Endothelial peroxiredoxin-4 is indispensable for blood-brain barrier integrity and long-term functional recovery after ischemic stroke.

The endothelial lining of cerebral microvessels is damaged relatively early after cerebral ischemia/reperfusion (I/R) injury and mediates blood-brain barrier (BBB) disruption, neurovascular injury, and long-term neurological deficits. I/R induces BBB leakage within 1 h due to subtle structural alterations in endothelial cells (ECs), including reorganization of the actin cytoskeleton and subcellular redistribution of junctional proteins. Herein, we show that the protein peroxiredoxin-4 (Prx4) is an endogenous protectant against endothelial dysfunction and BBB damage in a murine I/R model. We observed a transient upregulation of Prx4 in brain ECs 6 h after I/R in wild-type (WT) mice, whereas tamoxifen-induced, selective knockout of Prx4 from endothelial cells (eKO) mice dramatically raised vulnerability to I/R. Specifically, eKO mice displayed more BBB damage than WT mice within 1 to 24 h after I/R and worse long-term neurological deficits and focal brain atrophy by 35 d. Conversely, endothelium-targeted transgenic (eTG) mice overexpressing Prx4 were resistant to I/R-induced early BBB damage and had better long-term functional outcomes. As demonstrated in cultures of human brain endothelial cells and in animal models of I/R, Prx4 suppresses actin polymerization and stress fiber formation in brain ECs, at least in part by inhibiting phosphorylation/activation of myosin light chain. The latter cascade prevents redistribution of junctional proteins and BBB leakage under conditions of Prx4 repletion. Prx4 also tempers microvascular inflammation and infiltration of destructive neutrophils and proinflammatory macrophages into the brain parenchyma after I/R. Thus, the evidence supports an indispensable role for endothelial Prx4 in safeguarding the BBB and promoting functional recovery after I/R brain injury.

Leveraging single-cell RNA sequencing to unravel the impact of aging on stroke recovery mechanisms in mice.

Aging compromises the repair and regrowth of brain vasculature and white matter during stroke recovery, but the underlying mechanisms remain elusive. To understand how aging jeopardizes brain tissue repair after stroke, we performed single-cell transcriptomic profiling of young adult and aged mouse brains at acute (3 d) and chronic (14 d) stages after ischemic injury, focusing a priori on the expression of angiogenesis- and oligodendrogenesis-related genes. We identified unique subsets of endothelial cells (ECs) and oligodendrocyte (OL) progenitors in proangiogenesis and pro-oligodendrogenesis phenotypic states 3 d after stroke in young mice. However, this early prorepair transcriptomic reprogramming was negligible in aged stroke mice, consistent with the impairment of angiogenesis and oligodendrogenesis observed during the chronic injury stages after ischemia. In the stroke brain, microglia and macrophages (MG/MΦ) may drive angiogenesis and oligodendrogenesis through a paracrine mechanism. However, this reparative cell-cell cross talk between MG/MΦ and ECs or OLs is impeded in aged brains. In support of these findings, permanent depletion of MG/MΦ via antagonism of the colony-stimulating factor 1 receptor resulted in remarkably poor neurological recovery and loss of poststroke angiogenesis and oligodendrogenesis. Finally, transplantation of MG/MΦ from young, but not aged, mouse brains into the cerebral cortices of aged stroke mice partially restored angiogenesis and oligodendrogenesis and rejuvenated sensorimotor function and spatial learning and memory. Together, these data reveal fundamental mechanisms underlying the age-related decay in brain repair and highlight MG/MΦ as effective targets for promoting stroke recovery.

Interleukin-4 mitigates anxiety-like behavior and loss of neurons and fiber tracts in limbic structures in a microglial PPARγ-dependent manner after traumatic brain injury.

