Single-cell RNA sequencing reveals the evolution of the immune landscape during perihematomal edema progression after intracerebral hemorrhage.

BACKGROUND: Perihematomal edema (PHE) after post-intracerebral hemorrhage (ICH) has complex pathophysiological mechanisms that are poorly understood. The complicated immune response in the post-ICH brain constitutes a crucial component of PHE pathophysiology. In this study, we aimed to characterize the transcriptional profiles of immune cell populations in human PHE tissue and explore the microscopic differences between different types of immune cells. METHODS: 9 patients with basal ganglia intracerebral hemorrhage (hematoma volume 50-100 ml) were enrolled in this study. A multi-stage profile was developed, comprising Group1 (n = 3, 0-6 h post-ICH, G1), Group2 (n = 3, 6-24 h post-ICH, G2), and Group3 (n = 3, 24-48 h post-ICH, G3). A minimal quantity of edematous tissue surrounding the hematoma was preserved during hematoma evacuation. Single cell RNA sequencing (scRNA-seq) was used to map immune cell populations within comprehensively resected PHE samples collected from patients at different stages after ICH. RESULTS: We established, for the first time, a comprehensive landscape of diverse immune cell populations in human PHE tissue at a single-cell level. Our study identified 12 microglia subsets and 5 neutrophil subsets in human PHE tissue. What's more, we discovered that the secreted phosphoprotein-1 (SPP1) pathway served as the basis for self-communication between microglia subclusters during the progression of PHE. Additionally, we traced the trajectory branches of different neutrophil subtypes. Finally, we also demonstrated that microglia-produced osteopontin (OPN) could regulate the immune environment in PHE tissue by interacting with CD44-positive cells. CONCLUSIONS: As a result of our research, we have gained valuable insight into the immune-microenvironment within PHE tissue, which could potentially be used to develop novel treatment modalities for ICH.

Subclonal Cancer Driver Mutations Are Prevalent in the Unresected Peritumoral Edema of Adult Diffuse Gliomas.

Adult diffuse gliomas commonly recur regardless of therapy. As recurrence typically arises from the peritumoral edema adjacent to the resected bulk tumor, the profiling of somatic mutations from infiltrative malignant cells within this critical, unresected region could provide important insights into residual disease. A key obstacle has been the inability to distinguish between next-generation sequencing (NGS) noise and the true but weak signal from tumor cells hidden among the noncancerous brain tissue of the peritumoral edema. Here, we developed and validated True2 sequencing to reduce NGS-associated errors to <1 false positive/100 kb panel positions while detecting 97.6% of somatic mutations with an allele frequency ≥0.1%. True2 was then used to study the tumor and peritumoral edema of 22 adult diffuse gliomas including glioblastoma, astrocytoma, oligodendroglioma, and NF1-related low-grade neuroglioma. The tumor and peritumoral edema displayed a similar mutation burden, indicating that surgery debulks these cancers physically but not molecularly. Moreover, variants in the peritumoral edema included unique cancer driver mutations absent in the bulk tumor. Finally, analysis of multiple samples from each patient revealed multiple subclones with unique mutations in the same gene in 17 of 22 patients, supporting the occurrence of convergent evolution in response to patient-specific selective pressures in the tumor microenvironment that may form the molecular foundation of recurrent disease. Collectively, True2 enables the detection of ultralow frequency mutations during molecular analyses of adult diffuse gliomas, which is necessary to understand cancer evolution, recurrence, and individual response to therapy. SIGNIFICANCE: True2 is a next-generation sequencing workflow that facilitates unbiased discovery of somatic mutations across the full range of variant allele frequencies, which could help identify residual disease vulnerabilities for targeted adjuvant therapies.

Refining the phenotypic spectrum of CCDC88A-related PEHO-like syndrome.

Progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) and PEHO-like syndromes are very rare infantile disorders characterized by profound intellectual disability, hypotonia, convulsions, optic, and progressive brain atrophy. Many causative genes for PEHO and PEHO-like syndromes have been identified including CCDC88A. So far, only five patients from two unrelated families with biallelic CCDC88A variants have been reported in the literature. Herein, we describe a new family from Egypt with a lethal epileptic encephalopathy. Our patient was the youngest child born to a highly consanguineous couple and had a family history of five deceased sibs with the same condition. She presented with postnatal microcephaly, poor visual responsiveness, and epilepsy. Her brain MRI showed abnormal cortical gyration with failure of opercularization of the insula, hypogenesis of corpus callosum, colpocephaly, reduced white matter, hypoplastic vermis, and brain stem. Whole exome sequencing identified a new homozygous frameshift variant in CCDC88A gene (c.1795_1798delACAA, p.Thr599ValfsTer4). Our study presents the third reported family with this extremely rare disorder. We also reviewed all described cases to better refine the phenotypic spectrum associated with biallelic loss of function variants in the CCDC88A gene.

