Long-term neurological dysfunction associated with COVID-19: Lessons from influenza and inflammatory diseases?

As the COVID-19 pandemic persists, SARS-CoV-2 infection is increasingly associated with long-term neurological side effects including cognitive impairment, fatigue, depression, and anxiety, colloquially known as "long-COVID." While the full extent of long-COVID neuropathology across years or even decades is not yet known, we can perhaps take direction from long-standing research into other respiratory diseases, such as influenza, that can present with similar long-term neurological consequences. In this review, we highlight commonalities in the neurological impacts of influenza and COVID-19. We first focus on the common potential mechanisms underlying neurological sequelae of long-COVID and influenza, namely (1) viral neurotropism and (2) dysregulated peripheral inflammation. The latter, namely heightened peripheral inflammation leading to central nervous system dysfunction, is emerging as a shared mechanism in various peripheral inflammatory or inflammation-associated diseases and conditions. We then discuss historical and modern examples of influenza- and COVID-19-associated cognitive impairment, depression, anxiety, and fatigue, revealing key similarities in their neurological sequelae. Although we are learning that the effects of influenza and COVID differ somewhat in terms of their influence on the brain, as the impacts of long-COVID grow, such comparisons will likely prove valuable in guiding ongoing research into long-COVID, and perhaps foreshadow what could be in store for individuals with COVID-19 and their brain health.

Overlap in synaptic neurological condition susceptibility pathways and the neural pannexin 1 interactome revealed by bioinformatics analyses.

Many neurological conditions exhibit synaptic impairments, suggesting mechanistic convergence. Additionally, the pannexin 1 (PANX1) channel and signaling scaffold is linked to several of these neurological conditions and is an emerging regulator of synaptic development and plasticity; however, its synaptic pathogenic contributions are relatively unexplored. To this end, we explored connections between synaptic neurodevelopmental disorder and neurodegenerative disease susceptibility genes discovered by genome-wide association studies (GWASs), and the neural PANX1 interactome (483 proteins) identified from mouse Neuro2a (N2a) cells. To identify shared susceptibility genes, we compared synaptic suggestive GWAS candidate genes amongst autism spectrum disorders, schizophrenia, Parkinson's disease, and Alzheimer's disease. To further probe PANX1 signaling pathways at the synapse, we used bioinformatics tools to identify PANX1 interactome signaling pathways and protein-protein interaction clusters. To shed light on synaptic disease mechanisms potentially linking PANX1 and these four neurological conditions, we performed additional cross-analyses between gene ontologies enriched for the PANX1 synaptic and disease-susceptibility gene sets. Finally, to explore the regional specificity of synaptic PANX1-neurological condition connections, we identified brain region-specific elevations of synaptic PANX1 interactome and GWAS candidate gene set transcripts. Our results confirm considerable overlap in risk genes for autism spectrum disorders and schizophrenia and identify potential commonalities in genetic susceptibility for neurodevelopmental disorders and neurodegenerative diseases. Our findings also pinpointed novel putative PANX1 links to synaptic disease-associated pathways, such as regulation of vesicular trafficking and proteostasis, warranting further validation.

Mechanisms underlying the role of ankyrin-B in cardiac and neurological health and disease.