Traumatic brain injury (TBI) is commonly followed by intractable psychiatric disorders and long-term changes in affect, such as anxiety. The present study sought to investigate the effect of repetitive intranasal delivery of interleukin-4 (IL-4) nanoparticles on affective symptoms after TBI in mice. Adult male C57BL/6 J mice (10-12 weeks of age) were subjected to controlled cortical impact (CCI) and assessed by a battery of neurobehavioral tests up to 35 days after CCI. Neuron numbers were counted in multiple limbic structures, and the integrity of limbic white matter tracts was evaluated using ex vivo diffusion tensor imaging (DTI). As STAT6 is a critical mediator of IL-4-specific transcriptional activation, STAT6 knockout mice were used to explore the role of endogenous IL-4/STAT6 signaling axis in TBI-induced affective disorders. We also employed microglia/macrophage (Mi/Mϕ)-specific PPARγ conditional knockout (mKO) mice to test if Mi/Mϕ PPARγ critically contributes to IL-4-afforded beneficial effects. We observed anxiety-like behaviors up to 35 days after CCI, and these measures were exacerbated in STAT6 KO mice but mitigated by repetitive IL-4 delivery. We discovered that IL-4 protected against neuronal loss in limbic structures, such as the hippocampus and the amygdala, and improved the structural integrity of fiber tracts connecting the hippocampus and amygdala. We also observed that IL-4 boosted a beneficial Mi/Mϕ phenotype (CD206+/Arginase 1+/PPARγ+ triple-positive) in the subacute injury phase, and that the numbers of Mi/Mϕ appositions with neurons were robustly correlated with long-term behavioral performances. Remarkably, PPARγ-mKO completely abolished IL-4-afforded protection. Thus, CCI induces long-term anxiety-like behaviors in mice, but these changes in affect can be attenuated by transnasal IL-4 delivery. IL-4 prevents the long-term loss of neuronal somata and fiber tracts in key limbic structures, perhaps due to a shift in Mi/Mϕ phenotype. Exogenous IL-4 therefore holds promise for future clinical management of mood disturbances following TBI.

Microglia-specific deletion of histone deacetylase 3 promotes inflammation resolution, white matter integrity, and functional recovery in a mouse model of traumatic brain injury.

BACKGROUND: Histone deacetylases (HDACs) are believed to exacerbate traumatic brain injury (TBI) based on studies using pan-HDAC inhibitors. However, the HDAC isoform responsible for the detrimental effects and the cell types involved remain unknown, which may hinder the development of specific targeting strategies that boost therapeutic efficacy while minimizing side effects. Microglia are important mediators of post-TBI neuroinflammation and critically impact TBI outcome. HDAC3 was reported to be essential to the inflammatory program of in vitro cultured macrophages, but its role in microglia and in the post-TBI brain has not been investigated in vivo. METHODS: We generated HDAC3LoxP mice and crossed them with CX3CR1CreER mice, enabling in vivo conditional deletion of HDAC3. Microglia-specific HDAC3 knockout (HDAC3 miKO) was induced in CX3CR1CreER:HDAC3LoxP mice with 5 days of tamoxifen treatment followed by a 30-day development interval. The effects of HDAC3 miKO on microglial phenotype and neuroinflammation were examined 3-5 days after TBI induced by controlled cortical impact. Neurological deficits and the integrity of white matter were assessed for 6 weeks after TBI by neurobehavioral tests, immunohistochemistry, electron microscopy, and electrophysiology. RESULTS: HDAC3 miKO mice harbored specific deletion of HDAC3 in microglia but not in peripheral monocytes. HDAC3 miKO reduced the number of microglia by 26%, but did not alter the inflammation level in the homeostatic brain. After TBI, proinflammatory microglial responses and brain inflammation were markedly alleviated by HDAC3 miKO, whereas the infiltration of blood immune cells was unchanged, suggesting a primary effect of HDAC3 miKO on modulating microglial phenotype. Importantly, HDAC3 miKO was sufficient to facilitate functional recovery for 6 weeks after TBI. TBI-induced injury to axons and myelin was ameliorated, and signal conduction by white matter fiber tracts was significantly enhanced in HDAC3 miKO mice. CONCLUSION: Using a novel microglia-specific conditional knockout mouse model, we delineated for the first time the role of microglial HDAC3 after TBI in vivo. HDAC3 miKO not only reduced proinflammatory microglial responses, but also elicited long-lasting improvement of white matter integrity and functional recovery after TBI. Microglial HDAC3 is therefore a promising therapeutic target to improve long-term outcomes after TBI.

