Bone marrow hematopoiesis drives multiple sclerosis progression.

Multiple sclerosis (MS) is a T cell-mediated autoimmune disease of the central nervous system (CNS). Bone marrow hematopoietic stem and progenitor cells (HSPCs) rapidly sense immune activation, yet their potential interplay with autoreactive T cells in MS is unknown. Here, we report that bone marrow HSPCs are skewed toward myeloid lineage concomitant with the clonal expansion of T cells in MS patients. Lineage tracing in experimental autoimmune encephalomyelitis, a mouse model of MS, reveals remarkable bone marrow myelopoiesis with an augmented output of neutrophils and Ly6Chigh monocytes that invade the CNS. We found that myelin-reactive T cells preferentially migrate into the bone marrow compartment in a CXCR4-dependent manner. This aberrant bone marrow myelopoiesis involves the CCL5-CCR5 axis and augments CNS inflammation and demyelination. Our study suggests that targeting the bone marrow niche presents an avenue to treat MS and other autoimmune disorders.

Sphingosine-1-phosphate receptor modulation improves neurogenesis and functional recovery after stroke.

Cerebral ischemia activates neural progenitors that participate in brain remodeling following acute injury. Sphingosine-1-phosphate receptor (S1PR) signaling governs cell proliferation and mobilization, yet its potential impact on neural progenitors and stroke recovery remains poorly understood. The goal of this study was to investigate the impact of S1PR modulation on post-stroke neurogenesis and functional recovery, using a S1PR modulator BAF312. Mice were subjected to 60 min middle cerebral artery occlusion (MCAO) and received BAF312 starting from day 3 after MCAO until the end of experiment. BAF312 facilitated motor function recovery in MCAO mice until day 14 after surgery. Flow cytometry analysis revealed that BAF312 treatment led to an increase of type A cells in subventricular zone (SVZ), but not other progenitor cell subsets in MCAO mice. We found an increase of BrdU incorporation in SVZ DCX+ cells from MCAO mice receiving BAF312 and augmented proliferation of DCX+ cells in cultured neurospheres isolated from SVZ tissues. Notably, a S1PR1 antagonist W146 abolished BAF312-induced increase of SVZ type A cells from MCAO mice and proliferation of DCX+ cells in cultured neurospheres. Additionally, the benefit of BAF312 to improve neurogenesis and stroke recovery remains in Rag2-/- mice lacking of T and B cells. Our results demonstrate that S1PR modulation improves neurogenesis and functional recovery following brain ischemia.

NF-κB Signaling-Mediated Activation of WNK-SPAK-NKCC1 Cascade in Worsened Stroke Outcomes of Ang II-Hypertensive Mice.

BACKGROUND: Worsened stroke outcomes with hypertension comorbidity are insensitive to blood pressure-lowering therapies. In an experimental stroke model with comorbid hypertension, we investigated causal roles of ang II (angiotensin II)-mediated stimulation of the brain WNK (with no lysine [K] kinases)-SPAK (STE20/SPS1-related proline/alanine-rich kinase)-NKCC1 (Na-K-Cl cotransporter) complex in worsened outcomes. METHODS: Saline- or ang II-infused C57BL/6J male mice underwent stroke induced by permanent occlusion of the distal branches of the middle cerebral artery. Mice were randomly assigned to receive either vehicle dimethyl sulfoxide/PBS (2 mL/kg body weight/day, IP), a novel SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (ZT-1a' 5 mg/kg per day, IP) or a NF-κB (nuclear factor-κB) inhibitor TAT-NBD (transactivator of transcription-NEMO-binding domain' 20 mg/kg per day, IP). Activation of brain NF-κB and WNK-SPAK-NKCC1 cascade as well as ischemic stroke outcomes were examined. RESULTS: Stroke triggered a 2- to 5-fold increase of WNK (isoforms 1, 2, 4), SPAK/OSR1 (oxidative stress-responsive kinase 1), and NKCC1 protein in the ang II-infused hypertensive mouse brains at 24 hours after stroke, which was associated with increased nuclear translocation of phospho-NF-κB protein in the cortical neurons (a Pearson correlation r of 0.77, P<0.005). The upregulation of WNK-SPAK-NKCC1 cascade proteins resulted from increased NF-κB recruitment on Wnk1, Wnk2, Wnk4, Spak, and Nkcc1 gene promoters and was attenuated by NF-κB inhibitor TAT-NBD. Poststroke administration of SPAK inhibitor ZT-1a significantly reduced WNK-SPAK-NKCC1 complex activation, brain lesion size, and neurological function deficits in the ang II-hypertensive mice without affecting blood pressure and cerebral blood flow. CONCLUSIONS: The ang II-induced stimulation of NF-κB transcriptional activity upregulates brain WNK-SPAK-NKCC1 cascade and contributes to worsened ischemic stroke outcomes, illustrating the brain WNK-SPAK-NKCC1 complex as a therapeutic target for stroke with comorbid hypertension.