Novel KCNJ16 variants identified in a Chinese patient with hypokalemic metabolic acidosis.

BACKGROUND: Biallelic pathogenic variants in the KCNJ16 gene result in hypokalemic tubulopathy and deafness (HKTD) (MIM #619406), which is a rare autosomal recessive disease characterized by hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Currently, nine individuals with HKTD have been reported, and seven pathogenic variants in KCNJ16 have been revealed. METHODS: A 5-year-6-month-old Chinese female patient displayed hypokalemic metabolic acidosis, salt wasting, renin-angiotensin-aldosterone system (RAAS) activation, arrhythmia, myocardial damage, cardiogenic shock and secondary diffuse brain oedema. Trio-based whole-exome sequencing (WES) was applied to detect the genetic cause. RESULTS: Novel compound heterozygous variants, c.190A>C (p.Thr64Pro) and c.628C>G (p.His210Asp), in KCNJ16 were detected in the patient, and these variants were inherited from the patient's mother and father, respectively. Then, we systematically reviewed the available clinical manifestations of individuals with HKTD. We found that HKTD patients are at risk of cardiogenic shock and secondary diffuse brain oedema, which urges clinicians to make early diagnoses with prompt treatments. CONCLUSION: These findings expand the variant spectrum of KCNJ16, enrich the clinical characteristics of HKTD, and provide a solid base for the genetic counseling, diagnosis and treatment of this condition.

Cation flux through SUR1-TRPM4 and NCX1 in astrocyte endfeet induces water influx through AQP4 and brain swelling after ischemic stroke.

Brain swelling causes morbidity and mortality in various brain injuries and diseases but lacks effective treatments. Brain swelling is linked to the influx of water into perivascular astrocytes through channels called aquaporins. Water accumulation in astrocytes increases their volume, which contributes to brain swelling. Using a mouse model of severe ischemic stroke, we identified a potentially targetable mechanism that promoted the cell surface localization of aquaporin 4 (AQP4) in perivascular astrocytic endfeet, which completely ensheathe the brain's capillaries. Cerebral ischemia increased the abundance of the heteromeric cation channel SUR1-TRPM4 and of the Na+/Ca2+ exchanger NCX1 in the endfeet of perivascular astrocytes. The influx of Na+ through SUR1-TRPM4 induced Ca2+ transport into cells through NCX1 operating in reverse mode, thus raising the intra-endfoot concentration of Ca2+. This increase in Ca2+ stimulated calmodulin-dependent translocation of AQP4 to the plasma membrane and water influx, which led to cellular edema and brain swelling. Pharmacological inhibition or astrocyte-specific deletion of SUR1-TRPM4 or NCX1 reduced brain swelling and improved neurological function in mice to a similar extent as an AQP4 inhibitor and was independent of infarct size. Thus, channels in astrocyte endfeet could be targeted to reduce postischemic brain swelling in stroke patients.

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

Trifluoperazine reduces apoptosis and inflammatory responses in traumatic brain injury by preventing the accumulation of Aquaporin4 on the surface of brain cells.