The ANK2 gene encodes for ankyrin-B (ANKB), one of 3 members of the ankyrin family of proteins, whose name is derived from the Greek word for anchor. ANKB was originally identified in the brain (B denotes "brain") but has become most widely known for its role in cardiomyocytes as a scaffolding protein for ion channels and transporters, as well as an interacting protein for structural and signaling proteins. Certain loss-of-function ANK2 variants are associated with a primarily cardiac-presenting autosomal-dominant condition with incomplete penetrance and variable expressivity characterized by a predisposition to supraventricular and ventricular arrhythmias, arrhythmogenic cardiomyopathy, congenital and adult-onset structural heart disease, and sudden death. Another independent group of ANK2 variants are associated with increased risk for distinct neurological phenotypes, including epilepsy and autism spectrum disorders. The mechanisms underlying ANKB's roles in cells in health and disease are not fully understood; however, several clues from a range of molecular and cell biological studies have emerged. Notably, ANKB exhibits several isoforms that have different cell-type-, tissue-, and developmental stage- expression profiles. Given the conservation within ankyrins across evolution, model organism studies have enabled the discovery of several ankyrin roles that could shed important light on ANKB protein-protein interactions in heart and brain cells related to the regulation of cellular polarity, organization, calcium homeostasis, and glucose and fat metabolism. Along with this accumulation of evidence suggesting a diversity of important ANKB cellular functions, there is an on-going debate on the role of ANKB in disease. We currently have limited understanding of how these cellular functions link to disease risk. To this end, this review will examine evidence for the cellular roles of ANKB and the potential contribution of ANKB functional variants to disease risk and presentation. This contribution will highlight the impact of ANKB dysfunction on cardiac and neuronal cells and the significance of understanding the role of ANKB variants in disease.

Foundations and implications of astrocyte heterogeneity during brain development and disease.

Astrocytes play crucial roles in regulating brain circuit formation and physiology. Recent technological advances have revealed unprecedented levels of astrocyte diversity encompassing molecular, morphological, and functional differences. This diversification is initiated during embryonic specification events and (in rodents) continues into the early postnatal period where it overlaps with peak synapse development and circuit refinement. In fact, several lines of evidence suggest astrocyte diversity both influences and is a consequence of molecular crosstalk among developing astrocytes and other cell types, notably neurons and their synapses. Neurological disease states exhibit additional layers of astrocyte heterogeneity, which could help shed light on these cells' key pathological roles. This review highlights recent advances in clarifying astrocyte heterogeneity and molecular/cellular crosstalk and identifies key outstanding questions.

A Systematic, Open-Science Framework for Quantification of Cell-Types in Mouse Brain Sections Using Fluorescence Microscopy.

The ever-expanding availability and evolution of microscopy tools has enabled ground-breaking discoveries in neurobiology, particularly with respect to the analysis of cell-type density and distribution. Widespread implementation of many of the elegant image processing tools available continues to be impeded by the lack of complete workflows that span from experimental design, labeling techniques, and analysis workflows, to statistical methods and data presentation. Additionally, it is important to consider open science principles (e.g., open-source software and tools, user-friendliness, simplicity, and accessibility). In the present methodological article, we provide a compendium of resources and a FIJI-ImageJ-based workflow aimed at improving the quantification of cell density in mouse brain samples using semi-automated open-science-based methods. Our proposed framework spans from principles and best practices of experimental design, histological and immunofluorescence staining, and microscopy imaging to recommendations for statistical analysis and data presentation. To validate our approach, we quantified neuronal density in the mouse barrel cortex using antibodies against pan-neuronal and interneuron markers. This framework is intended to be simple and yet flexible, such that it can be adapted to suit distinct project needs. The guidelines, tips, and proposed methodology outlined here, will support researchers of wide-ranging experience levels and areas of focus in neuroscience research.

Treponema pallidum Disrupts VE-Cadherin Intercellular Junctions and Traverses Endothelial Barriers Using a Cholesterol-Dependent Mechanism.

Treponema pallidum subspecies pallidum, the causative agent of syphilis, traverses the vascular endothelium to gain access to underlying tissue sites. Herein, we investigate the mechanisms associated with T. pallidum traversal of endothelial barriers. Immunofluorescence microscopy reveals that a subpopulation of T. pallidum localizes to intercellular junctions and that viable T. pallidum, as well as a T. pallidum vascular adhesin (Tp0751), disrupts the architecture of the main endothelial junctional protein VE-cadherin. Intriguingly, in this study we show that T. pallidum traverses endothelial barriers with no disruption in barrier permeability. Furthermore, barrier traversal by T. pallidum is reduced by pretreatment of endothelial cells with filipin, an inhibitor that blocks cholesterol-mediated endocytosis. Collectively, these results suggest that T. pallidum can use a cholesterol-dependent, lipid raft-mediated endocytosis mechanism to traverse endothelial barriers. Further, treponemal localization to, and disruption of, intercellular junctions suggests that a paracellular route may also be utilized, a dual traversal strategy that has also been observed to occur for leukocytes and other invasive bacteria.

