Transplantation of astrocyte-derived mitochondria into injured astrocytes has a protective effect following stretch injury.

Traumatic brain injury (TBI) is a global public-health problem. Astrocytes, and their mitochondria, are important factors in the pathogenesis of TBI-induced secondary injury. Mitochondria extracted from healthy tissues and then transplanted have shown promise in models of a variety of diseases. However, the effect on recipient astrocytes is unclear. Here, we isolated primary astrocytes from newborn C57BL/6 mice, one portion of which was used to isolate mitochondria, and another was subjected to stretch injury (SI) followed by transplantation of the isolated mitochondria. After incubation for 12 h, cell viability, mitochondrial dysfunction, calcium overload, redox stress, inflammatory response, and apoptosis were improved. Live-cell imaging showed that the transplanted mitochondria were incorporated into injured astrocytes and fused with their mitochondrial networks, which was in accordance with the changes in the expression levels of markers of mitochondrial dynamics. The astrocytic IKK/NF-κB pathway was decelerated whereas the AMPK/PGC-1α pathway was accelerated by transplantation. Together, these results indicate that exogenous mitochondria from untreated astrocytes can be incorporated into injured astrocytes and fuse with their mitochondrial networks, improving cell viability by ameliorating mitochondrial dysfunction, redox stress, calcium overload, and inflammation.

Dynamics of N6-methyladenosine modification during Alzheimer's disease development.

N6-methyladenosine (m6A) modification is a common RNA modification in the central nervous system and has been linked to various neurological disorders, including Alzheimer's disease (AD). However, the dynamic of mRNA m6A modification and m6A enzymes during the development of AD are not well understood. Therefore, this study examined the expression profiles of m6A and its enzymes in the development of AD. The results showed that changes in the expression levels of m6A regulatory factors occur in the early stages of AD, indicating a potential role for m6A modification in the onset of the disease. Additionally, the analysis of mRNA m6A expression profiles using m6A-seq revealed significant differences in m6A modification between AD and control brains. The genes with differential methylation were found to be enriched in GO and KEGG terms related to processes such as inflammation response, immune system processes. And the differently expressed genes (DEGs) are negatively lryassociated with genes involved in microglia hemostasis, but positively associated with genes related to "disease-associated microglia" (DAM) associated genes. These findings suggest that dysregulation of mRNA m6A modification may contribute to the development of AD by affecting the function and gene expression of microglia.

Hemorrhagic stroke-induced subtype of inflammatory reactive astrocytes disrupts blood-brain barrier.

Astrocytes undergo disease-specific transcriptomic changes upon brain injury. However, phenotypic changes of astrocytes and their functions remain unclear after hemorrhagic stroke. Here we reported hemorrhagic stroke induced a group of inflammatory reactive astrocytes with high expression of Gfap and Vimentin, as well as inflammation-related genes lipocalin-2 (Lcn2), Complement component 3 (C3), and Serpina3n. In addition, we demonstrated that depletion of microglia but not macrophages inhibited the expression of inflammation-related genes in inflammatory reactive astrocytes. RNA sequencing showed that blood-brain barrier (BBB) disruption-related gene matrix metalloproteinase-3 (MMP3) was highly upregulated in inflammatory reactive astrocytes. Pharmacological inhibition of MMP3 in astrocytes or specific deletion of astrocytic MMP3 reduced BBB disruption and improved neurological outcomes of hemorrhagic stroke mice. Our study demonstrated that hemorrhagic stroke induced a group of inflammatory reactive astrocytes that were actively involved in disrupting BBB through MMP3, highlighting a specific group of inflammatory reactive astrocytes as a critical driver for BBB disruption in neurological diseases.

Endorepellin downregulation promotes angiogenesis after experimental traumatic brain injury.

Endorepellin plays a key role in the regulation of angiogenesis, but its effects on angiogenesis after traumatic brain injury are unclear. This study explored the effects of endorepellin on angiogenesis and neurobehavioral outcomes after traumatic brain injury in mice. Mice were randomly divided into four groups: sham, controlled cortical impact only, adeno-associated virus (AAV)-green fluorescent protein, and AAV-shEndorepellin-green fluorescent protein groups. In the controlled cortical impact model, the transduction of AAV-shEndorepellin-green fluorescent protein downregulated endorepellin while increasing the number of CD31+/Ki-67+ proliferating endothelial cells and the functional microvessel density in mouse brain. These changes resulted in improved neurological function compared with controlled cortical impact mice. Western blotting revealed increased expression of vascular endothelial growth factor and angiopoietin-1 in mice treated with AAV-shEndorepellin-green fluorescent protein. Synchrotron radiation angiography showed that endorepellin downregulation promoted angiogenesis and increased cortical neovascularization, which may further improve neurobehavioral outcomes. Furthermore, an in vitro study showed that downregulation of endorepellin increased tube formation by human umbilical vein endothelial cells compared with a control. Mechanistic analysis found that endorepellin downregulation may mediate angiogenesis by activating vascular endothelial growth factor- and angiopoietin-1-related signaling pathways.

Transcriptional and epigenetic decoding of the microglial aging process.

As important immune cells, microglia undergo a series of alterations during aging that increase the susceptibility to brain dysfunctions. However, the longitudinal characteristics of microglia remain poorly understood. In this study, we mapped the transcriptional and epigenetic profiles of microglia from 3- to 24-month-old mice. We first discovered unexpected sex differences and identified age-dependent microglia (ADEM) genes during the aging process. We then compared the features of aging and reactivity in female microglia at single-cell resolution and epigenetic level. To dissect functions of aged microglia excluding the influence from other aged brain cells, we established an accelerated microglial turnover model without directly affecting other brain cells. By this model, we achieved aged-like microglia in non-aged brains and confirmed that aged-like microglia per se contribute to cognitive decline. Collectively, our work provides a comprehensive resource for decoding the aging process of microglia, shedding light on how microglia maintain brain functions.

The role and potential therapeutic targets of astrocytes in central nervous system demyelinating diseases.

Astrocytes play vital roles in the central nervous system, contributing significantly to both its normal functioning and pathological conditions. While their involvement in various diseases is increasingly recognized, their exact role in demyelinating lesions remains uncertain. Astrocytes have the potential to influence demyelination positively or negatively. They can produce and release inflammatory molecules that modulate the activation and movement of other immune cells. Moreover, they can aid in the clearance of myelin debris through phagocytosis and facilitate the recruitment and differentiation of oligodendrocyte precursor cells, thereby promoting axonal remyelination. However, excessive or prolonged astrocyte phagocytosis can exacerbate demyelination and lead to neurological impairments. This review provides an overview of the involvement of astrocytes in various demyelinating diseases, emphasizing the underlying mechanisms that contribute to demyelination. Additionally, we discuss the interactions between oligodendrocytes, oligodendrocyte precursor cells and astrocytes as therapeutic options to support myelin regeneration. Furthermore, we explore the role of astrocytes in repairing synaptic dysfunction, which is also a crucial pathological process in these disorders.

Optogenetic Activation of Astrocytes Reduces Blood-Brain Barrier Disruption via IL-10 In Stroke.

Optogenetics has been used to regulate astrocyte activity and modulate neuronal function after brain injury. Activated astrocytes regulate blood-brain barrier functions and are thereby involved in brain repair. However, the effect and molecular mechanism of optogenetic-activated astrocytes on the change in barrier function in ischemic stroke remain obscure. In this study, adult male GFAP-ChR2-EYFP transgenic Sprague-Dawley rats were stimulated by optogenetics at 24, 36, 48, and 60 h after photothrombotic stroke to activate ipsilateral cortical astrocytes. The effects of activated astrocytes on barrier integrity and the underlying mechanisms were explored using immunostaining, western blotting, RT-qPCR, and shRNA interference. Neurobehavioral tests were performed to evaluate therapeutic efficacy. The results demonstrated that IgG leakage, gap formation of tight junction proteins, and matrix metallopeptidase 2 expression were reduced after optogenetic activation of astrocytes (p<0.05). Moreover, photo-stimulation of astrocytes protected neurons against apoptosis and improved neurobehavioral outcomes in stroke rats compared to controls (p<0.05). Notably, interleukin-10 expression in optogenetic-activated astrocytes significantly increased after ischemic stroke in rats. Inhibition of interleukin-10 in astrocytes compromised the protective effects of optogenetic-activated astrocytes (p<0.05). We found for the first time that interleukin-10 derived from optogenetic-activated astrocytes protected blood-brain barrier integrity by decreasing the activity of matrix metallopeptidase 2 and attenuated neuronal apoptosis, which provided a novel therapeutic approach and target in the acute stage of ischemic stroke.

Roles of Ependymal Cells in the Physiology and Pathology of the Central Nervous System.

Ependymal cells are indispensable components of the central nervous system (CNS). They originate from neuroepithelial cells of the neural plate and show heterogeneity, with at least three types that are localized in different locations of the CNS. As glial cells in the CNS, accumulating evidence demonstrates that ependymal cells play key roles in mammalian CNS development and normal physiological processes by controlling the production and flow of cerebrospinal fluid (CSF), brain metabolism, and waste clearance. Ependymal cells have been attached to great importance by neuroscientists because of their potential to participate in CNS disease progression. Recent studies have demonstrated that ependymal cells participate in the development and progression of various neurological diseases, such as spinal cord injury and hydrocephalus, raising the possibility that they may serve as a potential therapeutic target for the disease. This review focuses on the function of ependymal cells in the developmental CNS as well as in the CNS after injury and discusses the underlying mechanisms of controlling the functions of ependymal cells.

Classification, risk factors, and outcomes of patients with progressive hemorrhagic injury after traumatic brain injury.

BACKGROUND: According to the pathoanatomic classification system, progressive hemorrhagic injury (PHI) can be categorized into progressive intraparenchymal contusion or hematoma (pIPCH), epidural hematoma (pEDH), subdural hematoma (pSDH), and traumatic subarachnoid hemorrhage (ptSAH). The clinical features of each type differ greatly. The objective of this study was to determine the predictors, clinical management, and outcomes of PHI according to this classification. METHODS: Multivariate logistic regression analysis was used to identify independent risk factors for PHI and each subgroup. Patients with IPCH or EDH were selected for subgroup propensity score matching (PSM) to exclude confounding factors before evaluating the association of hematoma progression with the outcomes by classification. RESULTS: In the present cohort of 419 patients, 123 (29.4%) demonstrated PHI by serial CT scan. Of them, progressive ICPH (58.5%) was the most common type, followed by pEDH (28.5%), pSDH (9.8%), and ptSAH (3.2%). Old age (≥ 60 years), lower motor Glasgow Coma Scale score, larger primary lesion volume, and higher level of D-dimer were independent risk factors related to PHI. These factors were also independent predictors for pIPCH, but not for pEDH. The time to first CT scan and presence of skull linear fracture were robust risk factors for pEDH. After PSM, the 6-month mortality and unfavorable survival rates were significantly higher in the pIPCH group than the non-pIPCH group (24.2% vs. 1.8% and 12.1% vs. 7.3%, respectively, p < 0.001), but not significantly different between the pEDH group and the non-pEDH group. CONCLUSIONS: Understanding the specific patterns of PHI according to its classification can help early recognition and suggest targeted prevention or treatment strategies to improve patients' neurological outcomes.

PIEZO1-Related Physiological and Pathological Processes in CNS: Focus on the Gliomas.

PIEZO1 is ubiquitously expressed in cells in different kinds of tissues throughout the body, which can sense physical or mechanical stimuli and translate them into intracellular electrochemical signals to regulate organism functions. In particular, PIEZO1 appears in complex interactive regulatory networks as a central node, governing normal and pathological functions in the body. However, the effect and mechanism of the activation or expression of PIEZO1 in diseases of the central nervous system (CNS) remain unclear. On one hand, in CNS diseases, pathophysiological processes in neurons and glial are often accompanied by variations in the mechanical properties of the cellular and extracellular matrix stiffness. The expression of PIEZO1 can therefore be upregulated, in responding to mechanical stimulation, to drive the biological process in cells, which in turns indirectly affects the cellular microenvironment, resulting in alterations of the cellular status. On the other hand, it may have contradictory effects with the change of active patterns and/or subcellular location. This review highlights the biological processes involved with PIEZO1 in CNS cells, with special emphasis on its multiple roles in glioma-associated phenotypes. In conclusion, PIEZO1 can be used as an indicator to assess the malignancy and prognosis of patients with gliomas, as well as a therapeutic target for clinical application following fully exploring the potential mechanism of PIEZO1 in CNS diseases.

The roles of microglia and astrocytes in myelin phagocytosis in the central nervous system.

Myelination is an important process in the central nervous system (CNS). Oligodendrocytes (OLs) extend multiple layers to densely sheath on axons, composing the myelin to achieve efficient electrical signal conduction. The myelination during developmental stage maintains a balanced state. However, numerous CNS diseases including neurodegenerative and cerebrovascular diseases cause demyelination and disrupt the homeostasis, resulting in inflammation and white matter deficits. Effective clearance of myelin debris is needed in the region of demyelination, which is a key step for remyelination and tissue regeneration. Microglia and astrocytes are the major resident phagocytic cells in the brain, which may play different or collaborative roles in myelination. Microglia and astrocytes participate in developmental myelination through engulfing excessive unneeded myelin. They are also involved in the clearance of degenerated myelin debris for accelerating remyelination, or engulfing healthy myelin sheath for inhibiting remyelination. This review focuses on the roles of microglia and astrocytes in phagocytosing myelin in the developmental brain and diseased brain. In addition, the interaction between microglia and astrocytes to mediate myelin engulfment is also summarized.

Progranulin released from microglial lysosomes reduces neuronal ferroptosis after cerebral ischemia in mice.

The cellular redox state is essential for inhibiting ferroptosis. Progranulin (PGRN) plays an important role in maintaining the cellular redox state after ischemic brain injury. However, the effect of PGRN on ferroptosis and its underlying mechanism after cerebral ischemia remains unclear. This study assesses whether PGRN affects ferroptosis and explores its mechanism of action on ferroptosis after cerebral ischemia. We found endogenous PGRN expression in microglia increased on day 3 after ischemia. In addition, PGRN agonists chloroquine and trehalose upregulated PGRN expression, reduced brain infarct volume, and improved neurobehavioral outcomes after cerebral ischemia compared to controls (p < 0.05). Moreover, PGRN upregulation attenuated ferroptosis by decreasing malondialdehyde and increasing Gpx4, Nrf2, and Slc7a11 expression and glutathione content (p < 0.05). Furthermore, chloroquine induced microglial lysosome PGRN release, which was associated with increased neuron survival. Our results indicate that PGRN derived from microglial lysosomes effectively inhibits ferroptosis during ischemic brain injury, identifying it as a promising target for ischemic stroke therapy.

Inhibiting phosphatase and actin regulator 1 expression is neuroprotective in the context of traumatic brain injury.

Studies have found that the phosphatase actin regulatory factor 1 expression can be related to stroke, but it remains unclear whether changes in phosphatase actin regulatory factor 1 expression also play a role in traumatic brain injury. In this study we found that, in a mouse model of traumatic brain injury induced by controlled cortical impact, phosphatase actin regulatory factor 1 expression is increased in endothelial cells, neurons, astrocytes, and microglia. When we overexpressed phosphatase actin regulatory factor 1 by injection an adeno-associated virus vector into the contused area in the traumatic brain injury mice, the water content of the brain tissue increased. However, when phosphatase actin regulatory factor 1 was knocked down, the water content decreased. We also found that inhibiting phosphatase actin regulatory factor 1 expression regulated the nuclear factor kappa B signaling pathway, decreased blood-brain barrier permeability, reduced aquaporin 4 and intercellular adhesion molecule 1 expression, inhibited neuroinflammation, and neuronal apoptosis, thereby improving neurological function. The findings from this study indicate that phosphatase actin regulatory factor 1 may be a potential therapeutic target for traumatic brain injury.

Knockout of Sirt2 alleviates traumatic brain injury in mice.

Sirtuin 2 (SIRT2) inhibition or Sirt2 knockout in animal models protects against the development of neurodegenerative diseases and cerebral ischemia. However, the role of SIRT2 in traumatic brain injury (TBI) remains unclear. In this study, we found that knockout of Sirt2 in a mouse model of TBI reduced brain edema, attenuated disruption of the blood-brain barrier, decreased expression of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, reduced the activity of the effector caspase-1, reduced neuroinflammation and neuronal pyroptosis, and improved neurological function. Knockout of Sirt2 in a mechanical stretch injury cell model in vitro also decreased expression of the NLRP3 inflammasome and pyroptosis. Our findings suggest that knockout of Sirt2 is neuroprotective against TBI; therefore, Sirt2 could be a novel target for TBI treatment.

Dl-3-n-Butylphthalide Reduced Neuroinflammation by Inhibiting Inflammasome in Microglia in Mice after Middle Cerebral Artery Occlusion.

The inflammatory response is one of the key events in cerebral ischemia, causing secondary brain injury and neuronal death. Studies have shown that the NLRP3 inflammasome is a key factor in initiating the inflammatory response and that Dl-3-n-butylphthalide (NBP) can attenuate the inflammatory response and improve neuronal repair during ischemic stroke. However, whether NBP attenuates the inflammatory response via inhibition of NLRP3 remains unclear. A 90 min middle cerebral artery occlusion was induced in 62 2-month-old adult male ICR mice, and NBP was administered by gavage zero, one, or two days after ischemia. Brain infarct volume, neurological deficits, NLRP3, microglia, and neuronal death were examined in sacrificed mice to explore the correction between NBP effects and NLRP3 expression. NBP significantly reduced infarct volume and attenuated neurological deficits after ischemic stroke compared to controls (p < 0.05). Moreover, it inhibited ASC+ microglia activation and NLRP3 and CASP1 expression in ischemic mice. In addition, neuronal apoptosis was reduced in NBP-treated microglia cultures (p < 0.05). Our results indicate that NBP attenuates the inflammatory response in ischemic mouse brains, suggesting that NBP protects against microglia activation via the NLRP3 inflammasome.

ACT001 attenuates microglia-mediated neuroinflammation after traumatic brain injury via inhibiting AKT/NFκB/NLRP3 pathway.

BACKGROUND: Microglia-mediated neuroinflammatory response following traumatic brain injury (TBI) is considered as a vital secondary injury factor, which drives trauma-induced neurodegeneration and is lack of efficient treatment. ACT001, a sesquiterpene lactone derivative, is reportedly involved in alleviation of inflammatory response. However, little is known regarding its function in regulating innate immune response of central nervous system (CNS) after TBI. This study aimed to investigate the role and underlying mechanism of ACT001 in TBI. METHODS: Controlled cortical impact (CCI) models were used to establish model of TBI. Cresyl violet staining, evans blue extravasation, neurobehavioral function assessments, immunofluorescence and transmission electron microscopy were used to evaluate therapeutic effects of ACT001 in vivo. Microglial depletion was induced by administering mice with colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Cell-cell interaction models were established as co-culture system to simulate TBI conditions in vitro. Cytotoxic effect of ACT001 on cell viability was assessed by cell counting kit-8 and activation of microglia cells were induced by Lipopolysaccharides (LPS). Pro-inflammatory cytokines expression was determined by Real-time PCR and nitric oxide production. Apoptotic cells were detected by TUNEL and flow cytometry assays. Tube formation was performed to evaluate cellular angiogenic ability. ELISA and western blot experiments were used to determine proteins expression. Pull-down assay was used to analyze proteins that bound ACT001. RESULTS: ACT001 relieved the extent of blood-brain barrier integrity damage and alleviated motor function deficits after TBI via reducing trauma-induced activation of microglia cells. Delayed depletion of microglia with PLX5622 hindered therapeutic effect of ACT001. Furthermore, ACT001 alleviated LPS-induced activation in mouse and rat primary microglia cells. Besides, ACT001 was effective in suppressing LPS-induced pro-inflammatory cytokines production in BV2 cells, resulting in reduction of neuronal apoptosis in HT22 cells and improvement of tube formation in bEnd.3 cells. Mechanism by which ACT001 functioned was related to AKT/NFκB/NLRP3 pathway. ACT001 restrained NFκB nuclear translocation in microglia cells through inhibiting AKT phosphorylation, resulting in decrease of NLRP3 inflammasome activation, and finally down-regulated microglial neuroinflammatory response. CONCLUSIONS: Our study indicated that ACT001 played critical role in microglia-mediated neuroinflammatory response and might be a novel potential chemotherapeutic drug for TBI. Video Abstract.

Urolithin A alleviates blood-brain barrier disruption and attenuates neuronal apoptosis following traumatic brain injury in mice.

Urolithin A (UA) is a natural metabolite produced from polyphenolics in foods such as pomegranates, berries, and nuts. UA is neuroprotective against Parkinson's disease, Alzheimer's disease, and cerebral hemorrhage. However, its effect against traumatic brain injury remains unknown. In this study, we established adult C57BL/6J mouse models of traumatic brain injury by controlled cortical impact and then intraperitoneally administered UA. We found that UA greatly reduced brain edema; increased the expression of tight junction proteins in injured cortex; increased the immunopositivity of two neuronal autophagy markers, microtubule-associated protein 1A/B light chain 3A/B (LC3) and p62; downregulated protein kinase B (Akt) and mammalian target of rapamycin (mTOR), two regulators of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway; decreased the phosphorylation levels of inhibitor of NFκB (IκB) kinase alpha (IKKα) and nuclear factor kappa B (NFκB), two regulators of the neuroinflammation-related Akt/IKK/NFκB signaling pathway; reduced blood-brain barrier permeability and neuronal apoptosis in injured cortex; and improved mouse neurological function. These findings suggest that UA may be a candidate drug for the treatment of traumatic brain injury, and its neuroprotective effects may be mediated by inhibition of the PI3K/Akt/mTOR and Akt/IKK/NFκB signaling pathways, thus reducing neuroinflammation and enhancing autophagy.

Extracellular vesicles from adipose-derived stem cells promote microglia M2 polarization and neurological recovery in a mouse model of transient middle cerebral artery occlusion.

BACKGROUND: Adipose-derived stem cells (ADSCs) and their extracellular vesicles (EVs) have therapeutic potential in ischemic brain injury, but the underlying mechanism is poorly understood. The current study aimed to explore the contribution of miRNAs in ADSC-EVs to the treatment of cerebral ischemia. METHODS: After the intravenous injection of ADSC-EVs, therapeutic efficacy was evaluated by neurobehavioral tests and brain atrophy volume. The polarization of microglia was assessed by immunostaining and qPCR. We further performed miRNA sequencing of ADSC-EVs and analyzed the relationship between the upregulated miRNAs in ADSC-EVs and microglial polarization-related proteins using Ingenuity Pathway Analysis (IPA). RESULTS: The results showed that ADSC-EVs reduced brain atrophy volume, improved neuromotor and cognitive functions after mouse ischemic stroke. The loss of oligodendrocytes was attenuated after ADSC-EVs injection. The number of blood vessels, as well as newly proliferated endothelial cells in the peri-ischemia area were higher in the ADSC-EVs treated group than that in the PBS group. In addition, ADSC-EVs regulated the polarization of microglia, resulting in increased repair-promoting M2 phenotype and decreased pro-inflammatory M1 phenotype. Finally, STAT1 and PTEN were highlighted as two downstream targets of up-regulated miRNAs in ADSC-EVs among 85 microglia/macrophage polarization related proteins by IPA. The inhibition of STAT1 and PTEN by ADSC-EVs were confirmed in cultured microglia. CONCLUSIONS: In summary, ADSC-EVs reduced ischemic brain injury, which was associated with the regulation of microglial polarization. miRNAs in ADSC-EVs partly contributed to their function in regulating microglial polarization by targeting PTEN and STAT1.