Syed Ali Raza

A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

BackgroundKv1.3 potassium channels regulate microglial functions and are overexpressed in neuroinflammatory diseases. Kv1.3 blockade may selectively inhibit pro-inflammatory microglia in neurological diseases but the molecular and cellular mechanisms regulated by Kv1.3 channels are poorly defined.MethodsWe performed immunoblotting and flow cytometry to confirm Kv1.3 channel upregulation in lipopolysaccharide (LPS)-activated BV2 microglia and in brain mononuclear phagocytes freshly isolated from LPS-treated mice. Quantitative proteomics was performed on BV2 microglia treated with control, LPS, ShK-223 (highly selective Kv1.3 blocker), and LPS+ShK-223. Gene ontology (GO) analyses of Kv1.3-dependent LPS-regulated proteins were performed, and the most representative proteins and GO terms were validated. Effects of Kv1.3-blockade on LPS-activated BV2 microglia were studied in migration, focal adhesion formation, reactive oxygen species production, and phagocytosis assays. In vivo validation of protein changes and predicted molecular pathways were performed in a model of systemic LPS-induced neuroinflammation, employing antigen presentation and T cell proliferation assays. Informed by pathway analyses of proteomic data, additional mechanistic experiments were performed to identify early Kv1.3-dependent signaling and transcriptional events.ResultsLPS-upregulated cell surface Kv1.3 channels in BV2 microglia and in microglia and CNS-infiltrating macrophages isolated from LPS-treated mice. Of 144 proteins differentially regulated by LPS (of 3141 proteins), 21 proteins showed rectification by ShK-223. Enriched cellular processes included MHCI-mediated antigen presentation (TAP1, EHD1), cell motility, and focal adhesion formation. In vitro, ShK-223 decreased LPS-induced focal adhesion formation, reversed LPS-induced inhibition of migration, and inhibited LPS-induced upregulation of EHD1, a protein involved in MHCI trafficking. In vivo, intra-peritoneal ShK-223 inhibited LPS-induced MHCI expression by CD11b+CD45low microglia without affecting MHCI expression or trafficking of CD11b+CD45high macrophages. ShK-223 inhibited LPS-induced MHCI-restricted antigen presentation to ovalbumin-specific CD8+ T cells both in vitro and in vivo. Kv1.3 co-localized with the LPS receptor complex and regulated LPS-induced early serine (S727) STAT1 phosphorylation.ConclusionsWe have unraveled novel molecular and functional roles for Kv1.3 channels in pro-inflammatory microglial activation, including a Kv1.3 channel-regulated pathway that facilitates MHCI expression and MHCI-dependent antigen presentation by microglia to CD8+ T cells. We also provide evidence for neuro-immunomodulation by systemically administered ShK peptides. Our results further strengthen the therapeutic candidacy of microglial Kv1.3 channels in neurologic diseases.

Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer’s disease

BackgroundDisease-associated-microglia (DAM) represent transcriptionally-distinct and neurodegeneration-specific microglial profiles with unclear significance in Alzheimer’s disease (AD). An understanding of heterogeneity within DAM and their key regulators may guide pre-clinical experimentation and drug discovery.MethodsWeighted co-expression network analysis (WGCNA) was applied to existing microglial transcriptomic datasets from neuroinflammatory and neurodegenerative disease mouse models to identify modules of highly co-expressed genes. These modules were contrasted with known signatures of homeostatic microglia and DAM to reveal novel molecular heterogeneity within DAM. Flow cytometric validation studies were performed to confirm existence of distinct DAM sub-populations in AD mouse models predicted by WGCNA. Gene ontology analyses coupled with bioinformatics approaches revealed drug targets and transcriptional regulators of microglial modules predicted to favorably modulate neuroinflammation in AD. These guided in-vivo and in-vitro studies in mouse models of neuroinflammation and neurodegeneration (5xFAD) to determine whether inhibition of pro-inflammatory gene expression and promotion of amyloid clearance was feasible. We determined the human relevance of these findings by integrating our results with AD genome-wide association studies and human AD and non-disease post-mortem brain proteomes.ResultsWGCNA applied to microglial gene expression data revealed a transcriptomic framework of microglial activation that predicted distinct pro-inflammatory and anti-inflammatory phenotypes within DAM, which we confirmed in AD and aging models by flow cytometry. Pro-inflammatory DAM emerged earlier in mouse models of AD and were characterized by pro-inflammatory genes (Tlr2, Ptgs2, Il12b, Il1b), surface marker CD44, potassium channel Kv1.3 and regulators (NFkb, Stat1, RelA) while anti-inflammatory DAM expressed phagocytic genes (Igf1, Apoe, Myo1e), surface marker CXCR4 with distinct regulators (LXRα/β, Atf1). As neuro-immunomodulatory strategies, we validated LXRα/β agonism and Kv1.3 blockade by ShK-223 peptide that promoted anti-inflammatory DAM, inhibited pro-inflammatory DAM and augmented Aβ clearance in AD models. Human AD-risk genes were highly represented within homeostatic microglia suggesting causal roles for early microglial dysregulation in AD. Pro-inflammatory DAM proteins were positively associated with neuropathology and preceded cognitive decline confirming the therapeutic relevance of inhibiting pro-inflammatory DAM in AD.ConclusionsWe provide a predictive transcriptomic framework of microglial activation in neurodegeneration that can guide pre-clinical studies to characterize and therapeutically modulate neuroinflammation in AD.

Differential Phagocytic Properties of CD45low Microglia and CD45high Brain Mononuclear Phagocytes—Activation and Age-Related Effects

In the central nervous system (CNS), microglia are innate immune mononuclear phagocytes (CNS MPs) that can phagocytose infectious particles, apoptotic cells, neurons, and pathological protein aggregates, such as Aβ in Alzheimer’s disease (AD). While CD11b+CD45low microglia account for the majority of CNS MPs, a small population of CD11b+CD45high CNS MPs is also recognized in AD that surround Aβ plaques. These transcriptionally and pathologically unique CD45high cells have unclear origin and undefined phagocytic characteristics. We have comprehensively validated rapid flow cytometric assays of bulk-phase and amyloid β fibril (fAβ) phagocytosis and applied these to study acutely isolated CNS MPs. Using these methods, we provide novel insights into differential abilities of CD11b+ CD45low and CD45high CNS MPs to phagocytose macroparticles and fAβ under normal, acute, and chronic neuroinflammatory states. CD45high CNS MPs also highly upregulate TREM2, CD11c, and several disease-associated microglia signature genes and have a higher phagocytic capacity for Aβ as compared to CD45low microglia in the 5xFAD mouse model of AD that becomes more apparent with aging. Our data suggest an overall pro-phagocytic and protective role for CD11b+CD45high CNS MPs in neurodegeneration, which if promoted, could be beneficial.

Quantitative proteomics of acutely-isolated mouse microglia identifies novel immune Alzheimer’s disease-related proteins