Purinergic signaling in nervous system health and disease: Focus on pannexin 1.

Purinergic signaling encompasses the cycle of adenosine 5' triphosphate (ATP) release and its metabolism into nucleotide and nucleoside derivatives, the direct release of nucleosides, and subsequent receptor-triggered downstream intracellular pathways. Since the discovery of nerve terminal and glial ATP release into the neuropil, purinergic signaling has been implicated in the modulation of nervous system development, function, and disease. In this review, we detail our current understanding of the roles of the pannexin 1 (PANX1) ATP-release channel in neuronal development and plasticity, glial signaling, and neuron-glial-immune interactions. We additionally provide an overview of PANX1 structure, activation, and permeability to orientate readers and highlight recent research developments. We identify areas of convergence between PANX1 and purinergic receptor actions. Additional highlights include data on PANX1's participation in the pathophysiology of nervous system developmental, degenerative, and inflammatory disorders. Our aim in combining this knowledge is to facilitate the movement of our current understanding of PANX1 in the context of other nervous system purinergic signaling mechanisms one step closer to clinical translation.

Pannexin 1 Regulates Dendritic Protrusion Dynamics in Immature Cortical Neurons.

The integration of neurons into networks relies on the formation of dendritic spines. These specialized structures arise from dynamic filopodia-like dendritic protrusions. It was recently reported that cortical neurons lacking the channel protein pannexin 1 (PANX1) exhibited higher dendritic spine densities. Here, we expanded on those findings to investigate, at an earlier developmental time point (with more abundant dendritic protrusions), whether differences in the properties of dendritic protrusion dynamics could contribute to this previously discovered phenomenon. Using a fluorescent membrane tag (mCherry-CD9-10) to visualize dendritic protrusions in developing neurons [at 10 d in vitro (DIV10)], we confirmed that lack of PANX1 led to higher protrusion density, while transient transfection of Panx1 led to decreased protrusion density. To quantify the impact of PANX1 expression on protrusion formation, elimination, and motility, we used live cell imaging in DIV10 neurons (one frame every 5 s for 10 min). We discovered that at DIV10, loss of PANX1 stabilized protrusions. Notably, re-expression of PANX1 in Panx1 knock-out (KO) neurons resulted in a significant increase in protrusion motility and turnover. In summary, these new data revealed that PANX1 could regulate the development of dendritic spines, in part, by controlling dendritic protrusion dynamics.

PANX1 in inflammation heats up: New mechanistic insights with implications for injury and infection.

A new study by Yang and colleagues has revealed that TNF-alpha regulates PANX1 levels through an NF-kB-dependent mechanism in human endothelial cells. PANX1 modulates Ca2+ influx contributing to IL-1beta production independent of purinergic signaling. These novel findings expand our understanding of TNF-alpha-mediated upregulation of IL-1beta with implications for responses to tissue injury and infection.

Identification of the Neuroinvasive Pathogen Host Target, LamR, as an Endothelial Receptor for the Treponema pallidum Adhesin Tp0751.

