TYROBP/DAP12 knockout in Huntington's disease Q175 mice cell-autonomously decreases microglial expression of disease-associated genes and non-cell-autonomously mitigates astrogliosis and motor deterioration.

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.

Retinal dysfunction in Huntington's disease mouse models concurs with local gliosis and microglia activation.

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.

The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina.

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 Raxflox/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.

Neuronal APOE4 removal protects against tau-mediated gliosis, neurodegeneration and myelin deficits.

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.

SRY-box transcription factor 9 modulates Müller cell gliosis in diabetic retinopathy by upregulating TXNIP transcription.

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-1ß, 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.

Altered energy metabolism in Fatal Familial Insomnia cerebral organoids is associated with astrogliosis and neuronal dysfunction.

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.

Overexpressed ski efficiently promotes neurorestoration, increases neuronal regeneration, and reduces astrogliosis after traumatic brain injury.

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.

APOE2 Exacerbates TDP-43 Related Toxicity in the Absence of Alzheimer Pathology.

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.

Tenascin-C restricts reactive astrogliosis in the ischemic brain.

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.

Neuron-specific chromosomal megadomain organization is adaptive to recent retrotransposon expansions.

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 B2NeuN+ 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 B2NeuN+ contact frequencies at sites with ongoing insertions in Mus musculus. Neuronal ablation of the retrotransposon silencer Kmt1e/Setdb1 triggers B2NeuN+ 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.

Short-term high-fat diet favors the appearances of apoptosis and gliosis by activation of ERK1/2/p38MAPK pathways in brain.

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-1ß, 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-1ß 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-1ß production as well as gliosis in cerebral cortex and cerebellum, which may be related to enhancement of ERK1/2 and p38 MAPK activation.

High-fat diet increases gliosis and immediate early gene expression in APOE3 mice, but not APOE4 mice.

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.

Intramuscular insulin-like growth factor-1 gene therapy modulates reactive microglia after traumatic brain injury.

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.

RGC-32 Acts as a Hub to Regulate the Transcriptomic Changes Associated With Astrocyte Development and Reactive Astrocytosis.

Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-ß 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-ß. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-ß-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.

Alpha-synuclein pathology, microgliosis, and parvalbumin neuron loss in the amygdala associated with enhanced fear in the Thy1-aSyn model of Parkinson's disease.

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.

Analysis of Both Lipid Metabolism and Endocannabinoid Signaling Reveals a New Role for Hypothalamic Astrocytes in Maternal Caloric Restriction-Induced Perinatal Programming.

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.

Different Flavors of Astrocytes: Revising the Origins of Astrocyte Diversity and Epigenetic Signatures to Understand Heterogeneity after Injury.

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.

Acyl-CoA synthetase 6 is required for brain docosahexaenoic acid retention and neuroprotection during aging.

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.

The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery.

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 Gcdhki/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 Gcdhki/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. Gcdhki/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 Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/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, Gcdhki/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 Gcdhki/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.

Neuroinflammatory Response to TNFα and IL1ß Cytokines Is Accompanied by an Increase in Glycolysis in Human Astrocytes In Vitro.

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 1ß (IL1ß) 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.