT mice, many of which are involved in AD-related biological processes, such as activated immune response or suppressed synaptic activities. Many proteins in pathways, such as immune response were found to be suppressed in response to DUSP4 overexpression in male 5xFAD mice. In contrast, such a shift was absent in female mice. For the phosphoproteome, we detected an array of phosphorylation sites regulated in 5xFAD compared to WT and modulated via DUSP4 overexpression in each sex. Interestingly, 5xFAD- and DUSP4-associated phosphorylation changes occurred in opposite directions. Strikingly, both the 5xFAD- and DUSP4-associated phosphorylation changes were found to be mostly in neurons and play key roles in neuronal processes and synaptic functions. Site-centric pathway analysis revealed that both the 5xFAD- and DUSP4-associated phosphorylation sites were enriched for a number of kinase sets in females but only a limited number of sets of kinases in male mice. Taken together, our results suggest that male and female 5xFAD mice responded to DUSP4 overexpression via shared and sex-specific molecular mechanisms, which might underly similar reductions in amyloid pathology in both sexes while learning deficits were reduced in only females with DUSP4 overexpression. Finally, we validated our findings with the sex-specific AD-associated proteomes in human cohorts and further developed DUSP4-centric proteomic network models and signaling maps for each sex.

Identification of State-Specific Proteomic and Transcriptomic Signatures of Microglia-Derived Extracellular Vesicles.

Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impact EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and noncoding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and omics communities and provide novel insights into the role of microglia-derived EVs in neuroinflammation.

Entorhinal cortex vulnerability to human APP expression promotes hyperexcitability and tau pathology.

Preventative treatment for Alzheimer's Disease is of dire importance, and yet, cellular mechanisms underlying early regional vulnerability in Alzheimer's Disease remain unknown. In human patients with Alzheimer's Disease, one of the earliest observed pathophysiological correlates to cognitive decline is hyperexcitability1. In mouse models, early hyperexcitability has been shown in the entorhinal cortex, the first cortical region impacted by Alzheimer's Disease2-4. The origin of hyperexcitability in early-stage disease and why it preferentially emerges in specific regions is unclear. Using cortical-region and cell-type- specific proteomics and patch-clamp electrophysiology, we uncovered differential susceptibility to human-specific amyloid precursor protein (hAPP) in a model of sporadic Alzheimer's. Unexpectedly, our findings reveal that early entorhinal hyperexcitability may result from intrinsic vulnerability of parvalbumin interneurons, rather than the suspected layer II excitatory neurons. This vulnerability of entorhinal PV interneurons is specific to hAPP, as it could not be recapitulated with increased murine APP expression. Furthermore, the Somatosensory Cortex showed no such vulnerability to adult-onset hAPP expression, likely resulting from PV-interneuron variability between the two regions based on physiological and proteomic evaluations. Interestingly, entorhinal hAPP-induced hyperexcitability was quelled by co-expression of human Tau at the expense of increased pathological tau species. This study suggests early disease interventions targeting non-excitatory cell types may protect regions with early vulnerability to pathological symptoms of Alzheimer's Disease and downstream cognitive decline.

Identification of state-specific proteomic and transcriptomic signatures of microglia-derived extracellular vesicles.

Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impacts EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and non-coding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and glial communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation.

Profiling the neuroimmune cascade in 3xTg mice exposed to successive mild traumatic brain injuries.