BackgroundMicroglia are innate immune cells of the brain that perform phagocytic and inflammatory functions in disease conditions. Transcriptomic studies of acutely-isolated microglia have provided novel insights into their molecular and functional diversity in homeostatic and neurodegenerative disease states. State-of-the-art mass spectrometry methods can comprehensively characterize proteomic alterations in microglia in neurodegenerative disorders, potentially providing novel functionally relevant molecular insights that are not provided by transcriptomics. However, comprehensive proteomic profiling of adult primary microglia in neurodegenerative disease conditions has not been performed.MethodsWe performed quantitative mass spectrometry based proteomic analyses of purified CD11b+ acutely-isolated microglia from adult (6 mo) mice in normal, acute neuroinflammatory (LPS-treatment) and chronic neurodegenerative states (5xFAD model of Alzheimer’s disease [AD]). Differential expression analyses were performed to characterize specific microglial proteomic changes in 5xFAD mice and identify overlap with LPS-induced pro-inflammatory changes. Our results were also contrasted with existing proteomic data from wild-type mouse microglia and from existing microglial transcriptomic data from wild-type and 5xFAD mice. Neuropathological validation studies of select proteins were performed in human AD and 5xFAD brains.ResultsOf 4133 proteins identified, 187 microglial proteins were differentially expressed in the 5xFAD mouse model of AD pathology, including proteins with previously known (Apoe, Clu and Htra1) as well as previously unreported relevance to AD biology (Cotl1 and Hexb). Proteins upregulated in 5xFAD microglia shared significant overlap with pro-inflammatory changes observed in LPS-treated mice. Several proteins increased in human AD brain were also upregulated by 5xFAD microglia (Aβ peptide, Apoe, Htra1, Cotl1 and Clu). Cotl1 was identified as a novel microglia-specific marker with increased expression and strong association with AD neuropathology. Apoe protein was also detected within plaque-associated microglia in which Apoe and Aβ were highly co-localized, suggesting a role for Apoe in phagocytic clearance of Aβ.ConclusionsWe report a comprehensive proteomic study of adult mouse microglia derived from acute neuroinflammation and AD models, representing a valuable resource to the neuroscience research community. We highlight shared and unique microglial proteomic changes in acute neuroinflammation aging and AD mouse models and identify novel roles for microglial proteins in human neurodegeneration.

Pittsburgh response to endovascular therapy score as a pre-treatment prognostic tool: External validation in Trevo2.

Background Optimal patient selection is needed to maximize the therapeutic benefit of endovascular therapy for large vessel occlusion stroke. Aims To validate the Pittsburgh response to endovascular therapy (PRE) score in a randomized controlled trial (Trevo2) comparing stent retriever (Trevo) to the Merci device. Methods Trevo2 participants with internal carotid, M1 and M2 middle cerebral artery occlusions with prospectively collected baseline stroke severity (NIHSS), degree of hypodensity (CT ASPECTS), and three-month modified Rankin Scale (mRS) were included. Multivariable regression was used to confirm association between PRE score variables (age, NIHSS, and ASPECTS), medical comorbidities, randomization arm, and reperfusion status (mTICI2B/3) with good outcome (three-month modified Rankin Scale 0–2). Predictive power (area under the receiver operating characteristic curve) for good outcome of pre-treatment prognostic scores (PRE, THRIVE, HIAT2) was compared. Rates of good outcome were compared between successfully reperfused (mTICI2B/3) and non-reperfused (mTICI0-2A) patients across previously identified PRE score risk groups. Results Age, NIHSS, ASPECTS, reperfusion status, and randomization arm were independent predictors of good outcome. PRE score had moderate predictive power (AUC = 0.75) for good outcome and was comparable to other pre-treatment scores. Reperfusion resulted in maximal treatment benefit in patients with PRE score 0–24 (60% vs. 12.5%, p = 0.002) but not in those with PRE ≥50 (11.8% vs. 0.0%, p = 0.49). Conclusion The PRE score is a validated predictor of functional outcome and a tool for patient selection for endovascular therapy in anterior large vessel occlusion stroke. Our finding of limited benefit of reperfusion in patients with PRE score ≥50 needs to be prospectively validated.

A Review of Pre-Intervention Prognostic Scores for Early Prognostication and Patient Selection in Endovascular Management of Large Vessel Occlusion Stroke

