Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus of understanding ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglia-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus to understand ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglial-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

IFN-γ deficiency in the rostral ventrolateral medulla contributes to stress-induced hypertension by impairing microglial synaptic engulfment.

BACKGROUND: Dysfunctional neurons and microglia in the rostral ventrolateral medulla (RVLM) have been implicated in the pathogenesis of stress-induced hypertension (SIH). Functional perturbation of microglial synaptic engulfment can induce aberrant brain circuit activity. IFN-γ is a pleiotropic cytokine that plays a role in regulating neuronal activity. However, existing research on the exploration of the effects of microglia on synapses in the RVLM is lacking, particularly on the function of IFN-γ in microglial synaptic engulfment involved in SIH. METHODS: A SIH rat model was established by electric foot shocks combined with noise stimulation. The underlying mechanism of IFN-γ on synaptic density and microglial synaptic engulfment was investigated through in-vivo and in-vitro experiments involving gain of function, immunofluorescence, quantitative real-time PCR, western blot, and morphometric analysis. Furthermore, the function of IFN-γ in neuronal activity, renal sympathetic nerve activity (RSNA), and blood pressure (BP) regulation was determined through in-vivo and in-vitro experiments involving Ca 2+ imaging, immunofluorescence, platinum-iridium electrode recording, ELISA, the femoral artery cannulation test, and the tail-cuff method. RESULTS: The BP, heart rate, RSNA, plasma norepinephrine, and the number of c-Fos-positive neurons in SIH rats increased compared with those in control rats. Pre and postsynaptic densities in the RVLM also increased in SIH rats. IFN-γ and CCL2 expression levels were significantly reduced in the RVLM of the SIH group, whose microglia also exhibited an impaired capacity for synapse engulfment. IFN-γ elevation increased CCL2 expression and microglial synaptic engulfment and decreased synaptic density in vivo and in vitro . However, CCL2 inhibition reversed these effects. Moreover, the reduction of neuronal excitability, RSNA, plasma norepinephrine, and BP by IFN-γ was abrogated through CCL2 expression. CONCLUSION: IFN-γ deficiency in the RVLM impaired the microglial engulfment of synapses by inhibiting CCL2 expression and increasing synaptic density and neuronal excitability, thereby contributing to SIH progression. Targeting IFN-γ may be considered a potential strategy to combat SIH.

Microglia-derived TNF-α contributes to RVLM neuronal mitochondrial dysfunction via blocking the AMPK-Sirt3 pathway in stress-induced hypertension.

BACKGROUND: Neuroinflammation in the rostral ventrolateral medulla (RVLM) has been associated with the pathogenesis of stress-induced hypertension (SIH). Neuronal mitochondrial dysfunction is involved in many pathological and physiological processes. However, the impact of neuroinflammation on neuronal mitochondrial homeostasis and the involved signaling pathway in the RVLM during SIH are largely unknown. METHODS: The morphology and phenotype of microglia and the neuronal mitochondrial injury in vivo were analyzed by immunofluorescence, Western blot, RT-qPCR, transmission electron microscopy, and kit detection. The underlying mechanisms of microglia-derived tumor necrosis factor-α (TNF-α) on neuronal mitochondrial function were investigated through in vitro and in vivo experiments such as immunofluorescence and Western blot. The effect of TNF-α on blood pressure (BP) regulation was determined in vivo via intra-RVLM microinjection of TNF-α receptor antagonist R7050. RESULTS: The results demonstrated that BP, heart rate (HR), renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE), and electroencephalogram (EEG) power increased in SIH rats. Furthermore, the branching complexity of microglia in the RVLM of SIH rats decreased and polarized into M1 phenotype, accompanied by upregulation of TNF-α. Increased neuronal mitochondria injury was observed in the RVLM of SIH rats. Mechanistically, Sirtuin 3 (Sirt3) and p-AMPK expression were markedly downregulated in both SIH rats and TNF-α-treated N2a cells. AMPK activator A769662 upregulated AMPK-Sirt3 signaling pathway and consequently reversed TNF-α-induced mitochondrial dysfunction. Microinjection of TNF-α receptor antagonist R7050 into the RVLM of SIH rats significantly inhibited the biological activities of TNF-α, increased p-AMPK and Sirt3 levels, and alleviated neuronal mitochondrial injury, thereby reducing c-FOS expression, RSNA, plasma NE, and BP. CONCLUSIONS: This study revealed that microglia-derived TNF-α in the RVLM impairs neuronal mitochondrial function in SIH possibly through inhibiting the AMPK-Sirt3 pathway. Therefore, microglia-derived TNF-α in the RVLM may be a possible therapeutic target for the intervention of SIH.