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a human-specific sexually transmitted infection that causes a multistage disease with diverse clinical manifestations. Treponema pallidum undergoes rapid vascular dissemination to penetrate tissue, placental, and blood-brain barriers and gain access to distant tissue sites. The rapidity and extent of T. pallidum dissemination are well documented, but the molecular mechanisms have yet to be fully elucidated. One protein that has been shown to play a role in treponemal dissemination is Tp0751, a T. pallidum adhesin that interacts with host components found within the vasculature and mediates bacterial adherence to endothelial cells under shear flow conditions. In this study, we further explore the molecular interactions of Tp0751-mediated adhesion to the vascular endothelium. We demonstrate that recombinant Tp0751 adheres to human endothelial cells of macrovascular and microvascular origin, including a cerebral brain microvascular endothelial cell line. Adhesion assays using recombinant Tp0751 N-terminal truncations reveal that endothelial binding is localized to the lipocalin fold-containing domain of the protein. We also confirm this interaction using live T. pallidum and show that spirochete attachment to endothelial monolayers is disrupted by Tp0751-specific antiserum. Further, we identify the 67-kDa laminin receptor (LamR) as an endothelial receptor for Tp0751 using affinity chromatography, coimmunoprecipitation, and plate-based binding methodologies. Notably, LamR has been identified as a receptor for adhesion of other neurotropic invasive bacterial pathogens to brain endothelial cells, including Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae, suggesting the existence of a common mechanism for extravasation of invasive extracellular bacterial pathogens.IMPORTANCE Syphilis is a sexually transmitted infection caused by the spirochete bacterium Treponema pallidum subsp. pallidum. The continued incidence of syphilis demonstrates that screening and treatment strategies are not sufficient to curb this infectious disease, and there is currently no vaccine available. Herein we demonstrate that the T. pallidum adhesin Tp0751 interacts with endothelial cells that line the lumen of human blood vessels through the 67-kDa laminin receptor (LamR). Importantly, LamR is also a receptor for meningitis-causing neuroinvasive bacterial pathogens such as Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae Our findings enhance understanding of the Tp0751 adhesin and present the intriguing possibility that the molecular events of Tp0751-mediated treponemal dissemination may mimic the endothelial interaction strategies of other invasive pathogens.

Ankyrin-B p.S646F undergoes increased proteasome degradation and reduces cell viability in the H9c2 rat ventricular cardiomyoblast cell line.

Ankyrin-B (AnkB) is scaffolding protein that anchors integral membrane proteins to the cardiomyocyte cytoskeleton. We recently identified an AnkB variant, AnkB p.S646F (ANK2 c.1937 C>T) associated with a phenotype ranging from predisposition for cardiac arrhythmia to cardiomyopathy. AnkB p.S646F exhibited reduced expression levels in the H9c2 rat ventricular-derived cardiomyoblast cell line relative to wildtype AnkB. Here, we demonstrate that AnkB is regulated by proteasomal degradation and proteasome inhibition rescues AnkB p.S646F expression levels in H9c2 cells, although this effect is not conserved with differentiation. We also compared the impact of wildtype AnkB and AnkB p.S646F on cell viability and proliferation. AnkB p.S646F expression resulted in decreased cell viability at 30 h after transfection, whereas we observed a greater proportion of cycling, Ki67-positive cells at 48 h after transfection. Notably, the number of GFP-positive cells was low and was consistent between wildtype AnkB and AnkB p.S646F expressing cells, suggesting that AnkB and AnkB p.S646F affected paracrine communication between H9c2 cells differentially. This work reveals that AnkB levels are regulated by the proteasome and that AnkB p.S646F compromises cell viability. Together, these findings provide key new insights into the putative cellular and molecular mechanisms of AnkB-related cardiac disease.

Ankyrin B and Ankyrin B variants differentially modulate intracellular and surface Cav2.1 levels.