Single channel properties of pannexin-1 and connexin-43 hemichannels and P2X7 receptors in astrocytes cultured from rodent spinal cords.

Astrocytes express surface channels involved in purinergic signaling. Among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) release ATP that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, that is, open and close under physiological and pathological conditions, single channel properties of Px1 HCs in astrocytes have not been defined. Here, we developed a dual voltage clamp technique in HeLa cells expressing human Px1-YFP, and then applied this system to rodent spinal astrocytes to compare their single channel properties with other surface channels, that is, Cx43 HCs and P2X7 receptors (P2X7Rs). Channels were recorded in cell attached patches and evoked with ramp cycles applied through another pipette in whole cell voltage clamp. The mean unitary conductances of Px1 HCs were comparable in HeLa Px1-YFP cells and spinal astrocytes, ~42 and ~48 pS, respectively. Based on their unitary conductance, voltage-dependence, and unitary activity after pharmacological and gene silencing, Px1 HCs in astrocytes could be distinguished from Cx43 HCs and P2X7Rs. Channel activity of Px1 HCs and P2X7Rs was greater than that of Cx43 HCs in control astrocytes during ramps. Unitary activity of Px1 HCs was decreased and that of Cx43 HCs and P2X7Rs increased in astrocytes treated with fibroblast growth factor 1 (FGF-1). In summary, we resolved single channel properties of three different surface channels involved in purinergic signaling in spinal astrocytes, which were differentially modulated by FGF-1, a growth factor involved in neurodevelopment, inflammation and repair.

STAT1 contributes to microglial/macrophage inflammation and neurological dysfunction in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) triggers a plethora of inflammatory events in the brain that aggravate secondary injury and impede tissue repair. Resident microglia (Mi) and blood-borne infiltrating macrophages (MΦ) are major players of inflammatory responses in the post-TBI brain and possess high functional heterogeneity. However, the plasticity of these cells has yet to be exploited to develop therapies that can mitigate brain inflammation and improve the outcome after TBI. This study investigated the transcription factor STAT1 as a key determinant of proinflammatory Mi/MΦ responses and aimed to develop STAT1 as a novel therapeutic target for TBI using a controlled cortical impact model of TBI on adult male mice. TBI induced robust upregulation of STAT1 in the brain at the subacute injury stage, which occurred primarily in Mi/MΦ. Intraperitoneal administration of fludarabine, a selective STAT1 inhibitor, markedly alleviated proinflammatory Mi/MΦ responses and brain inflammation burden after TBI. Such phenotype-modulating effects of fludarabine on post-TBI Mi/MΦ were reproduced by tamoxifen-induced, selective knockout of STAT1 in Mi/MΦ (STAT1 mKO). By propelling Mi/MΦ away from a detrimental proinflammatory phenotype, STAT1 mKO was sufficient to reduce long-term neurological deficits and brain lesion size after TBI. Importantly, short-term fludarabine treatment after TBI elicited long-lasting improvement of TBI outcomes, but this effect was lost on STAT1 mKO mice. Together, our study provided the first line of evidence that STAT1 causatively determines the proinflammatory phenotype of brain Mi/MΦ after TBI. We also showed promising preclinical data supporting the use of fludarabine as a novel immunomodulating therapy to TBI.SIGNIFICANCE STATEMENTThe functional phenotype of microglia and macrophages (Mi/MΦ) critically influences brain inflammation and the outcome after traumatic brain injury (TBI); however, no therapies have been developed to modulate Mi/MΦ functions to treat TBI. Here we report for the first time that the transcription factor STAT1 is a key mediator of proinflammatory Mi/MΦ responses in the post-TBI brain, the specific deletion of which ameliorates neuroinflammation and improves long-term functional recovery after TBI. We also show excellent efficacy of a selective STAT1 inhibitor fludarabine against TBI-induced functional deficits and brain injury using a mouse model, presenting STAT1 as a promising therapeutic target for TBI.