Repetitive mild traumatic brain injuries (rmTBI) sustained within a window of vulnerability can result in long term cognitive deficits, depression, and eventual neurodegeneration associated with tau pathology, amyloid beta (Aß) plaques, gliosis, and neuronal and functional loss. However, we have limited understanding of how successive injuries acutely affect the brain to result in these devastating long-term consequences. In the current study, we addressed the question of how repeated injuries affect the brain in the acute phase of injury (<24hr) by exposing the 3xTg-AD mouse model of tau and Aß pathology to successive (1x, 3x, 5x) once-daily weight drop closed-head injuries and quantifying immune markers, pathological markers, and transcriptional profiles at 30min, 4hr, and 24hr after each injury. We used young adult mice (2-4 months old) to model the effects of rmTBI relevant to young adult athletes, and in the absence of significant tau and Aß pathology. Importantly, we identified pronounced sexual dimorphism, with females eliciting more differentially expressed proteins after injury compared to males. Specifically, females showed: 1) a single injury caused a decrease in neuron-enriched genes inversely correlated with inflammatory protein expression as well as an increase in AD-related genes within 24hr, 2) each injury significantly increased expression of a group of cortical cytokines (IL-1α, IL-1ß, IL-2, IL-9, IL-13, IL-17, KC) and MAPK phospho-proteins (phospho-Atf2, phospho-Mek1), several of which were co-labeled with neurons and correlated with phospho-tau, and 3) repetitive injury caused increased expression of genes associated with astrocyte reactivity and immune function. Collectively our data suggest that neurons respond to a single injury within 24h, while other cell types including astrocytes transition to inflammatory phenotypes within days of repetitive injury.

Factors associated with stroke after COVID-19 vaccination: a statewide analysis.

Background: The objective of our study was to evaluate vaccine type, COVID-19 infection, and their association with stroke soon after COVID-19 vaccination. Methods: In a retrospective cohort study, we estimated the 21-day post-vaccination incidence of stroke among the recipients of the first dose of a COVID-19 vaccine. We linked the Georgia Immunization Registry with the Georgia Coverdell Acute Stroke Registry and the Georgia State Electronic Notifiable Disease Surveillance System data to assess the relative risk of stroke by the vaccine type. Results: Approximately 5 million adult Georgians received at least one COVID-19 vaccine between 1 December 2020 and 28 February 2022: 54% received BNT162b2, 41% received mRNA-1273, and 5% received Ad26.COV2.S. Those with concurrent COVID-19 infection within 21 days post-vaccination had an increased risk of ischemic (OR = 8.00, 95% CI: 4.18, 15.31) and hemorrhagic stroke (OR = 5.23, 95% CI: 1.11, 24.64) with no evidence for interaction between the vaccine type and concurrent COVID-19 infection. The 21-day post-vaccination incidence of ischemic stroke was 8.14, 11.14, and 10.48 per 100,000 for BNT162b2, mRNA-1273, and Ad26.COV2.S recipients, respectively. After adjusting for age, race, gender, and COVID-19 infection status, there was a 57% higher risk (OR = 1.57, 95% CI: 1.02, 2.42) for ischemic stroke within 21 days of vaccination associated with the Ad26.COV2.S vaccine compared to BNT162b2; there was no difference in stroke risk between mRNA-1273 and BNT162b2. Conclusion: Concurrent COVID-19 infection had the strongest association with early ischemic and hemorrhagic stroke after the first dose of COVID-19 vaccination. Although not all determinants of stroke, particularly comorbidities, were considered in this analysis, the Ad26.COV2.S vaccine was associated with a higher risk of early post-vaccination ischemic stroke than BNT162b2.

Telemedicine impact on post-stroke outpatient follow-up in an academic healthcare network during the COVID-19 pandemic.