Background: Endovascular therapy (ET) has emerged as a highly effective treatment for acute large vessel occlusion stroke (LVOS). Tools that facilitate optimal patient selection of patients for ET are needed in order to maximize therapeutic benefit in a cost-effective manner. Several pre-intervention prognostic scores for prediction of outcomes in LVOS patients and patient selection for ET have been developed and validated, but their clinical use has been limited. Here, we review existing pre-intervention prognostic scores, compare their prognostic accuracies and levels of validation and identify gaps in current knowledge. Summary: We have reviewed published literature pertinent to development, validation, and implementation of pre-intervention prognostic scores for LVOS. Using receiver operating characteristic curve analysis, the prognostic accuracies of validated pre-interventional scores (Pittsburgh Response to Endovascular therapy [PRE], Totaled Health Risks in Vascular Events [THRIVE], Houston Intra-Arterial Therapy-2 (HIAT-2), Stroke Prognostication using Age and NIHSS [SPAN-100]) were compared in published work. Pre-intervention scores predicted functional out comes at 3 months with moderate prognostic accuracies (area under the receiver operator characteristic curve range 0.68–0.73). Using successful reperfusion (mTICI 2B/3) as the therapeutic objective of ET and 3-month modified Rankin Score 0–2 as good clinical outcome, patients most likely to clinically benefit from endovascular reperfusion can be identified using the PRE and HIAT-2 scores. Scores that incorporate collateral imaging or perfusion-based estimation of core and penumbra have not been published. Existing scores are predominantly limited to anterior circulation LVOS, and implementation studies of pre-interventional scores are lacking. Key Messages: Pre-intervention prognostic scores can serve as useful adjuncts for patient selection in ET for acute LVOS. Pre-intervention scores including HIAT-2, THRIVE, SPAN-100, and PRE have comparable moderate prognostic accuracies for good 3-month outcomes and can identify patients who derive maximal benefit from successful reperfusion. Improvements in prognostic accuracy may be achieved by incorporating variables such as collateral status and perfusion imaging data. Implementation and impact studies using pre-intervention scores are needed to guide clinical application.

A TRANSCRIPTOMIC LANDSCAPE OF MICROGLIAL ACTIVATION IN ALZHEIMER’S DISEASE

Background:Homeostatic microglia adopt distinct disease-associatedmicroglial (DAM) transcriptomic profiles inAlzheimer’s disease (AD) and the key transcriptional regulators of microglial states and factors responsible for microglial state transitions in AD need to be defined. Using weighted correlation network analysis (WGCNA) of microglial transcriptomic data, we recently identified 20 distinct microglial networks including a homeostatic profile and pro-inflammatory and anti-inflammatory sub-profiles within DAM. Each network represents a distinct microglial activation state and the genes at the interface of two adjacent networks may represent transitional checkpoints (or bottlenecks). Application of bioinformatics approaches to this framework of microglial diversity in AD may help elucidate key transcriptional and molecular regulators of these diverse microglial activation states in AD. Methods: Log2 transformed normalized expression data for key members of the 20 WGCNA networks (gene-level kME 0.65) were used to generate viSNE plots using the Rtsne Barnes-Hut-Stochastic Neighbor Embedding R package. The promoter regions of top 25 genes (Kme 0.9) of homeostatic and DAM modules were searched for transcription factor binding sites using TRANSFAC and then shared module-specific TFs were identified. Transcriptional factors were also predicted for each network’s hubs using Metacore. DAVID Gene Ontology, TF prediction and canonical pathway analyses (Metacore) were performed for interface genes between two adjacent networks. Chemical and genetic perturbagens likely to yield specific transcriptional profiles were determined using Connectivity map analysis (CMAP, Broad Institute). Results: ViSNE (Fig 1a) revealed a framework of microglial activation whereby homeostatic microglia (Blue network) transition towards DAM via distinct proinflammatory and anti-inflammatory pathways containing intermediary transition states and potential bridges between pathways.Distinct TFs for microglial states were identified (Fig 1B; Homeostatic: Crem Tau 1&2, CEBPa/b; Pro-inflammatory DAM: En1, Unc-86, Pou1F1, NFkB, RelA; Anti-inflammatory DAM: p300, LXRa/b, Atf1). Upstream TFs and predicted regulators of interface genes were also identified (Fig 1C; Homeostatic: TGFb1, IGF1; Pro-inflammatory pathway: Il1b, ephrin A/B, estradiol, thyroxine; Anti-inflammatory pathway: PDGFa, IGF-1/2, FSHb, Noradrenaline, CSF1). CMAP analysis suggested that several chemotherapeutic agents can promote pro-inflammatory DAM activation. Conclusions: We have used comprehensive bioinformatics approaches to describe the transcriptomic landscape of microglial activation in neurodegeneration, serving as a framework for experimental and therapeutic target validation.