E.L., a modern-day Phineas Gage: Revisiting frontal lobe injury.

Background: How the prefrontal cortex (PFC) recovers its functionality following lesions remains a conundrum. Recent work has uncovered the importance of transient low-frequency oscillatory activity (LFO; < 4 Hz) for the recovery of an injured brain. We aimed to determine whether persistent cortical oscillatory dynamics contribute to brain capability to support 'normal life' following injury. Methods: In this 9-year prospective longitudinal study (08/2012-2021), we collected data from the patient E.L., a modern-day Phineas Gage, who suffered from lesions, impacting 11% of his total brain mass, to his right PFC and supplementary motor area after his skull was transfixed by an iron rod. A systematic evaluation of clinical, electrophysiologic, brain imaging, neuropsychological and behavioural testing were used to clarify the clinical significance of relationship between LFO discharge and executive dysfunctions and compare E.L.´s disorders to that attributed to Gage (1848), a landmark in the history of neurology and neuroscience. Findings: Selective recruitment of the non-injured left hemisphere during execution of unimanual right-hand movements resulted in the emergence of robust LFO, an EEG-detected marker for disconnection of brain areas, in the damaged right hemisphere. In contrast, recruitment of the damaged right hemisphere during contralateral hand movement, resulted in the co-activation of the left hemisphere and decreased right hemisphere LFO to levels of controls enabling performance, suggesting a target for neuromodulation. Similarly, transcranial magnetic stimulation (TMS), used to create a temporary virtual-lesion over E.L.'s healthy hemisphere, disrupted the modulation of contralateral LFO, disturbing behaviour and impairing executive function tasks. In contrast to Gage, reasoning, planning, working memory, social, sexual and family behaviours eluded clinical inspection by decreasing LFO in the delta frequency range during motor and executive functioning. Interpretation: Our study suggests that modulation of LFO dynamics is an important mechanism by which PFC accommodates neurological injuries, supporting the reports of Gage´s recovery, and represents an attractive target for therapeutic interventions. Funding: Fundação de Amparo Pesquisa Rio de Janeiro (FAPERJ), Universidade Federal do Rio de Janeiro (intramural), and Fiocruz/Ministery of Health (INOVA Fiocruz).

IL-4/STAT6 signaling facilitates innate hematoma resolution and neurological recovery after hemorrhagic stroke in mice.

Intracerebral hemorrhage (ICH) is a devastating form of stroke affecting millions of people worldwide. Parenchymal hematoma triggers a series of reactions leading to primary and secondary brain injuries and permanent neurological deficits. Microglia and macrophages carry out hematoma clearance, thereby facilitating functional recovery after ICH. Here, we elucidate a pivotal role for the interleukin (IL)-4)/signal transducer and activator of transcription 6 (STAT6) axis in promoting long-term recovery in both blood- and collagenase-injection mouse models of ICH, through modulation of microglia/macrophage functions. In both ICH models, STAT6 was activated in microglia/macrophages (i.e., enhanced expression of phospho-STAT6 in Iba1+ cells). Intranasal delivery of IL-4 nanoparticles after ICH hastened STAT6 activation and facilitated hematoma resolution. IL-4 treatment improved long-term functional recovery in young and aged male and young female mice. In contrast, STAT6 knockout (KO) mice exhibited worse outcomes than WT mice in both ICH models and were less responsive to IL-4 treatment. The construction of bone marrow chimera mice demonstrated that STAT6 KO in either the CNS or periphery exacerbated ICH outcomes. STAT6 KO impaired the capacity of phagocytes to engulf red blood cells in the ICH brain and in primary cultures. Transcriptional analyses identified lower level of IL-1 receptor-like 1 (ST2) expression in microglia/macrophages of STAT6 KO mice after ICH. ST2 KO diminished the beneficial effects of IL-4 after ICH. Collectively, these data confirm the importance of IL-4/STAT6/ST2 signaling in hematoma resolution and functional recovery after ICH. Intranasal IL-4 treatment warrants further investigation as a clinically feasible therapy for ICH.