Ankyrin B (AnkB) is an adaptor and scaffold for motor proteins and various ion channels that is ubiquitously expressed, including in the brain. AnkB has been associated with neurological disorders such as epilepsy and autism spectrum disorder, but understanding of the underlying mechanisms is limited. Cav2.1, the pore-forming subunit of P/Q type voltage gated calcium channels, is a known interactor of AnkB and plays a crucial role in neuronal function. Here we report that wildtype AnkB increased overall Cav2.1 levels without impacting surface Cav2.1 levels in HEK293T cells. An AnkB variant, p.S646F, which we recently discovered to be associated with seizures, further increased overall Cav2.1 levels, again with no impact on surface Cav2.1 levels. AnkB p.Q879R, on the other hand, increased surface Cav2.1 levels in the presence of accessory subunits α2δ1 and ß4. Additionally, AnkB p.E1458G decreased surface Cav2.1 irrespective of the presence of accessory subunits. In addition, we found that partial deletion of AnkB in cortex resulted in a decrease in overall Cav2.1 levels, with no change to the levels of Cav2.1 detected in synaptosome fractions. Our work suggests that depending on the particular variant, AnkB regulates intracellular and surface Cav2.1. Notably, expression of the AnkB variant associated with seizure (AnkB p.S646F) caused further increase in intracellular Cav2.1 levels above that of even wildtype AnkB. These novel findings have important implications for understanding the role of AnkB and Cav2.1 in the regulation of neuronal function in health and disease.

A novel motif in the proximal C-terminus of Pannexin 1 regulates cell surface localization.

The Pannexin 1 (Panx1) ion and metabolite channel is expressed in a wide variety of cells where it regulates a number of cell behaviours including proliferation and differentiation. Panx1 is expressed on the cell surface as well as intracellular membranes. Previous work suggests that a region within the proximal Panx1 C-terminus (Panx1CT) regulates cell surface localization. Here we report the discovery of a putative leucine-rich repeat (LRR) motif in the proximal Panx1CT necessary for Panx1 cell surface expression in HEK293T cells. Deletion of the putative LRR motif results in significant loss of Panx1 cell surface distribution. Outcomes of complementary cell surface oligomerization and glycosylation state analyses were consistent with reduced cell surface expression of Panx1 LRR deletion mutants. Of note, the oligomerization analysis revealed the presence of putative dimers and trimers of Panx1 at the cell surface. Expression of Panx1 increased HEK293T cell growth and reduced doubling time, while expression of a Panx1 LRR deletion mutant (highly conserved segment) did not reproduce this effect. In summary, here we discovered the presence of a putative LRR motif in the Panx1CT that impacts on Panx1 cell surface localization. Overall these findings provide new insights into the molecular mechanisms underlying C-terminal regulation of Panx1 trafficking and raise potential new lines of investigation with respect to Panx1 oligomerization and glycosylation.

Pannexin 1 Regulates Network Ensembles and Dendritic Spine Development in Cortical Neurons.

Dendritic spines are the postsynaptic targets of excitatory synaptic inputs that undergo extensive proliferation and maturation during the first postnatal month in mice. However, our understanding of the molecular mechanisms that regulate spines during this critical period is limited. Previous work has shown that pannexin 1 (Panx1) regulates neurite growth and synaptic plasticity. We therefore investigated the impact of global Panx1 KO on spontaneous cortical neuron activity using Ca2+ imaging and in silico network analysis. Panx1 KO increased both the number and size of spontaneous co-active cortical neuron network ensembles. To understand the basis for these findings, we investigated Panx1 expression in postnatal synaptosome preparations from early postnatal mouse cortex. Between 2 and 4 postnatal weeks, we observed a precipitous drop in cortical synaptosome protein levels of Panx1, suggesting it regulates synapse proliferation and/or maturation. At the same time points, we observed significant enrichment of the excitatory postsynaptic density proteins PSD-95, GluA1, and GluN2a in cortical synaptosomes from global Panx1 knock-out mice. Ex vivo analysis of pyramidal neuron structure in somatosensory cortex revealed a consistent increase in dendritic spine densities in both male and female Panx1 KO mice. Similar findings were observed in an excitatory neuron-specific Panx1 KO line (Emx1-Cre driven; Panx1 cKOE) and in primary Panx1 KO cortical neurons cultured in vitro. Altogether, our study suggests that Panx1 negatively regulates cortical dendritic spine development.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability.

