JR14a: A novel antagonist of C3aR attenuates neuroinflammation in cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI), a prevalent stroke-related complication, can lead to severe brain damage. Inflammation is a crucial factor in CIRI pathogenesis, and the complement component 3a receptor (C3aR) could be a key mediator in the post-CIRI inflammatory cascade. In this study, the role of C3aR in CIRI was investigated utilizing a middle cerebral artery occlusion (MCAO) model in C3aR knockout (KO) mice. Magnetic resonance imaging (MRI) and neurofunctional assessments revealed that C3aR KO mice exhibited significantly diminished cerebral infarction and improved neurological impairments. Consequently, the focus shifted to searching for a small molecule antagonist of C3aR. JR14a, a new potent thiophene antagonist of C3aR, was injected intraperitoneally into mice 1-h post-MCAO model implementation. The mass spectrometry (MS) results indicated the ability of JR14a to penetrate the blood-brain barrier. Subsequent TTC staining and neurofunctional assessments revealed the efficacy of JR14a in reducing cerebral infarct volume and neurological impairment following MCAO. In addition, immunofluorescence (IF) and immunohistochemistry (IHC) demonstrated attenuated microglial activation, neutrophil infiltration, and blood-brain barrier disruption by JR14a in the MCAO model. Furthermore, enzyme-linked immunosorbent assay (ELISA) and Western blotting supported the role of JR14a in downregulating the expression levels of C3aR, tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), as well as the phosphorylation of p65. In conclusion, the findings suggested that C3aR could be a potential therapeutic target for CIRI, and JR14a emerged as a promising treatment candidate.

NLRP6 deficiency inhibits neuroinflammation and ameliorates brain injury in ischemic stroke by blocking NLRs inflammasomes activation through proteasomal degradation of pro-caspase-1.

Innate inflammation is crucial for ischemic stroke development. NLRP6, a nucleotide-binding and oligomerization domain-like receptors (NLRs) family member, regulates innate inflammation. Whether NLRP6 regulates neurological damage and neuroinflammation during ischemic stroke remains unclear. We report that NLRP6 is abundantly expressed in microglia and significantly upregulated in the ischemic brain. The brain injury severity was alleviated in NLRP6-deficient mice after ischemic stroke, as evidenced by reduced cerebral infarct volume, decreased neurological deficit scores, improved histopathological morphological changes, ameliorated neuronal denaturation, and relief of sensorimotor dysfunction. In the co-culture OGD/R model, NLRP6 deficiency prevented neuronal death and attenuated microglial cell injury. NLRP6 deficiency blocked several NLRs inflammasomes' activation and abrogated inflammasome-related cytokine production by decreasing the expression of the common effector pro-caspase-1. NLRP6 deficiency reduced pro-caspase-1's protein level by inducing proteasomal degradation. These findings confirm the neuroprotective role of NLRP6 deficiency in ischemic stroke and its underlying regulation mechanism in neuroinflammation and provide a potential therapeutic target for ischemic stroke.

ß-1, 3-galactosyltransferase 2 ameliorates focal ischemic cerebral injury by maintaining blood-brain barrier integrity.

Blood-brain barrier (BBB) damage at the early stage of ischemic stroke is a vital cause of brain parenchymal injury. The mechanism of BBB disruption has been intensively investigated, but still not fully understood. ß-1, 3-galactosyltransferase 2 (B3galt2) is expressed in the brain, but its role in the pathogenesis of cerebral ischemia remains unknown. In this study, we investigated the role of B3galt2 in cerebral ischemia in mice. Focal cerebral ischemia was induced in mice by middle cerebral artery occlusion (MCAO). B3galt2 protein levels were determined in microvessels which were isolated from ischemic brain at 12, 24 and 72 h after MCAO. Mice were administered lentiviral vectors encoding B3galt2 (LV- B3galt2) or recombinant transforming growth factor-ß1 (r-TGF-ß1) by intracerebroventricular injection. We assessed infarct volume and neurologic deficits on days 1, 3, and 14 after MCAO, blood-brain barrier (BBB) integrity at 12 and 24 h after MCAO, and the levels of TGF-ß1, TGF-ßR(Ⅱ) and p-Smad2/3 at 24 and 72 h after MCAO. Our results indicated that B3galt2 was expressed in brain microvascular endothelial cells and increased in the ischemic microvessels. Overexpression of B3galt2 by LV- B3galt2 administration reduced infarct volume and improved functional outcome after cerebral ischemia. Moreover, the neuroprotective effects were associated with preventing BBB damage. Compared with wild-type (WT) mice, heterozygous B3galt2 knockout (B3galt2-/+) mice not only showed severe BBB damage, neurologic functional deficits, but also showed reduced expression of TGF-ß1, TGF-ßR(Ⅱ) and p-Smad2/3 in microvessels after cerebral ischemia. Pre-administration of r-TGF-ß1 reduced BBB damage, and improved the neurological outcomes in both WT mice and B3galt2-/+ mice after cerebral ischemia. Our results suggested B3galt2 protected against ischemic stroke in mice, and the underlying mechanism might include TGF-ß signaling pathway in brain microvascular endothelial cells.

ß-1, 3-galactosyltransferase 2 deficiency exacerbates brain injury after transient focal cerebral ischemia in mice.

Glycosyltransferases are enzymes that catalyze the formation of a variety of glycoconjugates. Glycoconjugates play vital roles in the nervous system. ß-1, 3-Galactosyltransferase 2 (B3galt2) is one of the major types of glycosyltransferases, which has not been reported in ischemia induced-brain injury. The purpose of this study was to explore the role of B3galt2 exerts and its underlying mechanism in cerebral ischemia in mice. Wild-type (WT) and heterozygous B3galt2 knockout (B3galt2-/+) mice were subjected to 90 min transient focal cerebral ischemia by middle cerebral artery occlusion (MCAO). The brain samples were analyzed at 24 h after reperfusion. The B3galt2 level in the peri-infarct penumbra was quantified. The cerebral infarct volume, neurological deficits, apoptosis and the levels of Reelin and Dab1 were assessed. Compared with control mice, B3galt2-/+ mice not only showed severe brain damage, neurologic functional deficits, but also showed severe neuronal apoptosis in the cortical penumbra after ischemia/reperfusion (I/R). The Caspase-3 activity was increased and the levels of Reelin and Dab1 were decreased in B3galt2-/+ mice. Recombinant human Reelin (rh-Reelin) administered intracerebroventricularly before MCAO significantly reduced infarct volume, and prevented neuronal loss in B3galt2-/+ mice after I/R. Our results suggest B3galt2 deficiency exacerbates ischemic brain damage in acute ischemic stroke in mice, and this was reversed by giving rh-Reelin. B3galt2 might play a beneficial role for neurons survival in the penumbra through modulation of Reelin pathway.

MicroRNA-30a regulates acute cerebral ischemia-induced blood-brain barrier damage through ZnT4/zinc pathway.

The mechanism of early blood-brain barrier (BBB) disruption after stroke has been intensively studied but still not fully understood. Here, we report that microRNA-30a (miR-30a) could mediate BBB damage using both cellular and animal models of ischemic stroke. In the experiments in vitro, inhibition of miR-30a decreased BBB permeability, prevented the degradation of tight junction proteins, and reduced intracellular free zinc in endothelial cells. We found that the zinc transporter ZnT4 was a direct target of negative regulation by miR-30a, and ZnT4/zinc signaling pathway contributed significantly to miR-30a-mediated BBB damage. Consistent with these in vitro findings, treatment with miR-30a inhibitor reduced zinc accumulation, increased the expression of ZnT4, and prevented the loss of tight junction proteins in microvessels of ischemic animals. Furthermore, inhibition of miR-30a, even at 90 min post onset of middle cerebral artery occlusion, prevented BBB damage, reduced infarct volume, and ameliorated neurological deficits. Together, our findings provide novel insights into the mechanisms of cerebral ischemia-induced BBB disruption and indicate miR-30a as a regulator of BBB function that can be an effective therapeutic target for ischemic stroke.

Genome-wide association analysis of stripe rust resistance in modern Chinese wheat.

BACKGROUND: Stripe rust (yellow rust) is a significant disease for bread wheat (Triticum aestivum L.) worldwide. A genome-wide association study was conducted on 240 Chinese wheat cultivars and elite lines genotyped with the wheat 90 K single nucleotide polymorphism (SNP) arrays to decipher the genetic architecture of stripe rust resistance in Chinese germplasm. RESULTS: Stripe rust resistance was evaluated at the adult plant stage in Pixian and Xindu in Sichuan province in the 2015-2016 cropping season, and in Wuhan in Hubei province in the 2013-2014, 2016-2017 and 2018-2019 cropping seasons. Twelve stable loci for stripe rust resistance were identified by GWAS using TASSEL and GAPIT software. These loci were distributed on chromosomes 1B, 1D, 2A, 2B, 3A, 3B, 4B (3), 4D, 6D, and 7B and explained 3.6 to 10.3% of the phenotypic variation. Six of the loci corresponded with previously reported genes/QTLs, including Sr2/Yr30/Lr27, while the other six (QYr.hbaas-1BS, QYr.hbaas-2BL, QYr.hbaas-3AL, QYr.hbaas-4BL.3, QYr.hbaas-4DL, and QYr.hbaas-6DS) are probably novel. The results suggest high genetic diversity for stripe rust resistance in this population. The resistance alleles of QYr.hbaas-2AS, QYr.hbaas-3BS, QYr.hbaas-4DL, and QYr.hbaas-7BL were rare in the present panel, indicating their potential use in breeding for stripe rust resistance in China. Eleven penta-primer amplification refractory mutation system (PARMS) markers were developed from SNPs significantly associated with seven mapped QTLs. Twenty-seven genes were predicted for mapped QTLs. Six of them were considered as candidates for their high relative expression levels post-inoculation. CONCLUSION: The resistant germplasm, mapped QTLs, and PARMS markers developed in this study are resources for enhancing stripe rust resistance in wheat breeding.