Protease-independent action of tissue plasminogen activator in brain plasticity and neurological recovery after ischemic stroke.

Emerging evidence suggests that tissue plasminogen activator (tPA), currently the only FDA-approved medication for ischemic stroke, exerts important biological actions on the CNS besides its well-known thrombolytic effect. In this study, we investigated the role of tPA on primary neurons in culture and on brain recovery and plasticity after ischemic stroke in mice. Treatment with recombinant tPA stimulated axonal growth in culture, an effect independent of its protease activity and achieved through epidermal growth factor receptor (EGFR) signaling. After permanent focal cerebral ischemia, tPA knockout mice developed more severe sensorimotor and cognitive deficits and greater axonal and myelin injury than wild-type mice, suggesting that endogenously expressed tPA promotes long-term neurological recovery after stroke. In tPA knockout mice, intranasal administration of recombinant tPA protein 6 hours poststroke and 7 more times at 2 d intervals mitigated white matter injury, improved axonal conduction, and enhanced neurological recovery. Consistent with the proaxonal growth effects observed in vitro, exogenous tPA delivery increased poststroke axonal sprouting of corticobulbar and corticospinal tracts, which might have contributed to restoration of neurological functions. Notably, recombinant mutant tPA-S478A lacking protease activity (but retaining the EGF-like domain) was as effective as wild-type tPA in rescuing neurological functions in tPA knockout stroke mice. These findings demonstrate that tPA improves long-term functional outcomes in a clinically relevant stroke model, likely by promoting brain plasticity through EGFR signaling. Therefore, treatment with the protease-dead recombinant tPA-S478A holds particular promise as a neurorestorative therapy, as the risk for triggering intracranial hemorrhage is eliminated and tPA-S478A can be delivered intranasally hours after stroke.

Activation of autophagy rescues synaptic and cognitive deficits in fragile X mice.

Fragile X syndrome (FXS) is the most frequent form of heritable intellectual disability and autism. Fragile X (Fmr1-KO) mice exhibit aberrant dendritic spine structure, synaptic plasticity, and cognition. Autophagy is a catabolic process of programmed degradation and recycling of proteins and cellular components via the lysosomal pathway. However, a role for autophagy in the pathophysiology of FXS is, as yet, unclear. Here we show that autophagic flux, a functional readout of autophagy, and biochemical markers of autophagy are down-regulated in hippocampal neurons of fragile X mice. We further show that enhanced activity of mammalian target of rapamycin complex 1 (mTORC1) and translocation of Raptor, a defining component of mTORC1, to the lysosome are causally related to reduced autophagy. Activation of autophagy by delivery of shRNA to Raptor directly into the CA1 of living mice via the lentivirus expression system largely corrects aberrant spine structure, synaptic plasticity, and cognition in fragile X mice. Postsynaptic density protein (PSD-95) and activity-regulated cytoskeletal-associated protein (Arc/Arg3.1), proteins implicated in spine structure and synaptic plasticity, respectively, are elevated in neurons lacking fragile X mental retardation protein. Activation of autophagy corrects PSD-95 and Arc abundance, identifying a potential mechanism by which impaired autophagy is causally related to the fragile X phenotype and revealing a previously unappreciated role for autophagy in the synaptic and cognitive deficits associated with fragile X syndrome.

Tissue plasminogen activator promotes white matter integrity and functional recovery in a murine model of traumatic brain injury.

