Microglia sense astrocyte dysfunction and prevent disease progression in an Alexander disease model.

Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. It is a primary astrocyte disease with a pathological hallmark of Rosenthal fibres within astrocytes. AxD astrocytes show several abnormal phenotypes. Our previous study showed that AxD astrocytes in model mice exhibit aberrant Ca2+ signals that induce AxD aetiology. Here, we show that microglia have unique phenotypes with morphological and functional alterations, which are related to the pathogenesis of AxD. Immunohistochemical studies of 60TM mice (AxD model) showed that AxD microglia exhibited highly ramified morphology. Functional changes in microglia were assessed by Ca2+ imaging using hippocampal brain slices from Iba1-GCaMP6-60TM mice and two-photon microscopy. We found that AxD microglia showed aberrant Ca2+ signals, with high frequency Ca2+ signals in both the processes and cell bodies. These microglial Ca2+ signals were inhibited by pharmacological blockade or genetic knockdown of P2Y12 receptors but not by tetrodotoxin, indicating that these signals are independent of neuronal activity but dependent on extracellular ATP from non-neuronal cells. Our single-cell RNA sequencing data showed that the expression level of Entpd2, an astrocyte-specific gene encoding the ATP-degrading enzyme NTPDase2, was lower in AxD astrocytes than in wild-type astrocytes. In situ ATP imaging using the adeno-associated virus vector GfaABC1D ATP1.0 showed that exogenously applied ATP was present longer in 60TM mice than in wild-type mice. Thus, the increased ATP level caused by the decrease in its metabolizing enzyme in astrocytes could be responsible for the enhancement of microglial Ca2+ signals. To determine whether these P2Y12 receptor-mediated Ca2+ signals in AxD microglia play a significant role in the pathological mechanism, a P2Y12 receptor antagonist, clopidogrel, was administered. Clopidogrel significantly exacerbated pathological markers in AxD model mice and attenuated the morphological features of microglia, suggesting that microglia play a protective role against AxD pathology via P2Y12 receptors. Taken together, we demonstrated that microglia sense AxD astrocyte dysfunction via P2Y12 receptors as an increase in extracellular ATP and alter their morphology and Ca2+ signalling, thereby protecting against AxD pathology. Although AxD is a primary astrocyte disease, our study may facilitate understanding of the role of microglia as a disease modifier, which may contribute to the clinical diversity of AxD.

Severity of Peripheral Infection Differentially Affects Brain Functions in Mice via Microglia-Dependent and -Independent Mechanisms.

Peripheral infection induces inflammation in peripheral tissues and the brain, impacting brain function. Glial cells are key players in this process. However, the effects of peripheral infection on glial activation and brain function remain unknown. Here, we showed that varying degrees of peripheral infection had different effects on the regulation of brain functions by microglia-dependent and -independent mechanisms. Acute mild infection (one-day LPS challenge: 1LPS) exacerbated middle cerebral artery occlusion (MCAO) injury, and severe infection (four-day LPS challenge: 4LPS) for one week suppressed it. MCAO injury was assessed by triphenyltetrazolium chloride staining. We observed early activation of microglia in the 1LPS and 4LPS groups. Depleting microglia with a colony-stimulating factor-1 receptor (CSF1R) antagonist had no effect on 1LPS-induced brain injury exacerbation but abolished 4LPS-induced protection, indicating microglial independence and dependence, respectively. Microglia-independent exacerbation caused by 1LPS involved peripheral immune cells including macrophages. RNA sequencing analysis of 4LPS-treated microglia revealed increased factors related to anti-inflammatory and neuronal tissue repair, suggesting their association with the protective effect. In conclusion, varying degrees of peripheral inflammation had contradictory effects (exacerbation vs. protection) on MCAO, which may be attributed to microglial dependence. Our findings highlight the significant impact of peripheral infection on brain function, particularly in relation to glial cells.

Transcriptional regulatory elements of hif1α in a distal locus of islet1 in Xenopus laevis.

Adult mammalian hearts are not regenerative. However, recent studies have evidenced that hypoxia enhances their regeneration. Islet1 (isl1) is known as a cardiac progenitor marker, which is quiescent in adult mammal hearts. In Xenopus hearts, transcriptional activation of isl1 was shown during cardiac regeneration of froglets at 3 months after metamorphosis. In this study, we examined transcriptional regulation of isl1 focusing on hypoxia-inducible factor 1α (hif1α) in Xenopus heart. We found that hif1α expression was increased in response to cardiac injury and overexpression of hif1α upregulated mRNA expression of isl1. Multiple conservation analysis including 9 species revealed that 8 multiple conserved regions (MCRs) were present upstream of isl1. DNA sequence analysis using JASPAR showed hif1α binding motifs in MCRs. By luciferase reporter assay and chromatin immunoprecipitation analysis, we found that hif1α directly bound to hif1α motifs in the most distant MCR8 and showed a specific transcriptional activity on the MCR8. In the luciferase assay using constructs carrying MCR8 without a responsive motif of hif1α, the reporter activity was lost. Pharmacologically inhibition of hif1α affected isl1 transcription and downstream events including cardiac phenotypes, suggesting functional defects of islet1. Contrarily in murine hearts, transcription of isl1 was unresponsive even after cryoinjury to adult hearts while hif1α mRNA was induced. In comparative analysis of multiple alignment, hif1α elements present in MCR8 of Xenopus or zebrafish were found to be disrupted as species are evolutionarily distant from Xenopus and zebrafish. Our results suggested an altered switch of isl1 transcription between mammals and Xenopus laevis.

Derivation of proliferative islet1-positive cells during metamorphosis and wound response in Xenopus.

In mammalian hearts, cardiomyocytes retain a transient capacity to proliferate and regenerate following injury before birth, whereas they lose proliferative capacity immediately after birth. It has also been known that cardiac progenitor cells including islet1-positive cells do not contribute to the cardiac repair and regeneration in mammals. In contrast, hearts of zebrafish, amphibians and reptiles maintain a regenerative ability throughout life. Here, we analyzed proliferative capacity of cardiac cells during cardiac development and post-ventricular resection using Xenopus laevis, especially focusing on islet1. Immunohistochemical examination showed that islet1-positive cells were present in a wide range of the ventricle and maintained high dividing ability after metamorphosis. Interestingly, the islet1-positive cells were preserved even at 1 year after metamorphosis, some of which showed tropomyosin expression. To assess the possibility of islet1-positive cells as a cellular resource, islet1 response to cardiac resection was analyzed, using adult hearts of 3 months after metamorphosis. Transient gene activation of islet1 in apical region was detected within 1 day after amputation. Histological analyses revealed that islet1-positive cells appeared in the vicinity of resection plane at 1 day post-amputation (dpa) and increased at 3 dpa in both tropomyosin-positive and tropomyosin-negative regions. Vascular labeling analysis by biotinylated dextran amine (BDA) indicated that the islet1-positive cells in a tropomyosin-negative region were closely associated with cardiac vessels. Moreover, dividing ability at this time point was peaked. The resected region was healed with tropomyosin-positive cardiomyocytes until 3 months post-amputation. These results suggest a role of islet1-positive cells as a cellular resource for vascularization and cardiogenesis in Xenopus laevis.

Sialic acid deficiency is associated with oxidative stress leading to muscle atrophy and weakness in GNE myopathy.

Sialic acids are monosaccharides found in terminal sugar chains of cell surfaces and proteins; they have various biological functions and have been implicated in health and disease. Genetic defects of the GNE gene which encodes a critical bifunctional enzyme for sialic acid biosynthesis, lead to GNE myopathy, a disease manifesting with progressive muscle atrophy and weakness. The likely mechanism of disease is a lack of sialic acids. There remains, however, an unexplained link between hyposialylation and the muscle atrophy and weakness. In this study, we found that muscle proteins were highly modified by S-nitrosylation, and that oxidative stress-responsive genes were significantly upregulated, in hyposialylated muscles from human GNE myopathy patients and model mice. In both in vitro and in vivo models, the production of reactive oxygen species (ROS) was elevated with cellular hyposialylation, and increasing overall sialylation by extrinsic sialic acid intake reduced ROS and protein S-nitrosylation. More importantly, the antioxidant, oral N-acetylcysteine led to amelioration of the muscle atrophy and weakness in Gne mutant mice. Our data provide evidence of additional important function of sialic acids as a ROS scavenger in skeletal muscles, expanding our understanding on how sialic acid deficiency contributes to disease pathology, and identify oxidative stress as a therapeutic target in GNE myopathy.

Conserved C272/278 in b domain regulate the function of PDI-P5 to make lysozymes trypsin-resistant forms via significant intermolecular disulfide cross-linking.

Protein disulfide isomerase-P5 (P5) is thought to have important functions as an oxidoreductase, however, molecular functions of P5 have not been fully elucidated. We have reported that P5 has insulin reductase activity and inhibits lysozyme refolding by formation of lysozyme multimers with hypermolecular mass inactivated by intermolecular disulfides (hyLYS) in oxidative refolding of reduced denatured lysozyme. To explore the role of each domain of P5, we investigated the effects of domain deletion and Cys-Ala mutants of P5 on insulin reduction and the oxidative refolding of the lysozyme. The mutants of catalytic cysteines, C36/39A, C171/174A, and C36/39/171/174A inhibited the lysozyme refolding almost similarly to P5, and even b domain without catalytic cysteines showed moderate inhibitory effect, suggesting that the b domain played a key role in the inhibition. Western blotting analysis of the refolding products indicated that the catalytic cysteines in both the a and a' domains cross-linked lysozyme comparably to form hyLYS resistant to trypsin, in which b domain was suggested to capture lysozyme for the significant sulfhydryl oxidation. The mutant of the conserved cysteines in b domain, C272/278A, did not form hyLYS, however, showed predominant reductase activity, implying that P5 functioned as a potent sulfhydryl oxidase and a predominant reductase depending on the circumstance around C272/278. These results provide new insight into the molecular basis of P5 function.

Angiotensin II receptor antagonist reduces subsequent uterine arterial dysfunction in pregnant offspring of protein-restricted rat dams.

AIM: A low-protein diet (LPD) during pregnancy induces vascular dysfunction and hypertension in the offspring, prevented by administration of an angiotensin II type 1 (AT(1)) receptor antagonist in early life to the offspring. Whether such protection extends to subsequent pregnancy is unknown; we therefore hypothesized that administration of a specific AT(1) receptor antagonist (losartan) in early life to offspring of LPD dams would improve vascular dysfunction in their uterine arteries when they, in turn, were pregnant. METHODS: Pregnant rats were randomly divided into two dietary groups fed a control (C) or protein-restricted (R) diet throughout pregnancy. Between two and 10 weeks postnatally, female offspring (F(1)) were randomly assigned to drink either pure tap water (CO, RO) or water with losartan (CL, RL). Offspring were mated and killed on gestational day 19 or 20 in order to investigate uterine artery function. RESULTS: In pregnant offspring, vasoconstriction of the uterine arteries to phenylephrine (PE) and the thromboxane A2 mimetic U46619 was greater in RO than CO (F(1)). Responses to both antagonists were suppressed in RL (F(1)). Relaxation to sodium nitroprusside was increased in RO versus CO and suppressed in RL versus RO (F(1)). CONCLUSION: Administration of an AT(1) receptor antagonist to offspring during the suckling and juvenile period improves the uterine vascular dysfunction in pregnancy induced by prior maternal LPD during their development. Such treatment may contribute to decreasing the transmitted risks of maternal malnutrition from offspring to the subsequent generation.

Vascular, renal and placental effects on pregnant offspring of protein-restricted rat dams.

AIM: Our previous study showed that a maternal low-protein diet induced hypertension and vascular dysfunction in rat offspring after day 175. In the present study, we hypothesized that these female offspring would develop hypertension in their own pregnancies even at ages less than 175 days because potential vascular dysfunction is exacerbated by the circulatory demands of pregnancy. MATERIAL AND METHODS: Wistar rats were fed an isocaloric diet containing either 18% (control group) or 9% (low-protein group) casein throughout pregnancy. The female offspring were fed standard chow and mated between days 70 and 125. At the end of pregnancy, blood pressure was measured, and the uterine arteries were dissected and investigated with a wire myograph. RESULTS: Placental weights were significantly lower in offspring of the low-protein group versus control. There were no significant differences in blood pressure. Renal expression of AT1 receptor mRNA was greater, and of AT2 receptor was less, in the low-protein group versus control. Vasoconstriction of uterine arteries to phenylephrine and U46619 was increased in the low-protein group, and vasodilatation to sodium nitroprusside was also increased. CONCLUSION: Low-protein diet induces vascular effects on female offspring in their pregnancy, in terms of increased uterine artery vasoconstriction. This may be compensated for by increased sensitivity to nitric oxide (NO), maintaining blood pressure normal in the face of the demands of pregnancy. Such renal and vascular effects, combined with placental size, may transmit risk of vascular dysfunction to subsequent generations.