A Novel Na+-K+-Cl- Cotransporter 1 Inhibitor STS66* Reduces Brain Damage in Mice After Ischemic Stroke.

Background and Purpose- Inhibition of brain NKCC1 (Na+-K+-Cl- cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods- Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)-induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results- Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II-induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions- The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

Group 2 innate lymphoid cells suppress neuroinflammation and brain injury following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) mobilizes circulating leukocytes that contribute to neuroinflammation and neural injury. However, little is known about the endogenous regulatory immune mechanisms to restrict neuroinflammation following ICH. We examined the role of group 2 innate lymphoid cells (ILC2) that are a specialized subset of innate immune modulators in a mouse model of ICH. We found accumulation of ILC2 in the brain following acute ICH and a concomitant increase of ILC2 within the peripheral lymph nodes. Depletion of ILC2 exacerbated neurodeficits and brain edema after ICH in male and female mice. This aggravated ICH injury was accompanied by augmented microglia activity and leukocyte infiltration. In contrast, expansion of ILC2 using IL-33 led to reduced ICH injury, microglia activity and leukocyte infiltration. Notably, elimination of microglia using a colony stimulating factor 1 receptor inhibitor diminished the exacerbation of ICH injury induced by depletion of ILC2. Brain-infiltrating ILC2 had upregulation of IL-13 after ICH. Results from in vitro assays revealed that ILC2 suppressed thrombin-induced inflammatory activity in microglia-like BV2 cells. Thus, our findings demonstrate that ILC2 suppress neuroinflammation and acute ICH injury.

Translocator Protein Ligand Etifoxine Attenuates MPTP-Induced Neurotoxicity.

Parkinson's disease (PD) is a neurodegenerative disease, but the currently available treatments for this disease are symptomatic treatments. There is evidence that translocator protein (18 kDa) (TSPO) expression is upregulated in some neurodegenerative diseases, and TSPO ligands have obvious neuroprotective effects. However, the neuroprotective effects and other potential effects of the TSPO ligand etifoxine in PD remain unclear. Therefore, the present study was designed to explore the impacts of etifoxine on a mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We found that etifoxine significantly reduced motor function deficits, decreased the loss of tyrosine hydroxylase-positive neurons in the substantia nigra, and attenuated the decrease in striatal dopamine levels in mice that received MPTP. Etifoxine diminished the production of inflammatory mediators and infiltration of leukocytes in the brain after MPTP exposure. In vitro studies suggested that microglia contribute to etifoxine's neuroprotective effect. The results showed that etifoxine can alleviate MPTP-induced neurotoxicity and neuroinflammation, providing a new idea for the treatment of PD.

Inhibition of Colony Stimulating Factor 1 Receptor Suppresses Neuroinflammation and Neonatal Hypoxic-Ischemic Brain Injury.

Hypoxic-ischemic (HI) brain injury is a major cause of neonatal death or lifetime disability without widely accepted effective pharmacological treatments. It has been shown that the survival of microglia requires colony-stimulating factor 1 receptor (CSF1R) signaling and microglia participate in neonatal HI brain injury. We therefore hypothesize that microglia depletion during a HI insult period could reduce immature brain injury. In this study, CD1 mouse pups were treated with a CSF1R inhibitor (PLX3397, 25 mg/kg/daily) or a vehicle from postnatal day 4 to day 11 (P4-11), and over 90% of total brain microglia were deleted at P9. Unilateral hemisphere HI injury was induced at P9 by permanently ligating the left common carotid arteries and exposing the pups to 10% oxygen for 30 min to produce moderate left hemisphere injury. We found that the PLX3397 treatment reduced HI brain injury by 46.4%, as evaluated by the percentage of brain infarction at 48 h after HI. Furthermore, CSF1R inhibition suppressed the infiltration of neutrophils (69.7% reduction, p = 0.038), macrophages (77.4% reduction, p = 0.009), and T cells (72.9% reduction, p = 0.008) to the brain, the production of cytokines and chemokines (such as CCL12, CCL6, CCL21, CCL22, CCL19, IL7, CD14, and WISP-1), and reduced neuronal apoptosis as indicated by active caspase-3 labeled cells at 48 h after HI (615.20 ± 156.84/mm2 vs. 1,205.00 ± 99.15/mm2, p = 0.013). Our results suggest that CSF1R inhibition suppresses neuroinflammation and neonatal brain injury after acute cerebral hypoxia-ischemia in neonatal mice.