Recombinant tissue plasminogen activator (tPA) is a Food and Drug Administration-approved thrombolytic treatment for ischemic stroke. tPA is also naturally expressed in glial and neuronal cells of the brain, where it promotes axon outgrowth and synaptic plasticity. However, there are conflicting reports of harmful versus neuroprotective effects of tPA in acute brain injury models. Furthermore, its impact on white matter integrity in preclinical traumatic brain injury (TBI) has not been thoroughly explored, although white matter disruption is a better predictor of long-term clinical outcomes than focal lesion volumes. Here we show that the absence of endogenous tPA in knockout mice impedes long-term recovery of white matter and neurological function after TBI. tPA-knockout mice exhibited greater asymmetries in forepaw use, poorer sensorimotor balance and coordination, and inferior spatial learning and memory up to 35 d after TBI. White matter damage was also more prominent in tPA knockouts, as shown by diffusion tensor imaging, histological criteria, and electrophysiological assessments of axon conduction properties. Replenishment of tPA through intranasal application of the recombinant protein in tPA-knockout mice enhanced neurological function, the structural and functional integrity of white matter, and postinjury compensatory sprouting in corticofugal projections. tPA also promoted neurite outgrowth in vitro, partly through the epidermal growth factor receptor. Both endogenous and exogenous tPA protected against white matter injury after TBI without increasing intracerebral hemorrhage volumes. These results unveil a previously unappreciated role for tPA in the protection and/or repair of white matter and long-term functional recovery after TBI.

Oxidative stress and DNA damage after cerebral ischemia: Potential therapeutic targets to repair the genome and improve stroke recovery.

The past two decades have witnessed remarkable advances in oxidative stress research, particularly in the context of ischemic brain injury. Oxidative stress in ischemic tissues compromises the integrity of the genome, resulting in DNA lesions, cell death in neurons, glial cells, and vascular cells, and impairments in neurological recovery after stroke. As DNA is particularly vulnerable to oxidative attack, cells have evolved the ability to induce multiple DNA repair mechanisms, including base excision repair (BER), nucleotide excision repair (NER) and non-homogenous endpoint jointing (NHEJ). Defective DNA repair is tightly correlated with worse neurological outcomes after stroke, whereas upregulation of DNA repair enzymes, such as APE1, OGG1, and XRCC1, improves long-term functional recovery following stroke. Indeed, DNA damage and repair are now known to play critical roles in fundamental aspects of stroke recovery, such as neurogenesis, white matter recovery, and neurovascular unit remodeling. Several DNA repair enzymes are essential for comprehensive neural repair mechanisms after stroke, including Polß and NEIL3 for neurogenesis, APE1 for white matter repair, Gadd45b for axonal regeneration, and DNA-PKs for neurovascular remodeling. This review discusses the emerging role of DNA damage and repair in functional recovery after stroke and highlights the contribution of DNA repair to regenerative elements after stroke. This article is part of the Special Issue entitled 'Cerebral Ischemia'.

Peroxisome proliferator-activated receptor γ (PPARγ): A master gatekeeper in CNS injury and repair.

Peroxisome proliferator-activated receptor γ (PPARγ) is a widely expressed ligand-modulated transcription factor that governs the expression of genes involved in inflammation, redox equilibrium, trophic factor production, insulin sensitivity, and the metabolism of lipids and glucose. Synthetic PPARγ agonists (e.g. thiazolidinediones) are used to treat Type II diabetes and have the potential to limit the risk of developing brain injuries such as stroke by mitigating the influence of comorbidities. If brain injury develops, PPARγ serves as a master gatekeeper of cytoprotective stress responses, improving the chances of cellular survival and recovery of homeostatic equilibrium. In the acute injury phase, PPARγ directly restricts tissue damage by inhibiting the NFκB pathway to mitigate inflammation and stimulating the Nrf2/ARE axis to neutralize oxidative stress. During the chronic phase of acute brain injuries, PPARγ activation in injured cells culminates in the repair of gray and white matter, preservation of the blood-brain barrier, reconstruction of the neurovascular unit, resolution of inflammation, and long-term functional recovery. Thus, PPARγ lies at the apex of cell fate decisions and exerts profound effects on the chronic progression of acute injury conditions. Here, we review the therapeutic potential of PPARγ in stroke and brain trauma and highlight the novel role of PPARγ in long-term tissue repair. We describe its structure and function and identify the genes that it targets. PPARγ regulation of inflammation, metabolism, cell fate (proliferation/differentiation/maturation/survival), and many other processes also has relevance to other neurological diseases. Therefore, PPARγ is an attractive target for therapies against a number of progressive neurological disorders.