CircRNA Galntl6 sponges miR-335 to ameliorate stress-induced hypertension through upregulating Lig3 in rostral ventrolateral medulla.

Rostral ventrolateral medulla (RVLM) is thought to serve as a major vasomotor center that participates in controlling the progression of stress-induced hypertension (SIH). Circular RNAs (circRNAs) perform important functions in the regulation of diverse physiological and pathological processes. However, information concerning the functions of RVLM circRNAs on SIH remains limited. RNA sequencing was performed to profile circRNA expression in RVLMs from SIH rats, which were induced by electric foot shocks and noises. The functions of circRNA Galntl6 in reducing blood pressure (BP) and its potential molecular mechanisms on SIH were investigated via various experiments, such as Western blot and intra-RVLM microinjection. A total of 12,242 circRNA transcripts were identified, among which circRNA Galntl6 was dramatically downregulated in SIH rats. The upregulation of circRNA Galntl6 in RVLM effectively decreased the BP, sympathetic outflow, and neuronal excitability in SIH rats. Mechanistically, circRNA Galntl6 directly sponged microRNA-335 (miR-335) and restrained it to reduce oxidative stress. Reintroduction of miR-335 observably reversed the circRNA Galntl6-induced attenuation of oxidative stress. Furthermore, Lig3 can be a direct target of miR-335. MiR-335 inhibition substantially increased the expression of Lig3 and suppressed oxidative stress, and these favorable effects were blocked by Lig3 knockdown. CircRNA Galntl6 is a novel factor that impedes SIH development, and the circRNA Galntl6/miR-335/Lig3 axis represents one of the possible mechanisms. These findings demonstrated circRNA Galntl6 as a possibly useful target for the prevention of SIH.

PDZD8-mediated endoplasmic reticulum-mitochondria associations regulate sympathetic drive and blood pressure through the intervention of neuronal mitochondrial homeostasis in stress-induced hypertension.

Neuronal hyperexcitation in the rostral ventrolateral medulla (RVLM) drives heightened sympathetic nerve activity and contributes to the etiology of stress-induced hypertension (SIH). Maintenance of mitochondrial functions is central to neuronal homeostasis. PDZD8, an endoplasmic reticulum (ER) transmembrane protein, tethers ER to mitochondria. However, the mechanisms of PDZD8-mediated ER-mitochondria associations regulating neuronal mitochondrial functions and thereby mediating blood pressure (BP) in the RVLM of SIH were largely unknown. SIH rats were subjected to intermittent electric foot shocks plus noise for 2 h twice daily for 15 consecutive days. The underlying mechanisms of PDZD8 were investigated through in vitro experiments by using small interfering RNA and through in vivo experiments, such as intra-RVLM microinjection and Western blot analysis. The function of PDZD8 on BP regulation in the RVLM was determined in vivo via the intra-RVLM microinjection of adeno-associated virus (AAV)2-r-Pdzd8. We found that the c-Fos-positive RVLM tyrosine hydroxylase (TH) neurons, renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE) level, BP, and heart rate (HR) were elevated in SIH rats. ER-mitochondria associations in RVLM neurons were significantly reduced in SIH rats. PDZD8 was mainly expressed in RVLM neurons, and mRNA and protein levels were markedly decreased in SIH rats. In N2a cells, PDZD8 knockdown disrupted ER-mitochondria associations and mitochondrial structure, decreased mitochondrial membrane potential (MMP) and respiratory metabolism, enhanced ROS levels, and reduced catalase (CAT) activity. These effects suggested that PDZD8 dysregulation induced mitochondrial malfunction. By contrast, PDZD8 upregulation in the RVLM of SIH rats could rescue neuronal mitochondrial function, thereby suppressing c-Fos expression in TH neurons and decreasing RSNA, plasma NE, BP, and HR. Our results indicated that the dysregulation of PDZD8-mediated ER-mitochondria associations led to the loss of the activity homeostasis of RVLM neurons by disrupting mitochondrial functions, thereby participating in the regulation of SIH pathology.

Ultrathin Niobium Carbide MXenzyme for Remedying Hypertension by Antioxidative and Neuroprotective Actions.

Hypertension, as a leading risk factor for cardiovascular diseases, is associated with oxidative stress and impairment of endogenous antioxidant mechanisms, but there is still a tremendous knowledge gap between hypertension treatment and nanomedicines. Herein, we report a specific nanozyme based on ultrathin two-dimensional (2D) niobium carbide (Nb2 C) MXene, termed Nb2 C MXenzyme, to fight against hypertension by achieving highly efficient reactive oxygen species elimination and inflammatory factors inhibition. The biocompatible Nb2 C MXenzyme displays multiple enzyme-mimicking activities, involving superoxide dismutase, catalase, glutathione peroxidase, and peroxidase, inducing cytoprotective effects by resisting oxidative stress, thereby alleviating inflammatory response and reducing blood pressure, which is systematically demonstrated in a stress-induced hypertension rat model. This strategy not only opens new opportunities for nanozymes to treat hypertension but also expands the potential biomedical applications of 2D MXene nanosystems.

LncRNA INPP5F ameliorates stress-induced hypertension via the miR-335/Cttn axis in rostral ventrolateral medulla.

AIMS: The rostral ventrolateral medulla (RVLM) is an essential vasomotor center responsible for regulating the development of stress-induced hypertension (SIH). Long non-coding RNAs (lncRNAs) play critical roles in various physiopathology processes, but existing research on the functions of RVLM lncRNAs on SIH has been lacking. In this study, we investigated the roles of RVLM lncRNAs in SIH. METHODS: Genome-wide lncRNA profiles in RVLM were determined by RNA sequencing in a SIH rat model established using electric foot shocks plus noises. The hypotensive effect of lncRNA INPP5F and the underlying mechanisms of lncRNA INPP5F on SIH were explored through in vivo and in vitro experiments, such as intra-RVLM microinjection and immunofluorescence. RESULTS: We discovered 10,179 lncRNA transcripts, among which the lncRNA INPP5F expression level was significantly decreased in SIH rats. Overexpression of lncRNA INPP5F in RVLM dramatically reduced the blood pressure, sympathetic nerve activity, and neuronal excitability of SIH rats. LncRNA INPP5F overexpression markedly increased Cttn expression and reduced neural apoptosis by activating the PI3K-AKT pathway, and its inhibition had opposite effects. Mechanistically, lncRNA INPP5F acted as a sponge of miR-335, which further regulated the Cttn expression. CONCLUSION: LncRNA INPP5F was a key factor that inhibited SIH progression, and the identified lncRNA INPP5F/miR-335/Cttn/PI3K-AKT/apoptosis axis represented one of the possible mechanisms. LncRNA INPP5F could serve as a therapeutic target for SIH.

A Multifunctional Photoacoustic/Fluorescence Dual-Mode-Imaging Gold-Based Theranostic Nanoformulation without External Laser Limitations.

Theranostics is a new type of biomedical technology that organically combines the diagnosis and therapy of diseases. Among molecular imaging techniques, the integration of photoacoustic (PA) and fluorescence (FL) imaging modes with high sensitivity and imaging depth provides precise diagnostic outcomes. Gold nanorods (Au NRs) are well-known contrast agents for PA imaging and photothermal therapy. However, their high toxicity, poor biocompatibility, rapid clearance, and the need for an external laser source limit their application. Therefore, modification of Au NRs with carbon-based nanomaterials (CBNs) is done to obtain a multifunctional dual-mode gold-based nanoformulation (mdGC), which preforms dual-mode imaging of PA and FL. The results show that mdGC promotes tumor cell apoptosis and exhibits good antitumor performance through the mitochondria-mediated apoptotic pathway by increasing the production of intracellular reactive oxygen species, reducing mitochondrial membrane potential, and regulating the expression of apoptosis-related genes. The targeting rate of mdGC to tumor tissue is up to 20.71 ± 1.94% ID g-1 ; the tumor growth inhibition rate is as high as 80.44% without external laser sources. In general, mdGC is a potential multifunctional diagnostic and therapy integrated nanoformulation.

Up-regulation of miR-335 and miR-674-3p in the rostral ventrolateral medulla contributes to stress-induced hypertension.

The rostral ventrolateral medulla (RVLM) is known as the vasomotor center that plays a crucial role in mediating the development of stress-induced hypertension (SIH). MicroRNAs (miRNAs) are involved in many different biological processes and diseases. However, studies that evaluated the roles of miRNAs in the RVLM during SIH do not exist. Here, we performed RNA sequencing to explore the genome-wide miRNA profiles in RVLM in an SIH rat model established by administering electric foot-shocks and noises. The function of miRNAs in blood pressure regulation was determined in vivo via the intra-RVLM microinjection of the agomir or antagomir. Furthermore, the underlying mechanisms of miRNAs on SIH were investigated through in vitro and in vivo experiments, like gain-of-function. We discovered 786 miRNA transcripts among which 4 were differentially expressed. The over-expression of miR-335 and miR-674-3p in RVLM dramatically increased the heart rate (HR), arterial blood pressure (ABP), systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) levels of normotensive rats, whereas the knockdown of miR-335 and miR-674-3p in RVLM markedly reduced the HR, ABP, SBP, DBP, and MAP levels of SIH rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation revealed that miR-335 and miR-674-3p participated in regulating the development of SIH from different aspects, like apoptosis-multiple species pathway. Sphk1, whose expression was markedly decreased in SIH, was identified as a novel target of miR-335. MiR-335 over-expression substantially reduced the expression of Sphk1 and promoted neural apoptosis, and its inhibition had opposite effects. Re-introduction of Sphk1 dramatically abrogated the apoptosis induced by miR-335. This study provides the first systematic dissection of the RVLM miRNA landscape in SIH. MiR-335 and miR-674-3p act as SIH promoters, and the identified miR-335/Sphk1/apoptosis axis represents one of the possible mechanisms. These miRNAs can be exploited as potential targets for the molecular-based therapy of SIH.

Overexpression of NaV1.6 in the rostral ventrolateral medulla in rats mediates stress-induced hypertension via glutamate regulation.

BACKGROUND: The rostral ventrolateral medulla (RVLM) plays a key role in mediating the development of stress-induced hypertension (SIH). Furthermore, enhanced glutamate transport within glutamatergic neurons in the RVLM mediates pressor responses. Data from our previous studies suggest that the voltage-gated sodium channel NaV1.6 is overexpressed in neurons in the RVLM in SIH model rats and participates in the resulting elevation of blood pressure. However, previous studies have not investigated the relationship between NaV1.6 expression and glutamatergic neurons. METHODS: Here, we constructed an SIH rat model by knocking down NaV1.6 via microinjection of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA into the RVLM. Glutamate-related markers were quantified by Western blotting and immunofluorescence, and blood pressure was measured in the rats. RESULTS: Our findings showed that vesicular glutamate transporter 1 (VGluT1) protein expression in the RVLM was higher in SIH rats than in Control rats, and GAD67 protein expression in SIH rats was lower than that in Control rats. Therefore, the number of VGluT1-positive neurons increased, while the number of GAD67-labeled neurons decreased after stress. After knocking down NaV1.6 expression in the RVLM, VGluT1 expression and the number of VGluT1-positive neurons decreased relative to those in SIH rats, while GAD67 protein expression and the number of GAD67-labeled neurons increased relative to those in SIH rats. CONCLUSIONS: These results indicate that overexpression of NaV1.6 in the RVLM may mediate the transport and transformation of glutamate in neurons, and NaV1.6 may participate in SIH.

PLIN2 Mediates Neuroinflammation and Oxidative/Nitrosative Stress via Downregulating Phosphatidylethanolamine in the Rostral Ventrolateral Medulla of Stressed Hypertensive Rats.

PURPOSE: Oxidative/nitrosative stress, neuroinflammation and their intimate interactions mediate sympathetic overactivation in hypertension. An immoderate inflammatory response is characterized not only by elevated proinflammatory cytokines (PICs) but by increases in mitochondrial dysfunction, reactive oxygen species (ROS), and nitric oxide (NO). Recent data pinpoint that both the phospholipid and lipid droplets (LDs) are potent modulators of microglia physiology. METHODS: Stress rats underwent compound stressors for 15 days with PLIN2-siRNA or scrambled-siRNA (SC-siRNA) administrated into the rostral ventrolateral medulla (RVLM). Lipids were analyzed by mass spectroscopy-based quantitative lipidomics. The phenotypes and proliferation of microglia, LDs, in the RVLM of rats were detected; blood pressure (BP) and myocardial injury in rats were evaluated. The anti-oxidative/nitrosative stress effect of phosphatidylethanolamine (PE) was explored in cultured primary microglia. RESULTS: Lipidomics analysis showed that 75 individual lipids in RVLM were significantly dysregulated by stress [PE was the most one], demonstrating that lipid composition changed with stress. In vitro, prorenin stress induced the accumulation of LDs, increased PICs, which could be blocked by siRNA-PLIN2 in microglia. PLIN2 knockdown upregulated the PE synthesis in microglia. Anti-oxidative/nitrosative stress effect of PE delivery was confirmed by the decrease of ROS and decrease in 3-NT and MDA in prorenin-treated microglia. PLIN2 knockdown in the RVLM blocked the number of iNOS+ and PCNA+ microglia, decreased BP, alleviated cardiac fibrosis and hypertrophy in stressed rats. CONCLUSION: PLIN2 mediates microglial polarization/proliferation via downregulating PE in the RVLM of stressed rats. Delivery of PE is a promising strategy for combating neuroinflammation and oxidative/nitrosative stress in stress-induced hypertension.

Identification of a ß-Arrestin 2 Mutation Related to Autism by Whole-Exome Sequencing.

Autism spectrum disorder (ASD) is a complex neurological disease characterized by impaired social communication and interaction skills, rigid behavior, decreased interest, and repetitive activities. The disease has a high degree of genetic heterogeneity, and the genetic cause of ASD in many autistic individuals is currently unclear. In this study, we report a patient with ASD whose clinical features included social interaction disorder, communication disorder, and repetitive behavior. We examined the patient's genetic variation using whole-exome sequencing technology and found new de novo mutations. After analysis and evaluation, ARRB2 was identified as a candidate gene. To study the potential contribution of the ARRB2 gene to the human brain development and function, we first evaluated the expression profile of this gene in different brain regions and developmental stages. Then, we used weighted gene coexpression network analysis to analyze the associations between ARRB2 and ASD risk genes. Additionally, the spatial conformation and stability of the ARRB2 wild type and mutant proteins were examined by simulations. Then, we further established a mouse model of ASD. The results showed abnormal ARRB2 expression in the mouse ASD model. Our study showed that ARRB2 may be a risk gene for ASD, but the contribution of de novo ARRB2 mutations to ASD is unclear. This information will provide references for the etiology of ASD and aid in the mechanism-based drug development and treatment.

Deletion of Kv10.2 Causes Abnormal Dendritic Arborization and Epilepsy Susceptibility.

The abnormal function of the voltage-gated potassium channel Kv10.2 can induce epilepsy. However, the physiological function of Kv10.2 in the central nervous system remains unclear. In this study, we found that Kv10.2 knockout (KO) increased the complexity of neurons in the CA3 subarea of hippocampus. Kv10.2 KO led to enlarged somata, elongated dendritic length, and increased the number of dendritic tips in cultured rat hippocampus neurons. Kv10.2 KO also increased Synapsin I and PSD95 protein density in cultured rat hippocampal neurons. Whole cell patch-clamp recordings of brain slices in the CA3 subarea of hippocampus revealed that Kv10.2 KO increased the amplitude of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC), depolarized the resting membrane potential and increased the action potential firing, reduced the rheobase and increased the input resistance, which results in enhanced neuronal excitability. Furthermore, we made electroencephalogram (EEG) recordings of brain activity in freely moving rats before and after inducing seizures by pentylenetetrazole (PTZ) injection. Kv10.2 KO rats dramatically increased the EEG amplitude during epilepsy. Behavioral observation after seizure induction revealed that Kv10.2 KO rats demonstrated shortened onset latency, prolonged duration, and increased seizure severity when compared with wild type rats. Therefore, this study provides a new link between Kv10.2 and neuronal morphology and higher intrinsic excitability.

Transplantation of ACE2- Mesenchymal Stem Cells Improves the Outcome of Patients with COVID-19 Pneumonia.

A coronavirus (HCoV-19) has caused the novel coronavirus disease (COVID-19) outbreak in Wuhan, China. Preventing and reversing the cytokine storm may be the key to save the patients with severe COVID-19 pneumonia. Mesenchymal stem cells (MSCs) have been shown to possess a comprehensive powerful immunomodulatory function. This study aims to investigate whether MSC transplantation improves the outcome of 7 enrolled patients with COVID-19 pneumonia in Beijing YouAn Hospital, China, from Jan 23, 2020 to Feb 16, 2020. The clinical outcomes, as well as changes of inflammatory and immune function levels and adverse effects of 7 enrolled patients were assessed for 14 days after MSC injection. MSCs could cure or significantly improve the functional outcomes of seven patients without observed adverse effects. The pulmonary function and symptoms of these seven patients were significantly improved in 2 days after MSC transplantation. Among them, two common and one severe patient were recovered and discharged in 10 days after treatment. After treatment, the peripheral lymphocytes were increased, the C-reactive protein decreased, and the overactivated cytokine-secreting immune cells CXCR3+CD4+ T cells, CXCR3+CD8+ T cells, and CXCR3+ NK cells disappeared in 3-6 days. In addition, a group of CD14+CD11c+CD11bmid regulatory DC cell population dramatically increased. Meanwhile, the level of TNF-α was significantly decreased, while IL-10 increased in MSC treatment group compared to the placebo control group. Furthermore, the gene expression profile showed MSCs were ACE2- and TMPRSS2- which indicated MSCs are free from COVID-19 infection. Thus, the intravenous transplantation of MSCs was safe and effective for treatment in patients with COVID-19 pneumonia, especially for the patients in critically severe condition.

Rescuing Kv10.2 protein changes cognitive and emotional function in kainic acid-induced status epilepticus rats.

Voltage-gated potassium (Kv) channels are widely expressed in the central and peripheral nervous system and are crucial mediators of neuronal excitability. Importantly, these channels also actively participate in cellular and molecular signaling pathways that regulate the life and death processes of neurons. The current study used a kainic acid (KA)-induced temporal lobe epilepsy model to examine the role of the Kv10.2 gene in status epilepticus (SE). Lentiviral plasmids containing the coding sequence region of the KCNH5 gene (LV-KCNH5) were injected into the CA3 subarea of the right dorsal hippocampus within 24 h in post-SE rats to rescue Kv10.2 protein expression. Open-field and elevated plus maze test results indicated that anxiety-like behavior was ameliorated in the KA + LV-KCNH5 group rats compared with the SE group rats, and working memory was improved in the Y-maze test. However, the spatial reference memory of the LV-KCNH5 group rats did not improve in the Morris water maze test, and no difference was found in the light-dark transition box test. The results of this study indicate that Kv10.2 protein may play an important role in epilepsy, providing new potential therapeutic directions and drug targets for epilepsy treatment.

Increased Expression of Kv10.2 in the Hippocampus Attenuates Valproic Acid-Induced Autism-Like Behaviors in Rats.

The role of potassium channels provides suggestive evidence for the etiology of autism. The voltage-gated potassium channel Kv10.2 (KCNH5) is widely expressed in the brain. However, the inherent relationship between Kv10.2 and autism is still unclear. Herein, a rat valproic acid (VPA)-induced autism spectrum disorder model was established. The expression level of Kv10.2 was obviously decreased in the hippocampus of VPA rats. Kv10.2 was mainly localized in neurons. Subsequently, a recombinant lentivirus expressing Kv10.2 was used to upregulate the expression of Kv10.2 in the hippocampus of VPA-exposed rats. The results were promising as injection of the Kv10.2 lentivirus in the hippocampus relieved anxiety and stereotypical behavior, and improved the social and exploratory abilities of rats that were prenatally exposed to VPA. In addition, spectral analysis of electroencephalogram data revealed that animals exposed to VPA exhibited increased high-frequency activity compared with the control rats, and this activity recovered to a certain extent after upregulation of Kv10.2 expression by lentivirus injection. These results suggest that changes in Kv10.2 may play an important role in the etiology of autism, thus providing a promising direction for further research on autism.

Infiltrating T helper 17 cells in the paraventricular nucleus are pathogenic for stress-induced hypertension.

Central neuroinflammation produced by both innate and adaptive immunities plays a major role in the development of stress-induced hypertension (SIH), but successful T cell immunoregulation for SIH requires that the T cells can access brain tissues. So far, both the effects of T helper 17 (Th17) cells on SIH and the pathway for T cells entry into the brain were unknown. Here we show that the blood pressure (BP), heart rate (HR) and the norepinephrine(NE) of the SIH rats were considerably higher, the numbers of Th17 cells and IL-17 were higher, relative to control. Anti-IL-17 attenuated the elevation of BP and HR of the SIH rats when microinjected into the paraventricular nucleus (PVN).Alb-FITC, after infusion into the carotid artery, were found in the brain parenchyma of the PVN in the SIH rats. We concluded that Th17 cells infiltrated the parenchyma of the paraventricular nucleus (PVN) via a compromised blood brain barrier (BBB) in response to stress and Th17 cells and IL-17 play an important role in the pathophysiology of SIH.

Central changes in the Kv10.2 potassium channel in stress-induced hypertension rats.

Ion channels play as a pivotal role in hypertension in the processes of maintenance of vascular tone and sympathetic excitement of hypertension. The Kv10.2 channel (encoded by the Kcnh5 gene) belongs to the EAG voltage-gated superfamily. It is distributed widely in the brain, such as the hippocampus, the cortex, and the olfactory bulb. To date, the expression of Kv10.2 in central nervous system nuclei that regulates cardiovascular function and its inter-relationship with hypertension are still unclear. Here, electric foot-shock stressors with noise were used to establish the stress-induced hypertensive (SIH) rat model. The expression of Kv10.2 in the rostral ventrolateral medulla, the nucleus tractus solitarius, and the paraventricular nucleus (PVN) was examined by immunohistochemical staining and western blots. The following results were obtained: (a) the expression level of Kv10.2 was increased obviously in the paraventricular nucleus of SIH rats, whereas no significant difference was found in the rostral ventrolateral medulla and the nucleus tractus solitarius. (b) Kv10.2 was located in neurons. (c) Vesicular glutamate transporter 1 as a protein mark of glutamate neurons was increased in the paraventricular nucleus of the SIH group. (d) The expression of vesicular glutamate transporter 1 protein in neurons was significantly decreased when the Kcnh5 gene was knocked down by small interfering RNA in vitro. These findings indicate that the changes in Kv10.2 may be related to SIH, which may provide a potential avenue for further investigation of SIH.

Melatonin decreases M1 polarization via attenuating mitochondrial oxidative damage depending on UCP2 pathway in prorenin-treated microglia.

Accumulating evidence suggests that neuroinflammation and oxidative stress in cardiovascular center contribute to the pathological processes underlying hypertension. Microglia activation triggers the inflammation and oxidative stress. Melatonin is a documented potent anti-inflammatory regent and antioxidant, the underlying roles of melatonin in regulating microglia activation via mitochondria remain unclear. In present study, we investigated the protective role of melatonin in decreasing M1 phenotype switching via attenuating mitochondrial oxidative damage in dependence on uncoupling protein 2 (UCP2) pathway in microglia. Prorenin (20 nmol/L; 24 hr) was used to induce inflammation in cultured microglia. Mitochondrial morphology was detected by transmission electron microscope. The reactive oxygen species (ROS) production by using DCFH-DA fluorescence imaging and mitochondrial membrane potential (MMP, ΔΨm) was evaluated by JC-1 staining. The indicator of the redox status as the ratio of the amount of total NADP+ to total NADPH, and the expression of 6 subunits of NADPH oxidase is measured. The pro-inflammatory cytokines releasing was measured by qPCR. UCP2 and activated AMPKα (p-AMPKα) expression were examined by immunoblot. Melatonin (100 µM) markedly alleviated the M1 microglia phenotype shifting and abnormal mitochondria morphology. Melatonin attenuated prorenin-induced ΔΨm increasing and ROS overproduction. Melatonin decreased the redox ratio (NADP+/NADPH) and the p47phox and gp91phox subunits of NADPH oxidase expression in prorenin-treated microglia. These effects were reversed in the presence of UCP2 siRNA. Our results suggested that the protective effect of melatonin against prorenin-induced M1 phenotype switching via attenuating mitochondrial oxidative damage depending on UCP2 upregulation in prorenin-treated microglia.