Activation of endothelial Wnt/ß-catenin signaling by protective astrocytes repairs BBB damage in ischemic stroke.

The role of astrocytes in dysregulation of blood-brain barrier (BBB) function following ischemic stroke is not well understood. Here, we investigate the effects of restoring the repair properties of astrocytes on the BBB after ischemic stroke. Mice deficient for NHE1, a pH-sensitive Na+/H+ exchanger 1, in astrocytes have reduced BBB permeability after ischemic stroke, increased angiogenesis and cerebral blood flow perfusion, in contrast to wild-type mice. Bulk RNA-sequencing transcriptome analysis of purified astrocytes revealed that ∼177 genes were differentially upregulated in mutant astrocytes, with Wnt7a mRNA among the top genes. Using a Wnt reporter line, we confirmed that the pathway was upregulated in cerebral vessels of mutant mice after ischemic stroke. However, administration of the Wnt/ß-catenin inhibitor, XAV-939, blocked the reparative effects of Nhe1-deficient astrocytes. Thus, astrocytes lacking pH-sensitive NHE1 protein are transformed from injurious to "protective" by inducing Wnt production to promote BBB repair after ischemic stroke.

Modulation of brain cation-Cl- cotransport via the SPAK kinase inhibitor ZT-1a.

The SLC12A cation-Cl- cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.

Efficacy and safety of sonothombolysis versus non-sonothombolysis in patients with acute ischemic stroke: A meta-analysis of randomized controlled trials.

Recent studies have shown that inconsistent results of safety and efficacy between sonothombolysis vs. non-sonothombolysis in acute ischemic stroke (AIS). We implemented a meta-analysis to explore the value of sonothrombolysis in AIS treatment. The MEDLINE, EMBASE, and Cochrane Library databases were searched for randomized controlled trials (RCTs) which had evaluated sonothrombolysis or ultrasound thrombolysis in AIS. One hundred five studies were retrieved and analyzed, among them, 7 RCTs were included in the current meta-analysis. In comparison with the non-sonothombolysis, sonothrombolysis significantly improved complete recanalization (RR 2.16, 95% CI 1.51 to 3.08, P < 0.001), complete or partial recanalization (RR 1.90, 95% CI 1.26 to 2.88, P = 0.002), there is also a tendency to improvement of ≥ 4 points in NIHSS score (RR 1.43, 95% CI 0.99 to 2.07, P = 0.057). However, sonothrombolysis and non-sonothrombolysis had insignificant differences in neurological recovery and adverse events. In subgroup analysis, we found that "With t-PA", "NIHSS > 15", "Treatment time ≤ 150min", and "Age ≤ 65 years" are potential favorable factors for efficacy outcomes of sonothombolysis. Sonothrombolysis can significantly increase the rate of recanalization in patients with AIS compared with non-sonothrombolysis, but there is no significant effect on improving neurological functional recovery and avoiding complications.

Interleukin-33 reduces neuronal damage and white matter injury via selective microglia M2 polarization after intracerebral hemorrhage in rats.