Utility of Repeat Cerebrovascular Imaging among Hospitalized Stroke Patients

BACKGROUND AND PURPOSE The purpose of this study is to evaluate the frequency and clinical utility of repeat cerebrovascular imaging with computed tomography angiography (CTA) of the head after contrast-enhanced magnetic resonance angiography (CE-MRA) of the head in acute stroke patients. MATERIALS AND METHODS All stroke patients admitted to 2 academic medical centers from January 1, 2012 through December 31, 2014 were identified as part of prospective radiology database if they underwent CE-MRA of the head followed by subsequent CTA of the head within 7 days. Two vascular neurologists blinded to CTA results retrospectively reviewed medical records including documented indications for imaging studies and clinical changes in patients to determine necessity of CTA. RESULTS Of 1355 acute stroke patients who underwent CE-MRA of the head, 195 (14%) patients underwent subsequent CTA of the head within 7 days, including 33 patients with nondiagnostic CE-MRA because of motion artifact. Of the remaining 162 (12%) patients, 69 (43%) were considered to have an unnecessary CTA of the head. Multivariable logistic regression analysis identified (1) absence of new neurologic examination changes [OR 7.29; 95% CI 1.92-27.63] and (2) same documented indication for both studies [OR 6.47; 95% CI 3.04-13.78] as significant predictors of an unnecessary CTA. Changes in clinical management after CTA were significantly more common in necessary CTAs compared with studies determined to be unnecessary (42% versus 7%, P < .0001). CONCLUSION The utility of repeat cerebrovascular imaging with CTA of the head following a diagnostic CE-MRA is low when there is no change in neurologic examination or when ordered for the same indication.

Abbreviation of the Follow-Up NIH Stroke Scale Using Factor Analysis

Background: The NIH Stroke Scale (NIHSS) is a 15-item measure of stroke-related neurologic deficits that, when measured at 24 h, is highly predictive of long-term functional outcome. We hypothesized that a simplified 24-h scale that incorporates the most predictive components of the NIHSS can retain prognostic accuracy and have improved interrater reliability. Methods: In a post hoc analysis of the Interventional Management of Stroke-3 (IMS-3) trial, we performed principal component (PC) analysis to resolve the 24-h NIHSS into PCs. In the PCs that explained the largest proportions of variance, key variables were identified. Using these key variables, the prognostic accuracies (area under the curve [AUC]) for good outcome (3-month modified Rankin Scale [mRS] 0–2) and poor outcome (mRS 5–6) of various abbreviated NIHSS iterations were compared with the total 24-h NIHSS. The results were validated in the NINDS intravenous tissue plasminogen activator (NINDS-TPA) study cohort. Based on previously published data, interrater reliability of the abbreviated 24-h NIHSS (aNIHSS) was compared to the total 24-h NIHSS. Results: In 545 IMS-3 participants, 2 PCs explained 60.8% of variance in the 24-h NIHSS. The key variables in PC1 included neglect, arm and leg weakness; while PC2 included level-of-consciousness (LOC) questions, LOC commands, and aphasia. A 3-variable aNIHSS (aphasia, neglect, arm weakness) retained excellent prognostic accuracy for good outcome (AUC = 0.90) as compared to the total 24-h NIHSS (AUC = 0.91), and it was more predictive (p < 0.001) than the baseline NIHSS (AUC = 0.73). The prognostic accuracy of the aNIHSS for good outcome was validated in the NINDS-TPA trial cohort (aNIHSS: AUC = 0.89 vs. total 24-h NIHSS: 0.92). An aNIHSS >9 predicted very poor outcomes (mRS 0–2: 0%, mRS 4–6: 98.5%). The estimated interrater reliability of the aNIHSS was higher than that of the total 24-h NIHSS across 6 published datasets (mean weighted kappa 0.80 vs. 0.73, p < 0.001). Conclusions: At 24 h following ischemic stroke, aphasia, neglect, and arm weakness are the most prognostically relevant neurologic findings. The aNIHSS appears to have excellent prognostic accuracy with higher reliability and may be clinically useful.