Blood-brain barrier dysfunction and recovery after ischemic stroke.

The blood-brain barrier (BBB) plays a vital role in regulating the trafficking of fluid, solutes and cells at the blood-brain interface and maintaining the homeostatic microenvironment of the CNS. Under pathological conditions, such as ischemic stroke, the BBB can be disrupted, followed by the extravasation of blood components into the brain and compromise of normal neuronal function. This article reviews recent advances in our knowledge of the mechanisms underlying BBB dysfunction and recovery after ischemic stroke. CNS cells in the neurovascular unit, as well as blood-borne peripheral cells constantly modulate the BBB and influence its breakdown and repair after ischemic stroke. The involvement of stroke risk factors and comorbid conditions further complicate the pathogenesis of neurovascular injury by predisposing the BBB to anatomical and functional changes that can exacerbate BBB dysfunction. Emphasis is also given to the process of long-term structural and functional restoration of the BBB after ischemic injury. With the development of novel research tools, future research on the BBB is likely to reveal promising potential therapeutic targets for protecting the BBB and improving patient outcome after ischemic stroke.

HIV-Associated Cardiovascular Disease: Role of Connexin 43.

Chronic HIV infection due to effective antiretroviral treatment has resulted in a broad range of clinical complications, including accelerated heart disease. Individuals with HIV infection have a 1.5 to 2 times higher incidence of cardiovascular diseases than their uninfected counterparts; however, the underlying mechanisms are poorly understood. To explore the link between HIV infection and cardiovascular diseases, we used postmortem human heart tissues obtained from HIV-infected and control uninfected individuals to examine connexin 43 (Cx43) expression and distribution and HIV-associated inflammation. Here, we demonstrate that Cx43 is dysregulated in the hearts of HIV-infected individuals. In all HIV heart samples analyzed, there were areas where Cx43 was overexpressed and found along the lateral membrane of the cardiomyocyte and in the intercalated disks. Areas of HIV tissue with anomalous Cx43 expression and localization also showed calcium overload, sarcofilamental atrophy, and accumulation of collagen. All these changes were independent of viral replication, CD4 counts, inflammation, and type of antiretroviral treatment. Overall, we propose that HIV infection increases Cx43 expression in heart, resulting in tissue damage that likely contributes to the high rates of cardiovascular disease in HIV-infected individuals.

An Acute Mouse Spinal Cord Slice Preparation for Studying Glial Activation ex vivo.

Pathological conditions such as amyotrophic lateral sclerosis, spinal cord injury and chronic pain are characterized by activation of astrocytes and microglia in spinal cord and have been modeled in rodents. In vivo imaging at cellular level in these animal models is limited due to the spinal cord's highly myelinated funiculi. The preparation of acute slices may offer an alternative and valuable strategy to collect structural and functional information in vitro from dorsal, lateral and ventral regions of spinal cord. Here, we describe a procedure for preparing acute slices from mouse spinal cord (Garré et al., 2016). This preparation should allow further understanding of how glial cells in spinal cord respond acutely to various inflammatory challenges.

Endothelium-targeted overexpression of heat shock protein 27 ameliorates blood-brain barrier disruption after ischemic brain injury.

The damage borne by the endothelial cells (ECs) forming the blood-brain barrier (BBB) during ischemic stroke and other neurological conditions disrupts the structure and function of the neurovascular unit and contributes to poor patient outcomes. We recently reported that structural aberrations in brain microvascular ECs-namely, uncontrolled actin polymerization and subsequent disassembly of junctional proteins, are a possible cause of the early onset BBB breach that arises within 30-60 min of reperfusion after transient focal ischemia. Here, we investigated the role of heat shock protein 27 (HSP27) as a direct inhibitor of actin polymerization and protectant against BBB disruption after ischemia/reperfusion (I/R). Using in vivo and in vitro models, we found that targeted overexpression of HSP27 specifically within ECs-but not within neurons-ameliorated BBB impairment 1-24 h after I/R. Mechanistically, HSP27 suppressed I/R-induced aberrant actin polymerization, stress fiber formation, and junctional protein translocation in brain microvascular ECs, independent of its protective actions against cell death. By preserving BBB integrity after I/R, EC-targeted HSP27 overexpression attenuated the infiltration of potentially destructive neutrophils and macrophages into brain parenchyma, thereby improving long-term stroke outcome. Notably, early poststroke administration of HSP27 attached to a cell-penetrating transduction domain (TAT-HSP27) rapidly elevated HSP27 levels in brain microvessels and ameliorated I/R-induced BBB disruption and subsequent neurological deficits. Thus, the present study demonstrates that HSP27 can function at the EC level to preserve BBB integrity after I/R brain injury. HSP27 may be a therapeutic agent for ischemic stroke and other neurological conditions involving BBB breakdown.

Elevated ERK/p90 ribosomal S6 kinase activity underlies audiogenic seizure susceptibility in fragile X mice.

Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and a leading genetic form of autism. The Fmr1 KO mouse, a model of FXS, exhibits elevated translation in the hippocampus and the cortex. ERK (extracellular signal-regulated kinase) and mTOR (mechanistic target of rapamycin) signaling regulate protein synthesis by activating downstream targets critical to translation initiation and elongation and are known to contribute to hippocampal defects in fragile X. Here we show that the effect of loss of fragile X mental retardation protein (FMRP) on these pathways is brain region specific. In contrast to the hippocampus, ERK (but not mTOR) signaling is elevated in the neocortex of fragile X mice. Phosphorylation of ribosomal protein S6, typically a downstream target of mTOR, is elevated in the neocortex, despite normal mTOR activity. This is significant in that S6 phosphorylation facilitates translation, correlates with neuronal activation, and is altered in neurodevelopmental disorders. We show that in fragile X mice, S6 is regulated by ERK via the "alternative" S6 kinase p90-ribosomal S6 kinase (RSK), as evidenced by the site of elevated phosphorylation and the finding that ERK inhibition corrects elevated RSK and S6 activity. These findings indicate that signaling networks are altered in the neocortex of fragile X mice such that S6 phosphorylation receives aberrant input from ERK/RSK. Importantly, an RSK inhibitor reduces susceptibility to audiogenic seizures in fragile X mice. Our findings identify RSK as a therapeutic target for fragile X and suggest the therapeutic potential of drugs for the treatment of FXS may vary in a brain-region-specific manner.

APE1/Ref-1 facilitates recovery of gray and white matter and neurological function after mild stroke injury.

A major hallmark of oxidative DNA damage after stroke is the induction of apurinic/apyrimidinic (AP) sites and strand breaks. To mitigate cell loss after oxidative DNA damage, ischemic cells rapidly engage the base excision-repair proteins, such as the AP site-repairing enzyme AP endonuclease-1 (APE1), also named redox effector factor-1 (Ref-1). Although forced overexpression of APE1 is known to protect against oxidative stress-induced neurodegeneration, there is no concrete evidence demonstrating a role for endogenous APE1 in the long-term recovery of gray and white matter following ischemic injury. To address this gap, we generated, to our knowledge, the first APE1 conditional knockout (cKO) mouse line under control of tamoxifen-dependent Cre recombinase. Using a well-established model of transient focal cerebral ischemia (tFCI), we show that induced deletion of APE1 dramatically enlarged infarct volume and impaired the recovery of sensorimotor and cognitive deficits. APE1 cKO markedly increased postischemic neuronal and oligodendrocyte degeneration, demonstrating that endogenous APE1 preserves both gray and white matter after tFCI. Because white matter repair is instrumental in behavioral recovery after stroke, we also examined the impact of APE1 cKO on demyelination and axonal conduction and discovered that APE1 cKO aggravated myelin loss and impaired neuronal communication following tFCI. Furthermore, APE1 cKO increased AP sites and activated the prodeath signaling proteins, PUMA and PARP1, after tFCI in topographically distinct manners. Our findings provide evidence that endogenous APE1 protects against ischemic infarction in both gray and white matter and facilitates the functional recovery of the central nervous system after mild stroke injury.