Interleukin-33 (IL-33) is closely related to the regulation of immunological cells, and its receptor ST2 is a member of the interleukin-1 (IL-1) receptor family. Inflammatory responses play critical roles in neuronal damage and white matter injury (WMI) post intracerebral hemorrhage (ICH). In this study, we tried to explore the role of IL-33 in neuronal damage and WMI after ICH and the underlying mechanisms. The in vivo ICH model was performed by autologous whole blood injection into the right basal ganglia in rats. Immunoblotting, immunofluorescence, brain water content measurement, FJB staining, and TUNEL staining were applied in this study. IL-33 expression was increased in whole brain tissues post-ICH, mainly rapidly increased in ipsilateral astrocyte and microglia, but stayed at a low level in neurons. Intracerebroventricular infusion of IL-33 after ICH attenuated short-term and long-term neurological deficits, WMI, neuronal degeneration, cell death and promoted the transformation of microglia phenotype from M1 to M2 in brain tissues after ICH. These results suggest that IL-33 reduces neuronal damage and WMI by promoting microglia M2 polarization after ICH, thereby improving the outcomes of neurological function.

The WNK-SPAK/OSR1 Kinases and the Cation-Chloride Cotransporters as Therapeutic Targets for Neurological Diseases.

In recent years, cation-chloride cotransporters (CCCs) have drawn attention in the medical neuroscience research. CCCs include the family of Na+-coupled Cl- importers (NCC, NKCC1, and NKCC2), K+-coupled Cl- exporters (KCCs), and possibly polyamine transporters (CCC9) and CCC interacting protein (CIP1). For decades, CCCs have been the targets of several commonly used diuretic drugs, including hydrochlorothiazide, furosemide, and bumetanide. Genetic mutations of NCC and NKCC2 cause congenital renal tubular disorders and lead to renal salt-losing hypotension, secondary hyperreninemia, and hypokalemic metabolic alkalosis. New studies reveal that CCCs along with their regulatory WNK (Kinase with no lysine (K)), and SPAK (Ste20-related proline-alanine-rich kinase)/OSR1(oxidative stress-responsive kinase-1) are essential for regulating cell volume and maintaining ionic homeostasis in the nervous system, especially roles of the WNK-SPAK-NKCC1 signaling pathway in ischemic brain injury and hypersecretion of cerebrospinal fluid in post-hemorrhagic hydrocephalus. In addition, disruption of Cl- exporter KCC2 has an effect on synaptic inhibition, which may be involved in developing pain, epilepsy, and possibly some neuropsychiatric disorders. Interference with KCC3 leads to peripheral nervous system neuropathy as well as axon and nerve fiber swelling and psychosis. The WNK-SPAK/OSR1-CCCs complex emerges as therapeutic targets for multiple neurological diseases. This review will highlight these new findings.

Ischemic Injury-Induced CaMKIIδ and CaMKIIγ Confer Neuroprotection Through the NF-κB Signaling Pathway.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has long been implicated in neuronal injury caused by acute ischemia/reperfusion (I/R). However, its precise role and regulatory mechanisms remain obscure. Here, we investigated the role of the CaMKII family in neuronal survival during I/R. Our data indicated that CAMK2D/CaMKIIδ and CAMK2G/CaMKIIγ were selectively upregulated in a time-dependent manner at both transcriptional and protein levels after acute ischemia. Overexpression of CaMKIIδ promoted neuronal survival, while their depletion exacerbated ischemic neuronal death. Similar to CaMKIIδ, knockdown of CAMKIIγ resulted in significant neuronal death after I/R. We further identified CaMKIIδ2 as the subtype that is selectively induced by I/R in primary neurons. The induction of CaMKIIδ was controlled in part by a pair of long non-coding RNAs (lncRNAs), C2dat1 and C2dat2. C2dat2, similar to C2dat1, was upregulated by I/R and cooperated with C2dat1 to modulate CaMKIIδ expression. Knockdown of C2dat1/2 blocked OGD/R-induced CaMKIIδ expression and decreased neuronal survival but did not affect the levels of CaMKIIγ, indicating specific targeting of CAMK2D by C2dat1/2. Mechanistically, I/R-induced CaMKIIδ and CaMKIIγ caused the upregulation of IKKα/ß and further activation of the NF-κB signaling pathway to protect neurons from ischemic damage. Genetically, downregulating p65 subunit of NF-κB in mice increased I/R-induced neuronal death by blocking the activity of CaMKII/IKK/IκBα/NF-κB signaling axis. In summary, CaMKIIδ and CaMKIIγ are novel I/R-induced genes that promote neuronal survival during ischemic injury. The upregulation of these CaMKII kinases led to activation of the NF-κB signaling pathway, which protects neurons from ischemic damage.