Currently, no specific and standard treatment for traumatic brain injury (TBI) has been developed. Therefore, studies on new therapeutic drugs for TBI treatment are urgently needed. Trifluoperazine (TFP) is a therapeutic agent for the treatment of psychiatric disorders that reduces edema of the central nervous system. However, the specific working mechanism of TFP is not fully understood in TBI. In this study, the immunofluorescence co-localization analysis revealed that the area and intensity covered by Aquaporin4 (AQP4) on the surface of brain cells (astrocyte endfeet) increased significantly after TBI. In contrast, TFP treatment reversed these phenomena. This finding showed that TFP inhibited AQP4 accumulation on the surface of brain cells (astrocyte endfeet). The tunel fluorescence intensity and fluorescence area were lower in the TBI+TFP group compared to the TBI group. Additionally, the brain edema, brain defect area, and modified neurological severity score (mNSS) were lower in the TBI+TFP. The RNA-seq was performed on the cortical tissues of rats in the Sham, TBI, and TBI+TFP groups. A total of 3774 genes differently expressed between the TBI and the Sham group were identified. Of these, 2940 genes were up-regulated and 834 genes were down-regulated. A total of 1845 differently expressed genes between the TBI+TFP and TBI group were also identified, in which 621 genes were up-regulated and 1224 genes were down-regulated. Analysis of the common differential genes in the three groups showed that TFP could reverse the expression of apoptosis and inflammation genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the differentially expressed genes (DEGs) were highly enriched in the signaling pathways regulating inflammation. In conclusion, TFP alleviates brain edema after TBI by preventing the accumulation of AQP4 on the surface of brain cells. Generally, TFP alleviates apoptosis and inflammatory response induced by TBI, and promotes the recovery of nerve function in rats after TBI. Thus, TFP is a potential therapeutic agent for TBI treatment.

TRPM4 Drives Cerebral Edema by Switching to Alternative Splicing Isoform After Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) affects persons of all ages and is recognized as a major cause of death and disability worldwide; it also brings heavy life burden to patients and their families. The treatment of those with secondary injury after TBI is still scarce, however. Alternative splicing (AS) is a crucial post-transcriptional regulatory mechanism associated with various physiological processes, while the contribution of AS in treatment after TBI is poorly illuminated. In this study, we performed and analyzed the transcriptome and proteome datasets of brain tissue at multiple time points in a controlled cortical impact (CCI) mouse model. We found that AS, as an independent change against the transcriptional level, is a novel mechanism linked to cerebral edema after TBI. Bioinformatics analysis further indicated that the transformation of splicing isoforms after TBI was related to cerebral edema. Accordingly, we found that the fourth exon of transient receptor potential channel melastatin 4 (Trpm4) abrogated skipping at 72 h after TBI, resulting in a frameshift of the encoded amino acid and an increase in the proportion of spliced isoforms. Using magnetic resonance imaging (MRI), we have shown the numbers of 3nEx isoforms of Trpm4 may be positively correlated with volume of cerebral edema. Thus alternative splicing of Trpm4 becomes a noteworthy mechanism of potential influence on edema. In summary, alternative splicing of Trpm4 may drive cerebral edema after TBI. Trpm4 is a potential therapeutic targeting cerebral edema in patients with TBI.

CircBCL11B acts as a ceRNA to facilitate 1,2-dichloroethane-induced astrocyte swelling via miR-29b-3p/AQP4 axis in SVG p12 cells.

1,2-Dichloroethane (1,2-DCE) is a pervasive environmental pollutant found in ambient and residential air, as well as ground and drinking water. Brain edema is the primary pathological consequence of 1,2-DCE overexposure. We found that microRNA (miRNA)-29b dysregulation after 1,2-DCE exposure can aggravate brain edema by suppressing aquaporin 4 (AQP4). Moreover, circular RNAs (circRNAs) can regulate the expression of downstream target genes through miRNA, and affect protein function. However, circRNAs' role in 1,2-DCE-induced brain edema via miR-29b-3p/AQP4 axis remains unclear. To address the mechanism's bottleneck, we explored the circRNA-miRNA-mRNA network underlying 1,2-DCE-driven astrocyte swelling in SVG p12 cells by circRNA sequencing, electron microscopy and isotope 3H labeling combined with the 3-O-methylglucose uptake method. The results showed that 25 and 50 mM 1,2-DCE motivated astrocyte swelling, characterized by increased water content, enlarged cell vacuoles, and mitochondrial swelling. This was accompanied by miR-29b-3p downregulation and AQP4 upregulation. We verified that AQP4 were negatively regulated by miR-29b-3p in 1,2-DCE-induced astrocyte swelling. Also, circRNA sequencing highlighted that circBCL11B was upregulated by 1,2-DCE. This was manifested as circBCL11B overexpression playing an endogenous competitive role via upregulating AQP4 by binding to miR-29b-3p, thus leading to astrocyte swelling. Conversely, circBCL11B knockdown reversed the 1,2-DCE-motivated AQP4 upregulation and alleviated the cell swelling. Finally, we demonstrated that the circBCL11B was targeted to miR-29b-3p by fluorescence in situ hybridization and dual-luciferase reporter assay. In conclusion, our findings indicate that circBCL11B acts as a competing endogenous RNA to facilitate 1,2-DCE-caused astrocyte swelling via miR-29b-3p/AQP4 axis. These observations provide new insight into the epigenetic mechanisms underlying 1,2-DCE-induced brain edema.

