RESUMEN
Polypyrimidine tract-binding protein 1 (PTBP1) regulates numerous alternative splicing events during tumor progression and neurogenesis. Previously, PTBP1 downregulation was reported to convert astrocytes into functional neurons; however, how PTBP1 regulates astrocytic physiology remains unclear. In this study, we revealed that PTBP1 modulated glutamate uptake via ATP1a2, a member of Na+/K+-ATPases, and glutamate transporters in astrocytes. Ptbp1 knockdown altered mitochondrial function and energy metabolism, which involved PTBP1 regulating mitochondrial redox homeostasis via the succinate dehydrogenase (SDH)/Nrf2 pathway. The malfunction of glutamate transporters following Ptbp1 knockdown resulted in enhanced excitatory synaptic transmission in the cortex. Notably, we developed a biomimetic cationic triblock polypeptide system, i.e., polyethylene glycol44-polylysine30-polyleucine10 (PEG44-PLL30-PLLeu10) with astrocytic membrane coating to deliver Ptbp1 siRNA in vitro and in vivo, which approach allowed Ptbp1 siRNA to efficiently cross the blood-brain barrier and target astrocytes in the brain. Collectively, our findings suggest a framework whereby PTBP1 serves as a modulator in glutamate transport machinery, and indicate that biomimetic methodology is a promising route for in vivo siRNA delivery.
Asunto(s)
Astrocitos , Ácido Glutámico , Ribonucleoproteínas Nucleares Heterogéneas , Homeostasis , Factor 2 Relacionado con NF-E2 , Proteína de Unión al Tracto de Polipirimidina , ARN Interferente Pequeño , Animales , Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Factor 2 Relacionado con NF-E2/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ratones , Transducción de Señal , Membrana Celular/metabolismo , Ratones Endogámicos C57BL , Masculino , Humanos , Mitocondrias/metabolismoRESUMEN
Parkinson's disease (PD) is characterized by astrocyte activation and disruptions in circadian rhythm. Within the astrocyte population, two distinct reactive states exist: A1 and A2. A1 astrocytes are associated with neurotoxicity and inflammation, while A2 astrocytes exhibit neuroprotective functions. Our investigation focused on the role of REV-ERBα, a member of the nuclear receptor superfamily and a key regulator of the circadian clock, in astrocyte activation. We observed that REV-ERBα expression in A1 astrocytes was reduced to one-third of its normal level. Notably, activation of REV-ERBα prompted a transformation of astrocytes from A1 to A2. Mechanistically, REV-ERBα inhibition was linked to the classical NF-κB pathway, while it concurrently suppressed the STAT3 pathway. Furthermore, astrocytes with low REV-ERBα expression were associated with dopaminergic neurons apoptosis. Intriguingly, the opposite effect was observed when using a REV-ERBα agonist, which mitigated astrocyte activation and reduced dopaminergic neuron damage by 50%. In summary, our study elucidates the pivotal role of REV-ERBα in modulating astrocyte function and its potential implications in PD pathogenesis.
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Astrocitos , Neuronas Dopaminérgicas , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Animales , Neuronas Dopaminérgicas/metabolismo , Ratones , Células Cultivadas , FN-kappa B/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Apoptosis , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology.
Asunto(s)
Astrocitos , Corteza Cerebral , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Ratones Noqueados , Transducción de Señal , Animales , Astrocitos/metabolismo , Ratones , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Apirasa/genética , Apirasa/metabolismo , Células Cultivadas , Adenosina Trifosfato/metabolismo , Medios de Cultivo Condicionados , Adenosina/metabolismo , Adenosina/análogos & derivados , Receptores Purinérgicos P2Y/metabolismo , Receptores Purinérgicos P2Y/genética , Ratones Endogámicos C57BL , Antígenos CDRESUMEN
Sepsis-associated encephalopathy (SAE) is the most common neurological complication of sepsis, with an incidence of up to 70% in sepsis, and contributes to the increased mortality and disability in sepsis. To date, the exact pathogenesis of SAE is not clear. Most of current researches indicated that blood-brain barrier (BBB) dysfunction, active neuroinflammation, glial cell over activation as well as cerebral microcirculation dysfunction contributed to the pathophysiology of SAE. BBB, as a complex cellular structure between the central nervous system and the peripheral system, strictly controls the entrance and discharge of substances and plays an important role in maintaining the balance between biochemical system and immune system of central system. During the progress of sepsis, inflammatory cytokines and reactive oxygen species resulting from peripheral system directly or indirectly resulted in the damage to the integrity and structure of BBB, which helped above species easily enter into the central system. Above these damages caused glial cell activation (microglia and astrocyte), the imbalance of neurotransmitters, mitochondrial dysfunction and neural apoptosis, which also reversely contributed to the damage to the integrity and permeability of BBB via decreasing the expression of tight junctional protein between cells. Therefore, this review focuses on the structural and functional changes of BBB in SAE, and how these changes lead to the development of SAE, in order to seek a BBB-targeted therapy for SAE.
Asunto(s)
Barrera Hematoencefálica , Encefalopatía Asociada a la Sepsis , Sepsis , Humanos , Encefalopatía Asociada a la Sepsis/fisiopatología , Sepsis/complicaciones , Sepsis/fisiopatología , Animales , Especies Reactivas de Oxígeno/metabolismo , Citocinas/metabolismo , Astrocitos/metabolismoRESUMEN
AIMS: Type 2 diabetes mellitus (T2DM) is related to an increased risk of postoperative cognitive dysfunction (POCD), which may be caused by neuronal hyperexcitability. Astrocyte glutamate transporter 1 (GLT-1) plays a crucial role in regulating neuron excitability. We investigated if T2DM would magnify the increased neuronal excitability induced by anesthesia/surgery (A/S) and lead to POCD in young adult mice, and if so, determined whether these effects were associated with GLT-1 expression. METHODS: T2DM model was induced by high fat diet (HFD) and injecting STZ. Then, we evaluated the spatial learning and memory of T2DM mice after A/S with the novel object recognition test (NORT) and object location test (OLT). Western blotting and immunofluorescence were used to analyze the expression levels of GLT-1 and neuronal excitability. Oxidative stress reaction and neuronal apoptosis were detected with SOD2 expression, MMP level, and Tunel staining. Hippocampal functional synaptic plasticity was assessed with long-term potentiation (LTP). In the intervention study, we overexpressed hippocampal astrocyte GLT-1 in GFAP-Cre mice. Besides, AAV-Camkllα-hM4Di-mCherry was injected to inhibit neuronal hyperexcitability in CA1 region. RESULTS: Our study found T2DM but not A/S reduced GLT-1 expression in hippocampal astrocytes. Interestingly, GLT-1 deficiency alone couldn't lead to cognitive decline, but the downregulation of GLT-1 in T2DM mice obviously enhanced increased hippocampal glutamatergic neuron excitability induced by A/S. The hyperexcitability caused neuronal apoptosis and cognitive impairment. Overexpression of GLT-1 rescued postoperative cognitive dysfunction, glutamatergic neuron hyperexcitability, oxidative stress reaction, and apoptosis in hippocampus. Moreover, chemogenetic inhibition of hippocampal glutamatergic neurons reduced oxidative stress and apoptosis and alleviated postoperative cognitive dysfunction. CONCLUSIONS: These findings suggest that the adult mice with type 2 diabetes are at an increased risk of developing POCD, perhaps due to the downregulation of GLT-1 in hippocampal astrocytes, which enhances increased glutamatergic neuron excitability induced by A/S and leads to oxidative stress reaction, and neuronal apoptosis.
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Astrocitos , Diabetes Mellitus Tipo 2 , Regulación hacia Abajo , Transportador 2 de Aminoácidos Excitadores , Hipocampo , Ratones Endogámicos C57BL , Complicaciones Cognitivas Postoperatorias , Animales , Transportador 2 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/biosíntesis , Transportador 2 de Aminoácidos Excitadores/genética , Astrocitos/metabolismo , Complicaciones Cognitivas Postoperatorias/etiología , Complicaciones Cognitivas Postoperatorias/metabolismo , Hipocampo/metabolismo , Ratones , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Masculino , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa/efectos adversos , Ratones TransgénicosRESUMEN
Reactive astrocytes play critical roles in the occurrence of various neurological diseases such as multiple sclerosis. Activation of astrocytes is often accompanied by a glycolysis-dominant metabolic switch. However, the role and molecular mechanism of metabolic reprogramming in activation of astrocytes have not been clarified. Here, we found that PKM2, a rate-limiting enzyme of glycolysis, displayed nuclear translocation in astrocytes of EAE (experimental autoimmune encephalomyelitis) mice, an animal model of multiple sclerosis. Prevention of PKM2 nuclear import by DASA-58 significantly reduced the activation of mice primary astrocytes, which was observed by decreased proliferation, glycolysis and secretion of inflammatory cytokines. Most importantly, we identified the ubiquitination-mediated regulation of PKM2 nuclear import by ubiquitin ligase TRIM21. TRIM21 interacted with PKM2, promoted its nuclear translocation and stimulated its nuclear activity to phosphorylate STAT3, NF-κB and interact with c-myc. Further single-cell RNA sequencing and immunofluorescence staining demonstrated that TRIM21 expression was upregulated in astrocytes of EAE. TRIM21 overexpressing in mice primary astrocytes enhanced PKM2-dependent glycolysis and proliferation, which could be reversed by DASA-58. Moreover, intracerebroventricular injection of a lentiviral vector to knockdown TRIM21 in astrocytes or intraperitoneal injection of TEPP-46, which inhibit the nuclear translocation of PKM2, effectively decreased disease severity, CNS inflammation and demyelination in EAE. Collectively, our study provides novel insights into the pathological function of nuclear glycolytic enzyme PKM2 and ubiquitination-mediated regulatory mechanism that are involved in astrocyte activation. Targeting this axis may be a potential therapeutic strategy for the treatment of astrocyte-involved neurological disease.
Asunto(s)
Astrocitos , Encefalomielitis Autoinmune Experimental , Ribonucleoproteínas , Regulación hacia Arriba , Animales , Astrocitos/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Ratones , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hormonas Tiroideas/metabolismo , Hormonas Tiroideas/genética , Proteínas de Unión a Hormona Tiroide , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ratones Endogámicos C57BL , Piruvato Quinasa/metabolismo , Piruvato Quinasa/genética , Transporte Activo de Núcleo Celular , Femenino , Glucólisis , Ubiquitinación , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Núcleo Celular/metabolismoRESUMEN
OBJECTIVE: The objective of this study is to investigate the presence of astrocyte antibodies in patients, excluding aquaporin-4 or glial fibrillary acidic protein (GFAP) antibodies, while evaluating associated biomarkers and pathologies. METHODS: Patient serum and cerebrospinal fluid (CSF) were tested for antibodies using tissue- and cell-based assays. Neurofilament light chain (NFL) and GFAP in the CSF were detected using single-molecule array (SIMOA). RESULTS: 116 patients accepted SIMOA. Fifteen functional neurological disorders patients without antibodies were designated as controls. Thirty-five patients were positive for astrocyte antibodies (Anti-GFAP: 7; Anti-AQP4: 7; unknown antibodies: 21, designed as the double-negative group, DNAP). The most frequent phenotype of DNAP was encephalitis (42.9%), followed by myelitis (23.8%), movement disorders (19.0%), and amyotrophic lateral sclerosis-like (ALS-like) disease (14.2%). The levels of CSF GFAP and NFL in DNAP were higher than in the control (GFAP: 1967.29 [776.60-13214.47] vs 475.38 [16.80-943.60] pg/mL, p < 0.001; NFL: 549.11 [162.08-2462.61] vs 214.18 [81.60-349.60] pg/mL, p = 0.002). GFAP levels decreased in DNAP (n = 5) after immunotherapy (2446.75 [1583.45-6277.33] vs 1380.46 [272.16-2005.80] pg/mL, p = 0.043), while there was no difference in NFL levels (2273.78 [162.08-2462.61] vs 890.42 [645.06-3168.06] pg/mL, p = 0.893). Two brain biopsy patterns were observed: one exhibited prominent tissue proliferation and hypertrophic astrocytes, with local loss of astrocytes, while the other showed severe astrocyte depletion with loss of neurofilaments around the vessels. Eighteen patients received immunotherapy, and improved except one with ALS-like symptoms. We identified anti-vimentin in this patient. DISCUSSION: There are unidentified astrocyte antibodies. The manifestations of double-negativity are heterogeneous; nevertheless, the pathology and biomarkers remain consistent with astrocytopathy. Immunotherapy is effective.
Asunto(s)
Acuaporina 4 , Astrocitos , Biomarcadores , