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Introduction: Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat in the Huntingtin gene (HTT). Immune activation is abundant in the striatum of HD patients. Detection of active microglia at presymptomatic stages suggests that microgliosis is a key early driver of neuronal dysfunction and degeneration. Recent studies showed that deletion of Tyrobp, a microglial protein, ameliorates neuronal dysfunction in Alzheimer's disease amyloidopathy and tauopathy mouse models while decreasing components of the complement subnetwork., Objective: While TYROBP/DAP12-mediated microglial activation is detrimental for some diseases such as peripheral nerve injury, it is beneficial for other diseases. We sought to determine whether the TYROBP network is implicated in HD and whether Tyrobp deletion impacts HD striatal function and transcriptomics., Methods: To test the hypothesis that Tyrobp deficiency would be beneficial in an HD model, we placed the Q175 HD mouse model on a Tyrobp-null background. We characterized these mice with a combination of behavioral testing, immunohistochemistry, transcriptomic and proteomic profiling. Further, we evaluated the gene signature in isolated Q175 striatal microglia, with and without Tyrobp., Results: Comprehensive analysis of publicly available human HD transcriptomic data revealed that the TYROBP network is overactivated in the HD putamen. The Q175 mice showed morphologic microglial activation, reduced levels of post-synaptic density-95 protein and motor deficits at 6 and 9 months of age, all of which were ameliorated on the Tyrobp-null background. Gene expression analysis revealed that lack of Tyrobp in the Q175 model does not prevent the decrease in the expression of striatal neuronal genes but reduces pro-inflammatory pathways that are specifically active in HD human brain, including genes identified as detrimental in neurodegenerative diseases, e.g. C1q and members of the Ccr5 signaling pathway. Integration of transcriptomic and proteomic data revealed that astrogliosis and complement system pathway were reduced after Tyrobp deletion, which was further validated by immunofluorescence analysis., Conclusions: Our data provide molecular and functional support demonstrating that Tyrobp deletion prevents many of the abnormalities in the HD Q175 mouse model, suggesting that the Tyrobp pathway is a potential therapeutic candidate for Huntington's disease., (© 2024. The Author(s).)

Huntington's disease (HD) is caused by an aberrant expansion of CAG repeats in the HTT gene that mainly affects basal ganglia. Although striatal dysfunction has been widely studied in HD mouse models, other brain areas can also be relevant to the pathology. In this sense, we have special interest on the retina as this is the most exposed part of the central nervous system that enable health monitoring of patients using noninvasive techniques. To establish the retina as an appropriate tissue for HD studies, we need to correlate the retinal alterations with those in the inner brain, i.e., striatum. We confirmed the malfunction of the transgenic R6/1 retinas, which underwent a rearrangement of their transcriptome as extensive as in the striatum. Although tissue-enriched genes were downregulated in both areas, a neuroinflammation signature was only clearly induced in the R6/1 retina in which the observed glial activation was reminiscent of the situation in HD patient's brains. The retinal neuroinflammation was confirmed in the slow progressive knock-in zQ175 strain. Overall, these results demonstrated the suitability of the mouse retina as a research model for HD and its associated glial activation., (© 2024. The Author(s).)

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen-induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Rax flox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokinesignaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetic retinopathy (DR), a common complication of diabetes, involves excessive proliferation and inflammation of Muller cells and ultimately leads to vision loss and blindness. SRY-box transcription factor 9 (SOX9) has been reported to be highly expressed in Müller cells in light-induced retinal damage rats, but the functional role of SOX9 in DR remains unclear. To explore this issue, the DR rat model was successfully constructed via injection with streptozotocin (65 mg/kg) and the retinal thicknesses and blood glucose levels were evaluated. Müller cells were treated with 25 mmol/l glucose to create a cell model in vitro. The results indicated that SOX9 expression was significantly increased in DR rat retinas and in Müller cells stimulated with a high glucose (HG) concentration. HG treatment promoted the proliferation and migration capabilities of Müller cells, whereas SOX9 knockdown reversed those behaviors. Moreover, SOX9 knockdown provided protection against an HG-induced inflammatory response, as evidenced by reduced tumor necrosis factor-α, IL-1β, and IL-6 levels in serum and decreased NLRP3 inflammasome activation. Notably, SOX9 acted as a transcription factor that positively regulated thioredoxin-interacting protein (TXNIP), a positive regulator of Müller cells gliosis under HG conditions. A dual-luciferase assay demonstrated that SOX9 could enhance TXNIP expression at the transcriptional level through binding to the promoter of TXNIP. Moreover, TXNIP overexpression restored the effects caused by SOX9 silencing. In conclusion, these findings demonstrate that SOX9 may accelerate the progression of DR by promoting glial cell proliferation, metastasis, and inflammation, which involves the transcriptional regulation of TXNIP, providing new theoretical fundamentals for DR therapy.

Objective: Recent evidence supports a link between increased TDP-43 burden and the presence of an APOE4 gene allele in Alzheimer's disease (AD); however, it is difficult to conclude the direct effect of APOE on TDP-43 pathology due to the presence of mixed AD pathologies. The goal of this study is to address how APOE isoforms impact TDP-43 pathology and related neurodegeneration in the absence of typical AD pathologies., Methods: We overexpressed human TDP-43 via viral transduction in humanized APOE2, APOE3, APOE4 mice, and murine Apoe-knockout (Apoe-KO) mice. Behavior tests were performed across ages. Animals were harvested at 11 months of age and TDP-43 overexpression-related neurodegeneration and gliosis were assessed. To further address the human relevance, we analyzed the association of APOE with TDP-43 pathology in 160 postmortem brains from autopsy-confirmed amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with motor neuron disease (FTLD-MND) in the Mayo Clinic Brain Bank., Results: We found that TDP-43 overexpression induced motor function deficits, neuronal loss, and gliosis in the motor cortex, especially in APOE2 mice, with much milder or absent effects in APOE3, APOE4, or Apoe-KO mice. In the motor cortex of the ALS and FTLD-MND postmortem human brains, we found that the APOE2 allele was associated with more severe TDP-43-positive dystrophic neurites., Interpretation: Our data suggest a genotype-specific effect of APOE on TDP-43 proteinopathy and neurodegeneration in the absence of AD pathology, with the strongest association seen with APOE2. ANN NEUROL 2023;93:830-843., (© 2022 American Neurological Association.)

Apolipoprotein E4 (APOE4) is the strongest known genetic risk factor for late-onset Alzheimer's disease (AD). Conditions of stress or injury induce APOE expression within neurons, but the role of neuronal APOE4 in AD pathogenesis is still unclear. Here we report the characterization of neuronal APOE4 effects on AD-related pathologies in an APOE4-expressing tauopathy mouse model. The selective genetic removal of APOE4 from neurons led to a significant reduction in tau pathology, gliosis, neurodegeneration, neuronal hyperexcitability and myelin deficits. Single-nucleus RNA-sequencing revealed that the removal of neuronal APOE4 greatly diminished neurodegenerative disease-associated subpopulations of neurons, oligodendrocytes, astrocytes and microglia whose accumulation correlated to the severity of tau pathology, neurodegeneration and myelin deficits. Thus, neuronal APOE4 plays a central role in promoting the development of major AD pathologies and its removal can mitigate the progressive cellular and tissue alterations occurring in this model of APOE4-driven tauopathy., (© 2023. The Author(s).)

Traumatic brain injury (TBI) survivors suffer from long-term disability and neuropsychiatric sequelae due to irreparable brain tissue destruction. However, there are still few efficient therapies to promote neurorestoration in damaged brain tissue. This study aimed to investigate whether the pro-oncogenic gene ski can promote neurorestoration after TBI. We established a ski-overexpressing experimental TBI mouse model using adenovirus-mediated overexpression through immediate injection after injury. Hematoxylin-eosin staining, MRI-based 3D lesion volume reconstruction, neurobehavioral tests, and analyses of neuronal regeneration and astrogliosis were used to assess neurorestorative efficiency. The effects of ski overexpression on the proliferation of cultured immature neurons and astrocytes were evaluated using imaging flow cytometry. The Ski protein level increased in the perilesional region at 3 days post injury. ski overexpression further elevated Ski protein levels up to 14 days post injury. Lesion volume was attenuated by approximately 36-55% after ski overexpression, with better neurobehavioral recovery, more newborn immature and mature neurons, and less astrogliosis in the perilesional region. Imaging flow cytometry results showed that ski overexpression elevated the proliferation rate of immature neurons and reduced the proliferation rate of astrocytes. These results show that ski can be considered a novel neurorestoration-related gene that effectively promotes neurorestoration, facilitates neuronal regeneration, and reduces astrogliosis after TBI., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Fatal familial insomnia (FFI) is a rare neurodegenerative disease caused by a dominantly inherited single amino acid substitution (D178N) within the prion protein (PrP). No in vitro human brain tissue model for this disease has previously been available. Consequently, how this mutation exerts its damaging effect on brain cells is still unknown. Using CRISPR-Cas9 engineered induced pluripotent stem cells, we made D178N cerebral organoids and compared these with isotype control organoids. We found that, in the absence of other hallmarks of FFI, the D178N organoids exhibited astrogliosis with cellular oxidative stress. Abnormal post-translational processing of PrP was evident but no tissue deposition or propagation of mis-folded PrP isoforms were observed. Neuronal electrophysiological function was compromised and levels of neurotransmitters, particularly acetylcholine and GABA, altered. Underlying these dysfunctions were changes in cellular energy homeostasis, with substantially increased glycolytic and Krebs cycle intermediates, and greater mitochondrial activity. This increased energy demand in D178N organoids was associated with increased mitophagy and depletion of lipid droplets, in turn resulting in shifts of cellular lipid composition. Using a double mutation (178NN) we could confirm that most changes were caused by the presence of the mutation rather than interaction with PrP molecules lacking the mutation. Our data strongly suggests that shifting biosynthetic intermediates and oxidative stress, caused by an imbalance of energy supply and demand, results in astrogliosis with compromised neuronal activity in FFI organoids. They further support that many of the disease associated changes are due to a corruption of PrP function and do not require propagation of PrP mis-folding., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Cellular responses in glia play a key role in regulating brain remodeling post-stroke. However, excessive glial reactivity impedes post-ischemic neuroplasticity and hampers neurological recovery. While damage-associated molecular patterns and activated microglia were shown to induce astrogliosis, the molecules that restrain astrogliosis are largely unknown. We explored the role of tenascin-C (TnC), an extracellular matrix component involved in wound healing and remodeling of injured tissues, in mice exposed to ischemic stroke induced by transient intraluminal middle cerebral artery occlusion. In the healthy adult brain, TnC expression is restricted to neurogenic stem cell niches. We previously reported that TnC is upregulated in ischemic brain lesions. We herein show that the de novo expression of TnC post-stroke is closely associated with reactive astrocytes, and that astrocyte reactivity at 14 days post-ischemia is increased in TnC-deficient mice (TnC -/- ). By analyzing the three-dimensional morphology of astrocytes in previously ischemic brain tissue, we revealed that TnC -/- reduces astrocytic territorial volume, branching point number, and branch length, which are presumably hallmarks of the homeostatic regulatory astrocyte state, in the post-acute stroke phase after 42 days. Interestingly, TnC -/- moderately increased aggrecan, a neuroplasticity-inhibiting proteoglycan, in the ischemic brain tissue at 42 days post-ischemia. In vitro in astrocyte-microglia cocultures, we showed that TnC -/- reduces the microglial migration speed on astrocytes and elevates intercellular adhesion molecule 1 (ICAM1) expression. Post-stroke, TnC -/- did not alter the ischemic lesion size or neurological recovery, however microglia-associated ICAM1 was upregulated in TnC -/- mice during the first week post stroke. Our data suggest that TnC plays a central role in restraining post-ischemic astrogliosis and regulating astrocyte-microglial interactions., Competing Interests: Declaration of Competing Interests The authors have no competing interests related to this work, (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Regulatory mechanisms associated with repeat-rich sequences and chromosomal conformations in mature neurons remain unexplored. Here, we map cell-type specific chromatin domain organization in adult mouse cerebral cortex and report strong enrichment of Endogenous Retrovirus 2 (ERV2) repeat sequences in the neuron-specific heterochromatic B 2 NeuN+ megabase-scaling subcompartment. Single molecule long-read sequencing and comparative Hi-C chromosomal contact mapping in wild-derived SPRET/EiJ (Mus spretus) and laboratory inbred C57BL/6J (Mus musculus) reveal neuronal reconfigurations tracking recent ERV2 expansions in the murine germline, with significantly higher B 2 NeuN+ contact frequencies at sites with ongoing insertions in Mus musculus. Neuronal ablation of the retrotransposon silencer Kmt1e/Setdb1 triggers B 2 NeuN+ disintegration and rewiring with open chromatin domains enriched for cellular stress response genes, along with severe neuroinflammation and proviral assembly with infiltration of dendrites . We conclude that neuronal megabase-scale chromosomal architectures include an evolutionarily adaptive heterochromatic organization which, upon perturbation, results in transcriptional dysregulation and unleashes ERV2 proviruses with strong neuronal tropism., (© 2021. The Author(s).)

High-fat diet (HFD) has been associated with neuroinflammation and apoptosis in distinct brain regions. To explore the effect of short-term (7, 14 and 21 days) high-fat overfeeding on apoptosis, inflammatory signaling proteins, APP changes and glial cell activities in cerebral cortex and cerebellum. Mice were fed with HFD for different lengths (up to 21 days) and after each time body weights of mice was tested, then the apoptotic proteins, IL-1β, APP, BACE1and MAPKs, Akt and NF-κB signaling activity were evaluated by western blots. Results demonstrate that short period of high-fat overnutrition significantly promotes apoptosis, APP expression at day 21 of cerebral cortex and at day 7 of cerebellum compared to chow diet. In addition, increased GFAP + astrocytes, Iba-1 + microglia and IL-1β 30 were observed in cerebral cortex after 21 days HFD, but no changes for 7 days overfeeding of cerebellum. Serendipitously, ERK1/2 pathway was activated both in cerebral cortex and cerebellum for different time course of HFD. Furthermore, increased phospho-p38 MAPK level was observed in cerebellum only. In consistent with in vivo results, SH-SY5Y cells treatment with cholesterol (50 μM, 100 μM) for 48 h culture in vitro demonstrated that pro-apoptotic proteins were enhanced as well. In brief, short-term HFD consumption increases sensitivity to apoptosis, APP and IL-1β production as well as gliosis in cerebral cortex and cerebellum, which may be related to enhancement of ERK1/2 and p38 MAPK activation.

In Parkinson's disease (PD), the second most common neurodegenerative disorder, non-motor symptoms often precede the development of debilitating motor symptoms and present a severe impact on the quality of life. Lewy bodies containing misfolded α-synuclein progressively develop in neurons throughout the peripheral and central nervous system, which may be correlated with the early development of non-motor symptoms. Among those, increased fear and anxiety is frequent in PD and thought to result from pathology outside the dopaminergic system, which has been the focus of symptomatic treatment to alleviate motor symptoms. Alpha-synuclein accumulation has been reported in the amygdala of PD patients, a brain region critically involved in fear and anxiety. Here we asked whether α-synuclein overexpression alone is sufficient to induce an enhanced fear phenotype in vivo and which pathological mechanisms are involved. Transgenic mice expressing human wild-type α-synuclein (Thy1-aSyn), a well-established model of PD, were subjected to fear conditioning followed by extinction and then tested for extinction memory retention followed by histopathological analysis. Thy1-aSyn mice showed enhanced tone fear across acquisition and extinction compared to wild-type littermates, as well as a trend to less retention of fear extinction. Immunohistochemical analysis of the basolateral nucleus of the amygdala, a nucleus critically involved in tone fear learning, revealed extensive α-synuclein pathology, with accumulation, phosphorylation, and aggregation of α-synuclein in transgenic mice. This pathology was accompanied by microgliosis and parvalbumin neuron loss in this nucleus, which could explain the enhanced fear phenotype. Importantly, this non-motor phenotype was detected in the pre-clinical phase, prior to dopamine loss in Thy1-aSyn mice, thus replicating observations in patients. Results obtained in this study suggest a possible mechanism by which increased anxiety and maladaptive fear processing may occur in PD, opening a door for therapeutic options and further early biomarker research., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Reactive gliosis is a key feature and an important pathophysiological mechanism underlying chronic neurodegeneration following traumatic brain injury (TBI). In this study, we have explored the effects of intramuscular IGF-1 gene therapy on reactive gliosis and functional outcome after an injury of the cerebral cortex. Young adult male rats were intramuscularly injected with a recombinant adenoviral construct harboring the cDNA of human IGF-1 (RAd-IGF1), with a control vector expressing green fluorescent protein (RAd-GFP) or PBS as control. Three weeks after the intramuscular injections of adenoviral vectors, animals were subjected to a unilateral penetrating brain injury. The data revealed that RAd-IGF1 gene therapy significantly increased serum IGF1 levels and improved working memory performance after one week of TBI as compared to PBS or RAd-GFP lesioned animals. At the same time, when we analyzed the effects of therapy on glial scar formation, the treatment with RAd-IGF1 did not modify the number of glial fibrillary acidic protein (GFAP) positive cells, but we observed a decrease in vimentin immunoreactive astrocytes at 7 days post-lesion in the injured hemisphere compared to RAd-GFP group. Moreover, IGF-1 gene therapy reduced the number of Iba1+ cells with reactive phenotype and the number of MHCII + cells in the injured hemisphere. These results suggest that intramuscular IGF-1 gene therapy may represent a new approach to prevent traumatic brain injury outcomes in rats., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: APOE4 is the strongest genetic risk factor for Alzheimer's disease (AD), and obesity is a strong environmental risk factor for AD. These factors result in multiple central nervous system (CNS) disturbances and significantly increase chances of AD. Since over 20% of the US population carry the APOE4 allele and over 40% are obese, it is important to understand how these risk factors interact to affect neurons and glia in the CNS., Methods: We fed male and female APOE3 and APOE4 knock-in mice a high-fat diet (HFD-45% kcal fat) or a "control" diet (CD-10% kcal fat) for 12 weeks beginning at 6 months of age. At the end of the 12 weeks, brains were collected and analyzed for gliosis, neuroinflammatory genes, and neuronal integrity., Results: APOE3 mice on HFD, but not APOE4 mice, experienced increases in gliosis as measured by GFAP and Iba1 immunostaining. APOE4 mice on HFD showed a stronger increase in the expression of Adora2a than APOE3 mice. Finally, APOE3 mice on HFD, but not APOE4 mice, also showed increased neuronal expression of immediate early genes cFos and Arc., Conclusions: These findings demonstrate that APOE genotype and obesity interact in their effects on important processes particularly related to inflammation and neuronal plasticity in the CNS. During the early stages of obesity, the APOE3 genotype modulates a response to HFD while the APOE4 genotype does not. This supports a model where early dysregulation of inflammation in APOE4 brains could predispose to CNS damages from various insults and later result in the increased CNS damage normally associated with the APOE4 genotype., (© 2021. The Author(s).)

Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-β signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32's ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-β. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-β-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX) + and CD133 + radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tatomir, Beltrand, Nguyen, Courneya, Boodhoo, Cudrici, Muresanu, Rus, Badea and Rus.)

Astrocytes are a specific type of neuroglial cells that confer metabolic and structural support to neurons. Astrocytes populate all regions of the nervous system and adopt a variety of phenotypes depending on their location and their respective functions, which are also pleiotropic in nature. For example, astrocytes adapt to pathological conditions with a specific cellular response known as reactive astrogliosis, which includes extensive phenotypic and transcriptional changes. Reactive astrocytes may lose some of their homeostatic functions and gain protective or detrimental properties with great impact on damage propagation. Different astrocyte subpopulations seemingly coexist in reactive astrogliosis, however, the source of such heterogeneity is not completely understood. Altered cellular signaling in pathological compared to healthy conditions might be one source fueling astrocyte heterogeneity. Moreover, diversity might also be encoded cell-autonomously, for example as a result of astrocyte subtype specification during development. We hypothesize and propose here that elucidating the epigenetic signature underlying the phenotype of each astrocyte subtype is of high relevance to understand another regulative layer of astrocyte heterogeneity, in general as well as after injury or as a result of other pathological conditions. High resolution methods should allow enlightening diverse cell states and subtypes of astrocyte, their adaptation to pathological conditions and ultimately allow controlling and manipulating astrocyte functions in disease states. Here, we review novel literature reporting on astrocyte diversity from a developmental perspective and we focus on epigenetic signatures that might account for cell type specification.

Maternal malnutrition in critical periods of development increases the risk of developing short- and long-term diseases in the offspring. The alterations induced by this nutritional programming in the hypothalamus of the offspring are of special relevance due to its role in energy homeostasis, especially in the endocannabinoid system (ECS), which is involved in metabolic functions. Since astrocytes are essential for neuronal energy efficiency and are implicated in brain endocannabinoid signaling, here we have used a rat model to investigate whether a moderate caloric restriction (R) spanning from two weeks prior to the start of gestation to its end induced changes in offspring hypothalamic (a) ECS, (b) lipid metabolism (LM) and/or (c) hypothalamic astrocytes. Monitorization was performed by analyzing both the gene and protein expression of proteins involved in LM and ECS signaling. Offspring born from caloric-restricted mothers presented hypothalamic alterations in both the main enzymes involved in LM and endocannabinoids synthesis/degradation. Furthermore, most of these changes were similar to those observed in hypothalamic offspring astrocytes in culture. In conclusion, a maternal low caloric intake altered LM and ECS in both the hypothalamus and its astrocytes, pointing to these glial cells as responsible for a large part of the alterations seen in the total hypothalamus and suggesting a high degree of involvement of astrocytes in nutritional programming.

The omega-3 fatty acid docosahexaenoic acid (DHA) inversely relates to neurological impairments with aging; however, limited nondietary models manipulating brain DHA have hindered a direct linkage. We discovered that loss of long-chain acyl-CoA synthetase 6 in mice (Acsl6-/-) depletes brain membrane phospholipid DHA levels, independent of diet. Here, Acsl6-/- brains contained lower DHA compared with controls across the life span. The loss of DHA- and increased arachidonate-enriched phospholipids were visualized by MALDI imaging predominantly in neuron-rich regions where single-molecule RNA in situ hybridization localized Acsl6 to neurons. ACSL6 is also astrocytic; however, we found that astrocyte-specific ACSL6 depletion did not alter membrane DHA because astrocytes express a non-DHA-preferring ACSL6 variant. Across the life span, Acsl6-/- mice exhibited hyperlocomotion, impairments in working spatial memory, and increased cholesterol biosynthesis genes. Aging caused Acsl6-/- brains to decrease the expression of membrane, bioenergetic, ribosomal, and synaptic genes and increase the expression of immune response genes. With age, the Acsl6-/- cerebellum became inflamed and gliotic. Together, our findings suggest that ACSL6 promotes membrane DHA enrichment in neurons, but not in astrocytes, and is important for neuronal DHA levels across the life span. The loss of ACSL6 impacts motor function, memory, and age-related neuroinflammation, reflecting the importance of neuronal ACSL6-mediated lipid metabolism across the life span.

A disproportionate increase in activated nuclear factor-kappa B (NF-κB) has been shown to drive the Aβ deposition, neuroinflammation and neurodegeneration in Alzheimer's disease (AD). Hence, selective targeting of activated p65 represents an attractive therapeutic approach for AD. Glucocorticoid induced leucine zipper (GILZ) is a NF-κB interactant that binds and sequesters the activated p65 in the cytoplasm. The p65 binding domain of GILZ adopts a polyproline type II helical conformation, a motif that acts as an adaptable glove in the interface with the binding partner and constitutes an excellent template for drug design. Previously, peptide analogs of the p65 binding domain of GILZ, referred to as GA have been shown to suppress pathology in the lipopolysaccharide induced model of neuroinflammation. In this study, we investigated the CNS delivery of labeled GA administered intraperitoneally in adult mice for a period of upto 24 h. Further, we evaluated the suppressive potential of GA in 5xFAD mice, an aggressive model with five genetic mutations closely associated with human AD. Groups of 5xFAD mice administered GA or control peptide intraperitoneally on alternate days for six weeks were evaluated for Aβ deposition, microglia, inflammation and innate immune responses by immunohistochemistry and real time polymerase reaction. GA was observed in proximity with NeuN positive neurons suggesting that the compound crossed the blood brain barrier to reach the brain parenchyma. Further, GA treatment decreased Aβ load, reduced Iba1 + microglia and glial fibrillary acidic protein (GFAP)+ astrocytes, inhibited inflammatory cytokines and suppressed toll like receptor (TLR-2, TLR-4) expressions in 5xFAD mice., (Copyright © 2021. Published by Elsevier Masson SAS.)

Epilepsy is a neurological condition associated to significant brain damage produced by status epilepticus (SE) including neurodegeneration, gliosis and ectopic neurogenesis. Reduction of these processes constitutes a useful strategy to improve recovery and ameliorate negative outcomes after an initial insult. SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), has been shown to increase M-current density in neurons, leading to reduced excitability and protection against seizures. For this study, we used 4-5 months old male transgenic C57BL/6 J and FVB/NJ mice expressing near physiological levels of a constitutively active form of the kinase controlled by its endogenous promoter. Here we show that SGK1.1 activation potently reduces levels of neuronal death (assessed using Fluoro-Jade C staining) and reactive glial activation (reported by GFAP and Iba-1 markers) in limbic regions and cortex, 72 h after SE induced by kainate, even in the context of high seizure activity. This neuroprotective effect is not exclusively through M-current activation but is also directly linked to decreased apoptosis levels assessed by TUNEL assays and quantification of Bim and Bcl-x L by western blot of hippocampal protein extracts. Our results demonstrate that this newly described antiapoptotic role of SGK1.1 activation acts synergistically with the regulation of cellular excitability, resulting in a significant reduction of SE-induced brain damage in areas relevant to epileptogenesis., (Copyright © 2021. Published by Elsevier Inc.)

Cardiorespiratory collapse following a seizure is a suspected cause of sudden unexpected death in epilepsy (SUDEP), the leading cause of epilepsy-related mortality. In the commonly used Kcna1 gene knockout (Kcna1 -/- ) mouse model of SUDEP, cardiorespiratory profiling reveals an array of aberrant breathing patterns that could contribute to risk of seizure-related mortality. However, the brain structures mediating these respiratory abnormalities remain unknown. We hypothesize that Kv1.1 deficiency in respiratory control centers of the brain contribute to respiratory dysfunction in Kcna1 -/- mice leading to increased SUDEP risk. Thus, in this study, we first used immunohistochemistry to map expression of Kv1.1 protein in cardiorespiratory brain regions of wild-type Kcna1 +/+ (WT) mice. Next, GFAP and Iba1 immunostaining was used to test for the presence of astrogliosis and microgliosis, respectively, in the cardiorespiratory centers of Kcna1 -/- mice, which could be indicative of seizure-related brain injury that could impair breathing. In WT mice, we detected Kv1.1 protein in all cardiorespiratory centers examined, including the basolateral amygdala, dorsal respiratory group, dorsal motor nucleus of vagus, nucleus ambiguus, ventral respiratory column, and pontine respiratory group, as well as chemosensory centers including the retrotrapezoid and median raphae nuclei. Extensive gliosis was observed in the same areas in Kcna1 -/- mice suggesting that seizure-associated brain injury could contribute to respiratory abnormalities., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdh ki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdh ki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdh ki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdh ki/ki rats, but not in WT rats. It seems that Gcdh ki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdh ki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdh ki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdh ki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Astrogliosis has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism, and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes as a function of age have been reported, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected in supporting a functional switch of astrocytes from neurotrophic to neurotoxic. This study aimed to explore the metabolic changes occurring in astrocytes during their activation. Astrocytes were derived from human ReN cell neural progenitors and characterized. They were activated by exposure to tumor necrosis factor alpha (TNFα) or interleukin 1β (IL1β) for 24 h. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics and extracellular flux analyses. ReN-derived astrocytes reactivity was observed by the modifications of genes and proteins linked to inflammation (cytokines, nuclear factor-kappa B (NFκB), signal transducers and activators of transcription (STATs)) and immune pathways (major histocompatibility complex (MHC) class I). Increased NFκB1, NFκB2 and STAT1 expression, together with decreased STAT3 expression, suggest an activation towards the detrimental pathway. Strong modifications of astrocyte cytoskeleton were observed, including a glial fibrillary acidic protein (GFAP) decrease. Astrogliosis was accompanied by changes in energy metabolism characterized by increased glycolysis and lactate release. Increased glycolysis is reported for the first time during human astrocyte activation. Astrocyte activation is strongly tied to energy metabolism, and a possible association between NFκB signaling and/or MHC class I pathway and glycolysis is suggested.

Spinal cord injury (SCI) leads to permanent loss of motor and sensory function due to the complex mechanisms of the external microenvironment and internal neurobiochemistry that restrict neuronal plasticity and axonal regeneration. Chemokine CXCL12 was verified in regulating the development of central nervous system (CNS) and repairing of CNS disease. In the present study, CXCL12 was downregulated in the spinal cord after SCI. SCI also induced gliosis and loss of synapse. Intrathecal treatment of CXCL12 promoted the functional recovery of SCI by inducing the formation of neuronal connections and suppressing glia scar. To confirm whether CXCL12 promoted synapse formation and functional neuronal connections, the primary cortical neurons were treated with CXCL12 peptide, the synapse was examined using Immunofluorescence staining and the function of synapse was tested using a whole-cell patch clamp. The results indicated that CXCL12 peptide promoted axonal elongation, branche formation, dendrite generation and synaptogenesis. The electrophysiological results showed that CXCL12 peptide increased functional connections among neurons. Taken together, the present study illustrated an underlying mechanism of the development of SCI and indicated a potential approach to facilitate functional recovery of spinal cord after SCI., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Earlier, it was shown that reversing the downregulation of neuritin expression in the brain improves central neuropathy in diabetic rats. We investigated the protective mechanism of neuritin in diabetic cognitive dysfunction via astrocytes. Further, the impact of the overexpression of neuritin in the cortex and the hippocampus on diabetic cognitive dysfunction and astrogliosis in type 2 diabetic (db/db) mice was assessed. Antagonists were used to inhibit the JAK2/STAT3 signaling pathway in U-118MG, an astrocyte cell line. Immunofluorescence, Western blotting, and real-time PCR were performed. Neuritin overexpression in the hippocampus of db/db mice significantly ameliorated cognitive dysfunction, hippocampal neuronal impairment, and synaptic plasticity deterioration, and inhibited astrogliosis and the JAK2/STAT3 signaling pathway in the hippocampus. Neuritin suppressed the JAK2/STAT3 signaling pathway to inhibit lipopolysaccharide-induced gliosis in U-118MG cells. It was observed that neuritin regulates the JAK2/STAT3 signaling pathway in astrocytes to inhibit astrogliosis and improve diabetic cognitive dysfunction.

The homeostasis of protein palmitoylation and depalmitoylation is essential for proper physiological functions in various tissues, in particular the central nervous system (CNS). The dysfunction of PPT1 (PPT1-KI, infantile neuronal ceroid lipofuscinosis [INCL] mouse model), which catalyze the depalmitoylation process, results in serious neurodegeneration accompanied by severe astrogliosis in the brain. Endeavoring to determine critical factors that might account for the pathogenesis in CNS by palm-proteomics, glial fibrillary acidic protein (GFAP) was spotted, indicating that GFAP is probably palmitoylated. Questions concerning if GFAP is indeed palmitoylated in vivo and how palmitoylation of GFAP might participate in neural pathology remain unexplored and are waiting to be investigated. Here we show that GFAP is readily palmitoylated in vitro and in vivo; specifically, cysteine-291 is the unique palmitoylated residue in GFAP. Interestingly, it was found that palmitoylated GFAP promotes astrocyte proliferation in vitro. Furthermore, we showed that PPT1 depalmitoylates GFAP, and the level of palmitoylated GFAP is overwhelmingly up-regulated in PPT1-knockin mice, which lead us to speculate that the elevated level of palmitoylated GFAP might accelerate astrocyte proliferation in vivo and ultimately led to astrogliosis in INCL. Indeed, blocking palmitoylation by mutating cysteine-291 into alanine in GFAP attenuate astrogliosis, and remarkably, the concurrent neurodegenerative pathology in PPT1-knockin mice. Together, these findings demonstrate that hyperpalmitoylated GFAP plays critical roles in regulating the pathogenesis of astrogliosis and neurodegeneration in the CNS, and most importantly, pinpointing that cysteine-291 in GFAP might be a valuable pharmaceutical target for treating INCL and other potential neurodegenerative diseases., Competing Interests: Competing interest statement: E.K. is listed as inventor on pending patent covering the targeting of cysteine-291 in glial fibrillary acidic protein as intervention strategy for treating neurodegenerative diseases., (Copyright © 2021 the Author(s). Published by PNAS.)

The ε4 allele of the apolipoprotein E gene (APOE4) is a strong genetic risk factor for Alzheimer's disease (AD) and multiple vascular conditions. ApoE is abundantly expressed in multiple brain cell types, including astrocytes, microglia, and vascular mural cells (VMCs). Here, we show that VMC-specific expression of apoE4 in mice impairs behavior and cerebrovascular function. Expression of either apoE3 or apoE4 in VMCs was sufficient to rescue the hypercholesterolemia and atherosclerosis phenotypes seen in Apoe knockout mice. Intriguingly, vascular expression of apoE4, but not apoE3, reduced arteriole blood flow, impaired spatial learning, and increased anxiety-like phenotypes. Single-cell RNA sequencing of vascular and glial cells revealed that apoE4 in VMCs was associated with astrocyte activation, while apoE3 was linked to angiogenic signature in pericytes. Together, our data support cell-autonomous effects of vascular apoE on brain homeostasis in an isoform-dependent manner, suggesting a critical contribution of vascular apoE to AD pathogenesis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

CCCTC-binding factor (CTCF) is a genome organizer that regulates gene expression through transcription and chromatin structure regulation. CTCF also plays an important role during the developmental and adult stages. Cell-specific CTCF deletion studies have shown that a reduction in CTCF expression leads to the development of distinct clinical features and cognitive disorders. Therefore, we knocked out Ctcf (CTCF cKO) in the excitatory neurons of the forebrain in a Camk2a-Cre mouse strain to examine the role of CTCF in cell death and gliosis in the cortex. CTCF cKO mice were viable, but they demonstrated an age-dependent increase in reactive gliosis of astrocytes and microglia in the anterior cingulate cortex (ACC) from 16 weeks of age prior to neuronal loss observed at over 20 weeks of age. Consistent with these data, qRT-PCR analysis of the CTCF cKO ACC revealed changes in the expression of inflammation-related genes (Hspa1a, Prokr2 and Itga8) linked to gliosis and neuronal death. Our results suggest that prolonged Ctcf gene deficiency in excitatory neurons results in neuronal cell death and gliosis, possibly through functional changes in inflammation-related genes., (© 2020 International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Introduction: Presenilin enhancer2 (Pen-2) is an essential subunit of γ-secretase, which is a key protease responsible for the cleavage of amyloid precursor protein (APP) and Notch. Mutations on Pen-2 cause familial Alzheimer disease (AD). However, it remains unknown whether Pen-2 regulates neuronal survival and neuroinflammation in the adult brain., Methods: Forebrain neuron-specific Pen-2 conditional knockout (Pen-2 cKO) mice were generated for this study. Pen-2 cKO mice expressing Notch1 intracellular domain (NICD) conditionally in cortical neurons were also generated., Results: Loss of Pen-2 causes astrogliosis followed by age-dependent cortical atrophy and neuronal loss. Loss of Pen-2 results in microgliosis and enhanced inflammatory responses in the cortex. Expression of NICD in Pen-2 cKO cortices ameliorates neither neurodegeneration nor neuroinflammation., Conclusions: Pen-2 is required for neuronal survival in the adult cerebral cortex. The Notch signaling may not be involved in neurodegeneration caused by loss of Pen-2., (© 2020 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Aside from the classical motor symptoms, Parkinson's disease also has various non-classical symptoms. Interestingly, orexin neurons, involved in the regulation of exploratory locomotion, spontaneous physical activity, and energy expenditure, are affected in Parkinson's. In this study, we hypothesized that Parkinson's-disease-associated pathology affects orexin neurons and therefore impairs functions they regulate. To test this, we used a transgenic animal model of Parkinson's, the A53T mouse. We measured body composition, exploratory locomotion, spontaneous physical activity, and energy expenditure. Further, we assessed alpha-synuclein accumulation, inflammation, and astrogliosis. Finally, we hypothesized that chemogenetic inhibition of orexin neurons would ameliorate observed impairments in the A53T mice. We showed that aging in A53T mice was accompanied by reductions in fat mass and increases in exploratory locomotion, spontaneous physical activity, and energy expenditure. We detected the presence of alpha-synuclein accumulations in orexin neurons, increased astrogliosis, and microglial activation. Moreover, loss of inhibitory pre-synaptic terminals and a reduced number of orexin cells were observed in A53T mice. As hypothesized, this chemogenetic intervention mitigated the behavioral disturbances induced by Parkinson's disease pathology. This study implicates the involvement of orexin in early Parkinson's-disease-associated impairment of hypothalamic-regulated physiological functions and highlights the importance of orexin neurons in Parkinson's disease symptomology.

Mutations in the genes TARDBP (encoding the TDP-43 protein) and TBK1 can cause familial ALS. Neuronal cytoplasmatic accumulations of the misfolded, hyperphosphorylated RNA-binding protein TDP-43 are the pathological hallmark of most ALS cases and have been suggested to be a key aspect of ALS pathogenesis. Pharmacological induction of autophagy has been shown to reduce mutant TDP-43 aggregates and alleviate motor deficits in mice. TBK1 is exemplary for several other ALS genes that regulate autophagy. Consequently, we employed double mutant mice with both a heterozygous Tbk1 deletion and transgenic expression of human TDP-43 G298S to test the hypothesis that impaired autophagy reduces intracellular clearance of an aggregation-prone protein and enhances toxicity of mutant TDP-43. The heterozygous deletion of Tbk1 did not change expression or cellular distribution of TDP-43 protein, motor neuron loss or reactive gliosis in the spinal cord of double-mutant mice at the age of 19 months. However, it aggravated muscle denervation and, albeit to a small and variable degree, motor dysfunction in TDP-43 G298S transgenic mice, as similarly observed in the SOD1 G93A transgenic mouse model for ALS before. Conclusively, our findings suggest that TBK1 mutations can affect the neuromuscular synapse., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Expression of the 17β-estradiol (E2) synthesis enzyme aromatase is highly upregulated in astrocytes following brain injury. However, the precise role of astrocyte-derived E2 in the injured brain remains unclear. In the current study, we generated a glial fibrillary acidic protein (GFAP) promoter-driven aromatase knock-out (GFAP-ARO-KO) mouse model to deplete astrocyte-derived E2 in the brain and determine its roles after global cerebral ischemia (GCI) in male and female mice. GFAP-ARO-KO mice were viable and fertile, with normal gross brain structure, normal morphology, intensity and distribution of astrocytes, normal aromatase expression in neurons, and normal cognitive function basally. In contrast, after GCI, GFAP-ARO-KO mice: (1) lacked the normal elevation of astrocyte aromatase and hippocampal E2 levels; (2) had significantly attenuated reactive astrogliosis; and (3) displayed enhanced neuronal damage, microglia activation, and cognitive deficits. RNA-sequencing (RNA-seq) analysis revealed that the ischemic GFAP-ARO-KO mouse hippocampus failed to upregulate the "A2" panel of reactive astrocyte genes. In addition, the JAK-STAT3 pathway, which is critical for the induction of reactive astrogliosis, was significantly downregulated in the GFAP-ARO-KO hippocampus following GCI. Finally, exogenous E2 administration fully rescued the compromised JAK-STAT3 pathway and reactive astrogliosis, and reversed the enhanced neuronal damage and microglial activation in the GFAP-ARO-KO mice after GCI, suggesting that the defects in the KO mice are because of a loss of E2 rather than an increase in precursor androgens. In conclusion, the current study provides novel genetic evidence for a beneficial role of astrocyte-derived E2 in reactive astrogliosis, microglial activation, and neuroprotection following an ischemic injury to the brain. SIGNIFICANCE STATEMENT Following cerebral ischemia, reactive astrocytes express the enzyme aromatase and produce 17β-estradiol (E2), although the precise role of astrocyte-derived E2 is poorly understood. In this study, we generated a glial fibrillary acidic protein (GFAP) promoter-driven aromatase knock-out (GFAP-ARO-KO) mouse to deplete astrocyte-derived E2 and elucidate its roles after global cerebral ischemia (GCI). The GFAP-ARO-KO mice exhibited significantly attenuated reactive astrogliosis, as well as enhanced microglial activation, neuronal damage, and cognitive dysfunction after GCI. Transcriptome analysis further revealed that astrocyte-derived E2 was critical for the induction of the JAK-STAT3 signaling pathway, as well as the A2 reactive astrocyte phenotype after ischemia. Collectively, these findings indicate that astrocyte-derived E2 has a key role in the regulation of reactive astrogliosis, microglial activation, and neuroprotection after cerebral ischemia., (Copyright © 2020 the authors.)

Despite combined antiretroviral therapy (cART), HIV infection in the CNS persists with reported increases in activation of macrophages (MΦ), microglia, and surrounding astrocytes/neurons, conferring HIV-induced inflammation. Chronic inflammation results in HIV-associated neurocognitive disorders (HAND) with reported occurrence of up to half of individuals with HIV infection. The existing HAND mouse model used by laboratories including ours, and the effect of novel agents on its pathology present with labor-intensive and time-consuming limitations since brain sections and immunohistochemistry assays have to be performed and analyzed. A novel flow cytometry-based system to objectively quantify phenotypic effects of HIV using a SCID mouse HAND model was developed which demonstrated that the HIV-infected mice had significant increases in astrogliosis, loss of neuronal dendritic marker, activation of murine microglia, and human macrophage explants compared to uninfected control mice. HIV p24 could also be quantified in the brains of the infected mice. Correlation of these impairments with HIV-induced brain inflammation and previous behavioral abnormalities studies in mice suggests that this model can be used as a fast and relevant throughput methodology to quantify preclinical testing of novel treatments for HAND.

Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant neurodegenerative disease caused by CAG expansion in the gene encoding the TATA-binding protein (TBP). The neurological features of SCA17 are Purkinje cell loss and gliosis. We have generated SCA17 transgenic mice which recapitulate the patients' phenotypes and are suitable for the study of the SCA17 pathomechanism. Our previous study identified the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) occurred in the SCA17 cerebella, this study aims to study the role of ERK activation in SCA17. The levels of pERK, calbindin, and gliosis markers on the mouse cerebellum at 4-8 weeks old were analyzed to elucidate the correlation among behavioral performance, ERK activation and Purkinje cell degeneration. The motor incoordination was initiated in SCA17 mice at 6 weeks old. We found that the presence of TBP nuclear aggregation and microglia activation were observed at 4 weeks old. Gliosis of astrocytes and Bergmann glia, pERK, Bax/Bcl2 ratio, and caspase-3 were significantly increased in the 6-week-old SCA17 mouse cerebellum. In addition to the polyglutamine-protein aggregation in Purkinje cells caused apoptosis cell-autonomously, a significant body of evidence have shown that ERK pathways involves in neuronal apoptosis. Our study showed that the activation of ERK in the astrocytes and Bergmann glia was identified as preceding motor deficits, which suggest the elevated gliosis by ERK activation may contribute to neuronal apoptosis in SCA17 mice., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Degeneration of locus ceruleus (LC) neurons and dysregulation of noradrenergic signaling are ubiquitous features of Parkinson's disease (PD). The LC is among the first brain regions affected by α-synuclein (asyn) pathology, yet how asyn affects these neurons remains unclear. LC-derived norepinephrine (NE) can stimulate neuroprotective mechanisms and modulate immune cells, while dysregulation of NE neurotransmission may exacerbate disease progression, particularly nonmotor symptoms, and contribute to the chronic neuroinflammation associated with PD pathology. Although transgenic mice overexpressing asyn have previously been developed, transgene expression is usually driven by pan-neuronal promoters and thus has not been selectively targeted to LC neurons. Here we report a novel transgenic mouse expressing human wild-type asyn under control of the noradrenergic-specific dopamine β-hydroxylase promoter ( DBH-hSNCA ). These mice developed oligomeric and conformation-specific asyn in LC neurons, alterations in hippocampal and LC microglial abundance, upregulated GFAP expression, degeneration of LC fibers, decreased striatal DA metabolism, and age-dependent behaviors reminiscent of nonmotor symptoms of PD that were rescued by adrenergic receptor antagonists. These mice provide novel insights into how asyn pathology affects LC neurons and how central noradrenergic dysfunction may contribute to early PD pathophysiology. SIGNIFICANCE STATEMENT ɑ-Synuclein (asyn) pathology and loss of neurons in the locus ceruleus (LC) are two of the most ubiquitous neuropathologic features of Parkinson's disease (PD). Dysregulated norepinephrine (NE) neurotransmission is associated with the nonmotor symptoms of PD, including sleep disturbances, emotional changes such as anxiety and depression, and cognitive decline. Importantly, the loss of central NE may contribute to the chronic inflammation in, and progression of, PD. We have generated a novel transgenic mouse expressing human asyn in LC neurons to investigate how increased asyn expression affects the function of the central noradrenergic transmission and associated behaviors. We report cytotoxic effects of oligomeric and conformation-specific asyn, astrogliosis, LC fiber degeneration, disruptions in striatal dopamine metabolism, and age-dependent alterations in nonmotor behaviors without inclusions., (Copyright © 2020 the authors.)

Background: In human immunodeficiency virus-1 (HIV-1) infection, activation of astrocytes induces imbalance in physiological functions due to perturbed astrocytic functions that unleashes toxicity on neurons. This leads to inflammatory response finally culminating into neurocognitive dysfunction. In neuroAIDS, HIV-1 protein, transactivator of transcription (Tat) is detected in the cerebrospinal fluid of infected patients. Mortalin, a multifunctional protein, has anti-inflammatory role following its activation in various stress conditions. Recent studies demonstrate downregulation of mortalin in neurodegenerative diseases. Here, we explored the mechanisms of mortalin in modulating HIV-1 Tat-mediated neuroinflammation., Methods: Expression of mortalin in autopsy section in normal and diseased individuals were examined using immunohistochemistry. To decipher the role of mortalin in HIV-1 Tat-induced activation, human fetal brain-derived astrocytes were transiently transfected with Tat and mortalin using expression vectors. HIV-1 Tat-mediated damage was analyzed using RT-PCR and western blotting. Modulatory role of mortalin was examined by coexpressing it with Tat, followed by examination of mitochondrial morphodynamics using biochemical assay and confocal and electron microscopy. Extracellular ATP release was monitored using luciferase assay. Neuroinflammation in astrocytes was examined using flow cytometry, dye based study, immunocytochemistry, immunoprecipitation, and western blotting. Indirect neuronal damage was also analyzed., Results: HIV-1 Tat downregulates the expression of mortalin in astrocytes, and this is corroborated with autopsy sections of HIV-1 patients. We found that overexpression of mortalin with Tat reduced inflammation and also rescued astrocytic-mediated neuronal death. Using bioinformatics, we discovered that binding of mortalin with Tat leads to Tat degradation and rescues the cell from neuroinflammation. Blocking of proteosomal pathway rescued the Tat degradation and revealed the ubiquitination of Tat., Conclusion: Overall, our data demonstrated the protective role of mortalin in combating HIV-1 Tat-mediated damage. We also showed that mortalin could degrade Tat through direct binding with HIV-1 Tat. Overexpression of mortalin in the presence of Tat could significantly reduce cytotoxic effects of Tat in astrocytes. Indirect neuronal death was also found to be rescued. Our in vitro findings were validated as we found attenuated expression of mortalin in the autopsy sections of HIV-1 patients.

Alzheimer's disease (AD) is characterized by plaques containing amyloid-β (Aβ) and neurofibrillary tangles composed of aggregated, hyperphosphorylated tau. Beyond tau and Aβ, evidence suggests that microglia play an important role in AD pathogenesis. Rare variants in the microglia-expressed triggering receptor expressed on myeloid cells 2 (TREM2) gene increase AD risk 2- to 4-fold. It is likely that these TREM2 variants increase AD risk by decreasing the response of microglia to Aβ and its local toxicity. However, neocortical Aβ pathology occurs many years before neocortical tau pathology in AD. Thus, it will be important to understand the role of TREM2 in the context of tauopathy. We investigated the impact of the AD-associated TREM2 variant (R47H) on tau-mediated neuropathology in the PS19 mouse model of tauopathy. We assessed PS19 mice expressing human TREM2CV (common variant) or human TREM2R47H. PS19-TREM2R47H mice had significantly attenuated brain atrophy and synapse loss versus PS19-TREM2CV mice. Gene expression analyses and CD68 immunostaining revealed attenuated microglial reactivity in PS19-TREM2R47H versus PS19-TREM2CV mice. There was also a decrease in phagocytosis of postsynaptic elements by microglia expressing TREM2R47H in the PS19 mice and in human AD brains. These findings suggest that impaired TREM2 signaling reduces microglia-mediated neurodegeneration in the setting of tauopathy.

Astrocytes are the most populous glial cells in the central nervous system (CNS). They are essential to CNS physiology and play important roles in the maintenance of homeostasis, development of synaptic plasticity, and neuroprotection. Nevertheless, under the influence of certain factors, astrocytes may also exert detrimental effects through a process of reactive astrogliosis. Previous studies have shown that astrocytes have more than one type of polarization. Two types have been extensively researched. One is a damaging change that occurs under inflammation and has been termed A1 astrocyte, while the other is a restorative change that occurs under ischemic induction and was termed A2 astrocyte. Researchers are now increasingly paying attention to the role of astrocytes in spinal cord injury (SCI), degenerative diseases, chronic pain, neurological tumors, and other CNS disorders. In this review, we discuss (a) the characteristics of polarized astrocytes, (b) the relationship between astrocyte polarization and SCI, and (c) new implications of reactive astrogliosis for future SCI therapies., Competing Interests: The authors declare that they have no conflict of interest. B N is a Taishan scholar young expert and director of Jinan Institute of Biomechanics., (Copyright © 2020 Xinyu Li et al.)

Antibodies have been explored extensively as a potential therapeutic for Alzheimer's disease, where amyloid-β (Aβ) peptides and the tau protein deposit in patient brains. While the major focus of antibody-based therapy development was on Aβ, arguably with limited success in clinical trials, targeting tau has become an emerging strategy, possibly extending therapies to dementias with isolated tau pathology. Interestingly, low titres of autoantibodies to pathological tau have been described in humans and transgenic mouse models, but their pathophysiological relevance remained elusive. Here, we used two independent approaches to deplete the B-cell lineage and hence antibody formation in human P301S mutant tau transgenic mice, TAU58/2. TAU58/2 mice were either crossed with the B-cell-deficient Ighm knockout line (muMT -/- ) or treated with anti-CD20 antibodies that target B-cell precursors. In both models, B-cell depletion significantly reduced astrocytosis in TAU58/2 mice. Only when B-cells were absent throughout life, in TAU58/2.muMT -/- mice, were spatial learning deficits moderately aggravated while motor performance improved as compared to B-cell-competent TAU58/2 mice. This was associated with changes in brain region-specific tau solubility. No other relevant behavioural or neuropathological changes were observed in TAU58/2 mice in the absence of B-cells/antibodies. Taken together, our data suggests that the presence of antibodies throughout life contributes to astrocytosis in TAU58/2 mice and limits learning deficits, while other deficits and neuropathological changes appear to be independent of the presence of B-cells/antibodies.

Objectives: This tissue microarray (TMA) immunohistochemical (IHC) study elucidates the role of Wilms' tumor 1 protein (WT1) in diagnosis and prognostication of astrocytic tumors., Methods: IHC was applied to 75 astrocytic lesions (18 astrogliosis and 57 astrocytic tumors) using antibodies directed against WT1 clone 6F-H2, isocitrate dehydrogenase 1(IDH1), Bcl2 and Ki67. WT1 IHC staining was evaluated and scored blindly by 2 pathologists. Bcl2 and Ki67 scores and labelling indices were assessed and IDH1 status determined. Statistical analysis was performed using the appropriate methodology., Results: WT1 cytoplasmic expression was detected in 89.5% of astrocytic tumors but not in astrogliosis. Positive WT1 differentiated astrocytic tumors (92.6% accuracy) and grade II diffuse astrocytomas (93.5% accuracy) from astrogliosis with high sensitivity, specificity and positive predictive values (p<0.001). Increased WT1 score significantly associated higher Bcl2 and Ki67 labelling indices, increasing WHO tumor grade and tumor's histopathologic type (p<0.05) showing staining pattern variability by tumor entity and cell type. Glioblastomas, gliosarcomas and subependymal giant cell astrocytomas were the most frequently WT1 expressing tumors with frequent +3 WT1 score. About 21.4% of pilocytic astrocytomas had +3WT1 score in association with increased Bcl2 and Ki67 indices. Low WT1 scores in grade II and III diffuse astrocytomas were linked to the high frequency of IDH1 positivity, and were associated with low Bcl2 and Ki67 labelling indices. In glioblastomas, WT1 significantly associated poor prognostic variables: older age, negative-IDH1 status, high Bcl2 and Ki67 labelling indices (p=0.04, <0.001, =0.001 and.

Glioma is one of the most common primary malignant tumors of the central nervous system accounting for approximately 40% of all intracranial tumors. Temozolomide is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk gliomas, including grade II to grade IV. Our bioinformatic analysis of The Cancer Genome Atlas and Chinese Glioma Genome Atlas datasets and immunoblotting assay show that SLC12A2 gene and its encoded Na + -K + -2Cl - cotransporter isoform 1 (NKCC1) protein are abundantly expressed in grade II-IV gliomas. NKCC1 regulates cell volume and intracellular Cl - concentration, which promotes glioma cell migration, resistance to temozolomide, and tumor-related epilepsy in experimental glioma models. Using mouse syngeneic glioma models with intracranial transplantation of two different glioma cell lines (GL26 and SB28), we show that NKCC1 protein in glioma tumor cells as well as in tumor-associated reactive astrocytes was significantly upregulated in response to temozolomide monotherapy. Combination therapy of temozolomide with the potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated the cytotoxic effects of temozolomide, decreased tumor-associated reactive astrogliosis, and restored astrocytic GLT-1 and GLAST glutamate transporter expression. The combinatorial therapy also led to suppressed tumor growth and prolonged survival of mice bearing GL26 glioma cells. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in the pathogenesis of glioma tumors and presents as a therapeutic target for reducing temozolomide-mediated resistance and tumor-associated astrogliosis., (©2020 American Association for Cancer Research.)

The astrocytic response to injury is characterized on the cellular level, but our understanding of the molecular mechanisms controlling the cellular processes is incomplete. The astrocytic response to injury is similar to wound-healing responses in non-neural tissues that involve epithelial-to-mesenchymal transitions (EMTs) and upregulation in ZEB transcription factors. Here we show that injury-induced astrogliosis increases EMT-related genes expression, including Zeb2, and long non-coding RNAs, including Zeb2os, which facilitates ZEB2 protein translation. In mouse models of either contusive spinal cord injury or transient ischemic stroke, the conditional knockout of Zeb2 in astrocytes attenuates astrogliosis, generates larger lesions, and delays the recovery of motor function. These findings reveal ZEB2 as an important regulator of the astrocytic response to injury and suggest that astrogliosis is an EMT-like process, which provides a conceptual connection for the molecular and cellular similarities between astrogliosis and wound-healing responses in non-neural tissue., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: A growing body of evidence shows that hypothalamic inflammation is an important factor in the initiation of obesity. In particular, reactive gliosis accompanied by inflammatory responses in the hypothalamus are pivotal cellular events that elicit metabolic abnormalities. In this study, we examined whether MyD88 signaling in hypothalamic astrocytes controls reactive gliosis and inflammatory responses, thereby contributing to the pathogenesis of obesity., Methods: To analyze the role of astrocyte MyD88 in obesity pathogenesis, we used astrocyte-specific Myd88 knockout (KO) mice fed a high-fat diet (HFD) for 16 weeks or injected with saturated free fatty acids. Astrocyte-specific gene expression in the hypothalamus was determined using real-time PCR with mRNA purified by the Ribo-Tag system. Immunohistochemistry was used to detect the expression of glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, phosphorylated signal transducer and activator of transcription 3, and α-melanocyte-stimulating hormone in the hypothalamus. Animals' energy expenditure was measured using an indirect calorimetry system., Results: The astrocyte-specific Myd88 KO mice displayed ameliorated hypothalamic reactive gliosis and inflammation induced by injections of saturated free fatty acids and a long-term HFD. Accordingly, the KO mice were resistant to long-term HFD-induced obesity and showed an improvement in HFD-induced leptin resistance., Conclusions: These results suggest that MyD88 in hypothalamic astrocytes is a critical molecular unit for obesity pathogenesis that acts by mediating HFD signals for reactive gliosis and inflammation.

A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia 1,2 . The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration 3-9 . The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins 5 before its non-canonical translation into neural-toxic dipeptide proteins 3,4 . The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation 6-9 . Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria 10,11 protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system.

Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G > T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.

Competing Interests: Declaration of Competing Interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

A hallmark pathology of Alzheimer's disease (AD) is the formation of amyloid β (Aβ) deposits that exhibit diverse localization and morphologies, ranging from diffuse to cored-neuritic deposits in brain parenchyma, with cerebral vascular deposition in leptomeningeal and parenchymal compartments. Most AD brains exhibit the full spectrum of pathologic Aβ morphologies. In the course of studies to model AD amyloidosis, we have generated multiple transgenic mouse models that vary in the nature of the transgene constructs that are expressed; including the species origin of Aβ peptides, the levels and length of Aβ that is deposited, and whether mutant presenilin 1 (PS1) is co-expressed. These models recapitulate features of human AD amyloidosis, but interestingly some models can produce pathology in which one type of Aβ morphology dominates. In prior studies of mice that primarily develop cored-neuritic deposits, we determined that Aβ deposition is associated with changes in cytosolic protein solubility in which a subset of proteins become detergent-insoluble, indicative of secondary proteome instability. Here, we survey changes in cytosolic protein solubility across seven different transgenic mouse models that exhibit a range of Aβ deposit morphologies. We find a surprisingly diverse range of changes in proteome solubility across these models. Mice that deposit human Aβ40 and Aβ42 in cored-neuritic plaques had the most robust changes in proteome solubility. Insoluble cytosolic proteins were also detected in the brains of mice that develop diffuse Aβ42 deposits but to a lesser extent. Notably, mice with cored deposits containing only Aβ42 had relatively few proteins that became detergent-insoluble. Our data provide new insight into the diversity of biological effects that can be attributed to different types of Aβ pathology and support the view that fibrillar cored-neuritic plaque pathology is the more disruptive Aβ pathology in the Alzheimer's cascade.

Changes to neonatal nutrition result in long-lasting impairments in energy balance, which may be described as metabolic programing. Astrocytes, which are interconnected by gap junctions, have emerged as important players in the hypothalamic control of food intake. In order to study the effects of nutritional programming on glial morphology and protein expression, cross-fostered male Wistar rats at postnatal day 3 were assigned to three groups based on litter size: small litter (3 pups per dam, SL), normal litter (10 pups per dam, NL), and large litter (16 pups per dam, LL). Rats from the SL group exhibited higher body weight throughout the study and hyperphagia after weaning. LL animals exhibited hyperphagia, high energy efficiency and catch-up of body weight after weaning. Both the SL and LL groups at postnatal day 60 (PN60) exhibited increased levels of plasma leptin, the Lee index (as an index of obesity), adiposity content, immunoreactivity toward T-cell protein tyrosine phosphatase (TCPTP), and glial fibrillary acidic protein (GFAP) in the arcuate nucleus (ARC) of the hypothalamus. Astrocyte morphology was altered in the ARC of SL and LL animals, and this effect occurred in parallel with a reduction in immunoreactivity toward connexin 30 (CX30). The data obtained demonstrate that both neonatal over- and underfeeding promote not only alterations in the metabolic status but also morphological changes in glial cells in parallel with increasing TCPTP and changes in connexin expression., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Among the most challenging diagnostic issues in surgical neuropathology is the distinction between scant infiltration by diffuse gliomas and reactive gliosis. The best documented ancillary marker to establish a definitive diagnosis of glioma in this setting is the identification of hotspot mutations in the isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) genes, which is limited, however, by the low prevalence of these mutations in gliomas of elderly adults. Since telomerase reverse transcriptase (TERT) promoter mutations are present in the vast majority of IDH-wildtype diffuse gliomas, we hypothesized that combined analysis of IDH and TERT might overcome these limitations. For this purpose, we analyzed a series of non-neoplastic and neoplastic CNS samples for the prevalence of TERT hotspot mutations. TERT mutations were identified in none out of 58 (0%) reactive gliosis samples, and in 91 out of 117 (78%) IDH-wildtype gliomas. Based on a series of 200 consecutive diffuse gliomas, we found that IDH mutation analysis alone had a sensitivity of 28% (63% and 12%, respectively, in patients below and above age of 50) for detection of gliomas, whereas a combined analysis of IDH and TERT was 85% sensitive (87% and 84%, respectively, below and above age of 50). In sum, our findings suggest that TERT promoter mutation analysis contributes favorably to a molecular panel in cases equivocal for glioma versus gliosis on morphological grounds, especially in patients above age of 50, in which IDH analysis alone performs poorly., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Yes-associated protein (YAP) transcriptional coactivator is negatively regulated by the Hippo pathway and functions in controlling the size of multiple organs, such as liver during development. However, it is not clear whether YAP signaling participates in the process of the formation of glia scars after spinal cord injury (SCI). In this study, we found that YAP was upregulated and activated in astrocytes of C57BL/6 male mice after SCI in a Hippo pathway-dependent manner. Conditional knockout (KO) of yap in astrocytes significantly inhibited astrocytic proliferation, impaired the formation of glial scars, inhibited the axonal regeneration, and impaired the behavioral recovery of C57BL/6 male mice after SCI. Mechanistically, the bFGF was upregulated after SCI and induced the activation of YAP through RhoA pathways, thereby promoting the formation of glial scars. Additionally, YAP promoted bFGF-induced proliferation by negatively controlling nuclear distribution of p27 Kip1 mediated by CRM1. Finally, bFGF or XMU-MP-1 (an inhibitor of Hippo kinase MST1/2 to activate YAP) injection indeed activated YAP signaling and promoted the formation of glial scars and the functional recovery of mice after SCI. These findings suggest that YAP promotes the formation of glial scars and neural regeneration of mice after SCI, and that the bFGF-RhoA-YAP-p27 Kip1 pathway positively regulates astrocytic proliferation after SCI. SIGNIFICANCE STATEMENT Glial scars play critical roles in neuronal regeneration of CNS injury diseases, such as spinal cord injury (SCI). Here, we provide evidence for the function of Yes-associated protein (YAP) in the formation of glial scars after SCI through regulation of astrocyte proliferation. As a downstream of bFGF (which is upregulated after SCI), YAP promotes the proliferation of astrocytes through negatively controlling nuclear distribution of p27 Kip1 mediated by CRM1. Activation of YAP by bFGF or XMU-MP-1 injection promotes the formation of glial scar and the functional recovery of mice after SCI. These results suggest that the bFGF-RhoA-YAP-p27 Kip1 axis for the formation of glial scars may be a potential therapeutic strategy for SCI patients., (Copyright © 2020 the authors.)