Abcc8 (sulfonylurea receptor-1) knockout mice exhibit reduced axonal injury, cytotoxic edema and cognitive dysfunction vs. wild-type in a cecal ligation and puncture model of sepsis.

Sepsis-associated brain injury (SABI) is characterized by an acute deterioration of mental status resulting in cognitive impairment and acquisition of new and persistent functional limitations in sepsis survivors. Previously, we reported that septic mice had evidence of axonal injury, robust microglial activation, and cytotoxic edema in the cerebral cortex, thalamus, and hippocampus in the absence of blood-brain barrier disruption. A key conceptual advance in the field was identification of sulfonylurea receptor 1 (SUR1), a member of the adenosine triphosphate (ATP)-binding cassette protein superfamily, that associates with the transient receptor potential melastatin 4 (TRPM4) cation channel to play a crucial role in cerebral edema development. Therefore, we hypothesized that knockout (KO) of Abcc8 (Sur1 gene) is associated with a decrease in microglial activation, cerebral edema, and improved neurobehavioral outcomes in a murine cecal ligation and puncture (CLP) model of sepsis. Sepsis was induced in 4-6-week-old Abcc8 KO and wild-type (WT) littermate control male mice by CLP. We used immunohistochemistry to define neuropathology and microglial activation along with parallel studies using magnetic resonance imaging, focusing on cerebral edema on days 1 and 4 after CLP. Abcc8 KO mice exhibited a decrease in axonal injury and cytotoxic edema vs. WT on day 1. Abcc8 KO mice also had decreased microglial activation in the cerebral cortex vs. WT. These findings were associated with improved spatial memory on days 7-8 after CLP. Our study challenges a key concept in sepsis and suggests that brain injury may not occur merely as an extension of systemic inflammation. We advance the field further and demonstrate that deletion of the SUR1 gene ameliorates CNS pathobiology in sepsis including edema, axonal injury, neuroinflammation, and behavioral deficits. Benefits conferred by Abcc8 KO in the murine CLP model warrant studies of pharmacological Abcc8 inhibition as a new potential therapeutic strategy for SABI.

Transcriptome of pituitary function changes in rat model of high altitude cerebral edema.

High altitude cerebral edema (HACE) is a serious subtype of acute mountain sickness (AMS). Studies have suggested that increased expression of corticotropin releasing hormone receptor 1 (CRFR1) in pituitary is related to the development of HACE, but no study has revealed the molecular landscape of pituitary function changes in this process. Rat model of HACE was established by simulating the high-altitude hypobaric hypoxia environment. Then RNA-sequencing was performed of rat pituitary gland (PG) in HACE and non-HACE groups. The function annotations, enrichment analysis, protein-protein interaction (PPI) network, chromosome location and drug repositioning of differentially expressed genes (DEGs) were explored based on the transcriptomic data. And we found pituitary secretion function was disordered in HACE, which was partly due to activated inflammation and oxidative stress. In addition, we identified potential biomarkers for early recognition of pituitary dysfunction and potential protective drugs for pituitary function in HACE.

AQP4-A25Q Point Mutation in Mice Depolymerizes Orthogonal Arrays of Particles and Decreases Polarized Expression of AQP4 Protein in Astrocytic Endfeet at the Blood-Brain Barrier.