BACKGROUND: The expansion of telemedicine associated with the COVID-19 pandemic has influenced outpatient medical care. The objective of our study was to determine the impact of telemedicine on post-acute stroke clinic follow-up. METHODS: We retrospectively evaluated the impact of telemedicine in Emory Healthcare, an academic healthcare system of comprehensive and primary stroke centers in Atlanta, Georgia, on post-hospital stroke clinic follow-up. We compared the frequency of 90-day follow-up in a centralized subspecialty stroke clinic among patients hospitalized before the local COVID-19 pandemic (January 1, 2019- February 28, 2020), during (March 1- April 30, 2020) and after telemedicine implementation (May 1- December 31, 2020). A comparison was made across hospitals less than 1 mile, 10 miles, and 25 miles from the stroke clinic. RESULTS: Of 1096 ischemic stroke patients discharged home or to a rehab facility during the study period, 342 (31%) had follow-up in the Emory Stroke Clinic (comprehensive stroke center 46%, primary stroke center 10 miles away 18%, primary stroke center 25 miles away 14%). Overall, 90-day follow-up increased from 19% to 41% after telemedicine implementation (p<0.001) with telemedicine appointments amounting for up to 28% of all follow-up visits. In multivariable analysis, factors associated with teleneurology follow-up (vs no follow-up) included discharge from the comprehensive stroke center, thrombectomy treatment, private insurance, private transport to the hospital, NIHSS 0-5 and history of dyslipidemia. CONCLUSIONS: Despite telemedicine implementation at an academic healthcare network successfully increasing post-stroke discharge follow-up in a centralized subspecialty stroke clinic, the majority of patients did not complete 90-day follow-up during the COVID-19 pandemic.

Native-state proteomics of Parvalbumin interneurons identifies novel molecular signatures and metabolic vulnerabilities to early Alzheimer's disease pathology.

One of the earliest pathophysiological perturbations in Alzheimer's Disease (AD) may arise from dysfunction of fast-spiking parvalbumin (PV) interneurons (PV-INs). Defining early protein-level (proteomic) alterations in PV-INs can provide key biological and translationally relevant insights. Here, we use cell-type-specific in vivo biotinylation of proteins (CIBOP) coupled with mass spectrometry to obtain native-state proteomes of PV interneurons. PV-INs exhibited proteomic signatures of high metabolic, mitochondrial, and translational activity, with over-representation of causally linked AD genetic risk factors. Analyses of bulk brain proteomes indicated strong correlations between PV-IN proteins with cognitive decline in humans, and with progressive neuropathology in humans and mouse models of Aß pathology. Furthermore, PV-IN-specific proteomes revealed unique signatures of increased mitochondrial and metabolic proteins, but decreased synaptic and mTOR signaling proteins in response to early Aß pathology. PV-specific changes were not apparent in whole-brain proteomes. These findings showcase the first native state PV-IN proteomes in mammalian brain, revealing a molecular basis for their unique vulnerabilities in AD.

APOE expression and secretion are modulated by mitochondrial dysfunction.

Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer's disease risk factor apolipoprotein E (APOE) and other secretome components. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.

Advances in proteomic phenotyping of microglia in neurodegeneration.

Microglia are dynamic resident immune cells of the central nervous system (CNS) that sense, survey, and respond to changes in their environment. In disease states, microglia transform from homeostatic to diverse molecular phenotypic states that play complex and causal roles in neurologic disease pathogenesis, as evidenced by the identification of microglial genes as genetic risk factors for neurodegenerative disease. While advances in transcriptomic profiling of microglia from the CNS of humans and animal models have provided transformative insights, the transcriptome is only modestly reflective of the proteome. Proteomic profiling of microglia is therefore more likely to provide functionally and therapeutically relevant targets. In this review, we discuss molecular insights gained from transcriptomic studies of microglia in the context of Alzheimer's disease as a prototypic neurodegenerative disease, and highlight existing and emerging approaches for proteomic profiling of microglia derived from in vivo model systems and human brain.

Cellular Proteomic Profiling Using Proximity Labeling by TurboID-NES in Microglial and Neuronal Cell Lines.

Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity-dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without the need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole-cell proteome and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID labeled endolysosome, translation, vesicle, and signaling proteins in BV2 microglia and synaptic, neuron projection, and microtubule proteins in N2A neurons. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells and did not affect lipopolysaccharide-mediated cytokine production or resting cellular respiration in BV2 cells. MS analysis of the microglial biotin-labeled proteins captured the impact of lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory proteins (Il1a, Irg1, and Oasl1) and decreased anti-inflammatory proteins (Arg1 and Mgl2).