FK506 binding proteins (FKBPs) catalyze the interconversion of cis-trans proline conformers in proteins. Importantly, FK506 drugs have anti-cancer and neuroprotective properties, but the effectors and mechanisms underpinning these properties are not well understood because the cellular function(s) of most FKBP proteins are unclear. FKBP25 is a nuclear prolyl isomerase that interacts directly with nucleic acids and is associated with several DNA/RNA binding proteins. Here, we show the catalytic FKBP domain binds microtubules (MTs) directly to promote their polymerization and stabilize the MT network. Furthermore, FKBP25 associates with the mitotic spindle and regulates entry into mitosis. This interaction is important for mitotic spindle dynamics, as we observe increased chromosome instability in FKBP25 knockdown cells. Finally, we provide evidence that FKBP25 association with chromatin is cell-cycle regulated by Protein Kinase C phosphorylation. This disrupts FKBP25-DNA contacts during mitosis while maintaining its interaction with the spindle apparatus. Collectively, these data support a model where FKBP25 association with chromatin and MTs is carefully choreographed to ensure faithful genome duplication. Additionally, they highlight that FKBP25 is a MT-associated FK506 receptor and potential therapeutic target in MT-associated diseases.

Transcriptional and post-translational regulation of pannexins.

Pannexins are a 3-membered family of proteins that form large pore ion and metabolite channels in vertebrates. The impact of pannexins on vertebrate biology is intricately tied to where and when they are expressed, and how they are modified, once produced. The purpose of this review is therefore to outline our current understanding of transcriptional and post-translational regulation of pannexins. First, we briefly summarize their discovery and characteristics. Next, we describe several aspects of transcriptional regulation, including cell and tissue-specific expression, dynamic expression over development and disease, as well as new insights into the underlying molecular machinery involved. Following this, we delve into the role of post-translational modifications in the regulation of trafficking and channel properties, highlighting important work on glycosylation, phosphorylation, S-nitrosylation and proteolytic cleavage. Embedded throughout, we also highlight important knowledge gaps and avenues of future research. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Upregulation of inflammatory mediators in the ventricular zone after cortical stroke.

PURPOSE: After cortical stroke, neural precursor cells (NPCs) in the distal ventricular zone (VZ) proliferate more rapidly and migrate toward the injured cortex. While evidence suggests this can enhance stroke recovery, the underlying molecular mechanisms initiating the response are poorly understood. Here we identified changes in protein expression in the ipsilateral VZ early (4 h) after stroke to gain insight into the initial mechanisms involved in NPC activation post-stroke. EXPERIMENTAL DESIGN: Four hours after photothrombotic stroke (or sham surgery control) in the sensorimotor cortex, adult mice (10 stroke, 10 sham) were subjected to cardiac perfusion with PBS, and ipsilateral and contralateral VZ tissue was microdissected. Two separate sets of ipsilateral and contralateral VZ tissues (from 5 pooled surgery or 5 pooled sham mice) were analyzed simultaneously using 8-plex iTRAQ. We used Western blotting and confocal microscopy to confirm changes in protein expression in the VZ ipsilateral to stroke in a separate cohort of mice. RESULTS: We identified nine proteins which exhibited a significant mean increase (by ≥ 2-fold) in stroke ipsilateral compared to sham ipsilateral. Many of these proteins were antiproteases or cytokine/growth factor binding proteins that are known to act as inflammatory responders or effectors and play roles in modulating tissue growth and remodeling. CONCLUSION AND CLINICAL RELEVANCE: These novel findings support a growing body of literature that inflammatory signaling is involved in the NPC response to brain injury and identifies novel potential targets that could be exploited to better understand and to optimize this regenerative response.