Aquaporin-4 (AQP4) is characterized by the formation of orthogonal arrays of particles (OAPs) comprising its M1 and M23 isoforms in the plasma membrane. However, the biological importance of OAP formation is obscure. Here, we developed an OAP depolymerization male mouse model by transgenic knock-in of an AQP4-A25Q mutation. Analyses of the mutant brain tissue using blue native polyacrylamide gel electrophoresis, super-resolution imaging, and immunogold electron microscopy revealed remarkably reduced OAP structures and glial endfeet localization of the AQP4-A25Q mutant protein without effects on its overall mRNA and protein expression. AQP4A25Q/A25Q mice showed better survival and neurologic deficit scores when cerebral edema was induced by water intoxication or middle cerebral artery occlusion/reperfusion. The brain water content and swelling of pericapillary astrocytic endfeet processes in AQP4A25Q/A25Q mice were significantly reduced, functionally supporting decreased AQP4 protein expression at the blood-brain barrier. The infarct volume and neuronal damage were also reduced in AQP4A25Q/A25Q mice in the middle cerebral artery occlusion/reperfusion model. Astrocyte activation in the brain was alleviated in AQP4A25Q/A25Q mice, which may be associated with decreased cell swelling. We conclude that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.SIGNIFICANCE STATEMENT Aquaporin-4 (AQP4) is characterized by orthogonal arrays of particles (OAPs) comprising the M1 and M23 isoforms in the membrane. Here, an OAP depolymerization male mouse model induced by AQP4-A25Q mutation was first established, and the functions of OAP depolymerization in cerebral edema have been studied. The results revealed that AQP4 lost its OAP structure without affecting AQP4 mRNA and protein levels in AQP4-A25Q mice. AQP4-A25Q mutation mice has neuroprotective effects on cerebral edema induced by water intoxication and middle cerebral artery occlusion/reperfusion through relieving the activation of astrocytes and suppressed microglia-mediated neuroinflammation. We concluded that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.

Studies of mutations of assembly factor Hit1 in budding yeast suggest translation defects as the molecular basis for PEHO syndrome.

Regulation of protein synthesis is critical for control of gene expression in all cells. Ribosomes are ribonucleoprotein machines responsible for translating cellular proteins. Defects in ribosome production, function, or regulation are detrimental to the cell and cause human diseases, such as progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) syndrome. PEHO syndrome is a devastating neurodevelopmental disorder caused by mutations in the ZNHIT3 gene, which encodes an evolutionarily conserved nuclear protein. The precise mechanisms by which ZNHIT3 mutations lead to PEHO syndrome are currently unclear. Studies of the human zinc finger HIT-type containing protein 3 homolog in budding yeast (Hit1) revealed that this protein is critical for formation of small nucleolar ribonucleoprotein complexes that are required for rRNA processing and 2'-O-methylation. Here, we use budding yeast as a model system to reveal the basis for the molecular pathogenesis of PEHO syndrome. We show that missense mutations modeling those found in PEHO syndrome patients cause a decrease in steady-state Hit1 protein levels, a significant reduction of box C/D snoRNA levels, and subsequent defects in rRNA processing and altered cellular translation. Using RiboMethSeq analysis of rRNAs isolated from actively translating ribosomes, we reveal site-specific changes in the rRNA modification pattern of PEHO syndrome mutant yeast cells. Our data suggest that PEHO syndrome is a ribosomopathy and reveal potential new aspects of the molecular basis of this disease in translation dysregulation.

Variants in genes encoding the SUR1-TRPM4 non-selective cation channel and sudden infant death syndrome (SIDS): potentially increased risk for cerebral edema.

Increasing evidence suggests that brain edema might play an important role in the pathogenesis of sudden infant death syndrome (SIDS) and that variants of genes for cerebral water channels might be associated with SIDS. The role of the sulfonylurea receptor 1 (SUR1)-transient receptor potential melastatin 4 (TRPM4) non-selective cation channel in cerebral edema was demonstrated by extensive studies. Therefore, we hypothesized that variants at genes of the SUR1-TRPM4 channel complex might be linked to SIDS. Twenty-four polymorphisms in candidate genes involved in the SUR1-TRPM4 non-selective cation channel were investigated in 185 SIDS cases and 339 controls. One (rs11667393 in TRPM4) of these analyzed SNPs reached nominal significance regarding an association with SIDS in the overall analysis (additive model: p = 0.015, OR = 1.438, 95% CI = 1.074-1.925; dominant model: p = 0.036; OR = 1.468, 95% CI = 1.024-2.106). In the stratified analysis, further 8 variants in ABCC8 (encoding SUR1) or TRPM4 showed pronounced associations. However, none of the results remained significant after correction for multiple testing. This preliminary study has provided the first evidence for a genetic role of the SUR1-TRPM4 complex in the etiology of SIDS, and we suggest that our initial results should be evaluated by further studies.

CaV2.1 channel mutations causing familial hemiplegic migraine type 1 increase the susceptibility for cortical spreading depolarizations and seizures and worsen outcome after experimental traumatic brain injury.

Patients suffering from familial hemiplegic migraine type 1 (FHM1) may have a disproportionally severe outcome after head trauma, but the underlying mechanisms are unclear. Hence, we subjected knock-in mice carrying the severer S218L or milder R192Q FHM1 gain-of-function missense mutation in the CACNA1A gene that encodes the α1A subunit of neuronal voltage-gated CaV2.1 (P/Q-type) calcium channels and their wild-type (WT) littermates to experimental traumatic brain injury (TBI) by controlled cortical impact and investigated cortical spreading depolarizations (CSDs), lesion volume, brain edema formation, and functional outcome. After TBI, all mutant mice displayed considerably more CSDs and seizures than WT mice, while S218L mutant mice had a substantially higher mortality. Brain edema formation and the resulting increase in intracranial pressure were more pronounced in mutant mice, while only S218L mutant mice had larger lesion volumes and worse functional outcome. Here, we show that gain of CaV2.1 channel function worsens histopathological and functional outcome after TBI in mice. This phenotype was associated with a higher number of CSDs, increased seizure activity, and more pronounced brain edema formation. Hence, our results suggest increased susceptibility for CSDs and seizures as potential mechanisms for bad outcome after TBI in FHM1 mutation carriers.

The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner.

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1ß (IL-1ß), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

A novel homozygous missense variant in the NAXE gene in an Iranian family with progressive encephalopathy with brain edema and leukoencephalopathy.

Homozygous or compound heterozygous mutations in the NAD(P)HX epimerase (NAXE) gene, cause early-onset progressive encephalopathy with brain edema and/or leukoencephalopathy 1. This disorder is characterized by psychomotor regression, hypotonia, ataxia, respiratory insufficiency, tetraparesis, and seizures, leading to coma and death in early childhood. In this study, whole-exome sequencing was used to identify the pathogenic variant, followed by confirmation of identified variant in the proband and segregation analysis in the family by Sanger sequencing. Several in-silico prediction tools were employed to provide additional evidences on the pathogenicity of the identified variant. The proband was an affected 3-year-old boy presented with encephalopathy and developmental regression from Ardebil province, northwest of Iran. Additional clinical features were cognitive regression and a high level of lactate in CSF. The clinical presentation was suggestive of a mitochondrial disorder. In addition, his brother died at the age of 20 months old due to encephalopathy, seizures, developmental regression, and loss of consciousness. We found a novel homozygous missense variant within the NAXE gene, [NM_144772.3:c.565G > A; p.(Gly189Ser)]. Applying different in-silico prediction tools and bioinformatics databases analysis showed that this variant is damaging. So far, seven mutations have been reported in the NAXE gene. In this study, we report the first mutation in the Iranian population and the eighth one in total for this gene.

Peroxisome proliferator­activated receptor ß/δ regulates cerebral vasospasm after subarachnoid hemorrhage via modulating vascular smooth muscle cells phenotypic conversion.

Cerebral vasospasm (CVS) is a common complication of subarachnoid hemorrhage (SAH) with high deformity rates and cerebral vascular smooth muscle cells (VSMCs) phenotypic switch is considered to be involved in the regulation of CVS. However, to the best of the authors' knowledge, its underlying molecular mechanism remains to be elucidated. Peroxisome proliferator­activated receptor ß/δ (PPARß/δ) has been demonstrated to be involved in the modulation of vascular cells proliferation and maintains the autoregulation function of blood vessels. The present study investigated the potential effect of PPARß/δ on CVS following SAH. A model of SAH was established by endovascular perforation on male adult Sprague­Dawley rats, and the adenovirus PPARß/δ (Ad­PPARß/δ) was injected via intracerebroventricular administration prior to SAH. The expression levels of phenotypic markers α­smooth muscle actin and embryonic smooth muscle myosin heavy chain were measured via western blotting or immunofluorescence staining. The basilar artery diameter and vessel wall thickness were evaluated under fluorescence microscopy. SAH grade, neurological scores, brain water content and brain swelling were measured to study the mechanisms of PPARß/δ on vascular smooth muscle phenotypic transformation. It was revealed that the expression levels of synthetic proteins were upregulated in rats with SAH and this was accompanied by CVS. Activation of PPARß/δ using Ad­PPARß/δ markedly upregulated the contractile proteins elevation, restrained the synthetic proteins expression and attenuated SAH­induced CVS by regulating the phenotypic switch in VSMCs at 72 h following SAH. Furthermore, the preliminary study demonstrated that PPARß/δ downregulated ERK activity and decreased the expression of phosphorylated (p­)ETS domain­containing protein Elk­1 and p­p90 ribosomal S6 kinase, which have been demonstrated to serve an important role in VSMC phenotypic change. Additionally, it was revealed that Ad­PPARß/δ could positively improve CVS by ameliorating the diameter of the basilar artery and mitigating the thickness of the vascular wall. Furthermore, subsequent experiments demonstrated that Ad­PPARß/δ markedly reduced the brain water content and brain swelling and improved the neurological outcome. Taken together, the present study identified PPARß/δ as a useful regulator for the VSMCs phenotypic switch and attenuating CVS following SAH, thereby providing novel insights into the therapeutic strategies of delayed cerebral ischemia.

Attenuation of cerebral edema facilitates recovery of glymphatic system function after status epilepticus.

Status epilepticus (SE) is a neurological emergency usually accompanied by acute cerebral edema and long-term cognitive impairment, and is characterized by neurodegeneration and aberrant hyperphosphorylated tau protein (p-tau) aggregation. The glia-lymphatic (glymphatic) system plays a central role in facilitating the clearance of metabolic waste from the brain, but its relationship with cerebral edema and cognitive dysfunction after SE is unclear. We hypothesized that cerebral edema after SE might impair glymphatic system function through compression, thus leading to impaired removal of metabolic waste, and ultimately affecting long-term cognitive function. Our results showed that glymphatic system function was temporarily impaired, as evidenced by 2-photon imaging, MRI enhancement, imaging of brain sections, and astrocytic water channel aquaporin 4 (AQP4) protein polarization. The severity of cerebral edema on MRI correlated well with glymphatic system dysfunction within 8 days following SE. Moreover, when cerebral edema was alleviated by glibenclamide treatment or genetic deletion of Trpm4, post-SE glymphatic system function recovered earlier, along with fewer p-tau-deposited neurons and neuronal degeneration and better cognitive function. These findings suggest that SE-induced cerebral edema may cause glymphatic system dysfunction and render the post-SE brain vulnerable to p-tau aggregation and neurocognitive impairment.

1,2-Dichloroethane induces apoptosis in the cerebral cortexes of NIH Swiss mice through microRNA-182-5p targeting phospholipase D1 via a mitochondria-dependent pathway.

1,2-Dichloroethane (1,2-DCE) is a pervasive environmental pollutant found in ambient and residential air, as well as ground and drinking water. Overexposure to it results in cortex edema, in both animals and humans. 1,2-DCE induces apoptosis in the cerebellum, liver and testes. This promotes the hypothesis that 1,2-DCE may induce apoptosis in the cortex as brain edema progresses. To validate our hypothesis, 40 NIH male mice were exposed to 0, 100, 350, 700 mg/m3 1,2-DCE by whole-body dynamic inhalation for 28 consecutive days. MicroRNA (miRNA) and mRNA microarray combined with TdT-mediated dUTP nick-end labeling, flow cytometry, and mitochondrial membrane potential (mtΔΨ) measurement were applied to identify the cortex apoptosis pathways' specific responses to 1,2-DCE, in vitro and in vivo. The results showed that 1,2-DCE caused brain edema and increased apoptosis in the mouse cortexes. We confirmed that 1,2-DCE induced increased apoptosis via mitochondrial pathway, both in vitro and in vivo, as evidenced by increased Caspase-3, cleaved Caspase-3, Cytochrome c and Bax expression, and decreased Bcl-2 expression. Additionally, mtΔΨ decreased after 1,2-DCE treatment in vitro. 1,2-DCE exposure increased miR-182-5p and decreased phospholipase D1 (PLD1) in the cerebral cortex of mice. MiR-182-5p overexpression and PLD1 inhibition reduced mtΔΨ and increased astrocyte apoptosis, yet miR-182-5p inhibition alleviated the 1,2-DCE-induced PLD1 down-regulation and the increased apoptosis. Finally, PLD1 was confirmed to be a target of miR-182-5p by luciferase assay. Taken together, our findings indicate that 1,2-DCE exposure induces apoptosis in the cortex via a mitochondria-dependent pathway. This pathway is regulated by a miR-182-5p⊣PLD1 axie.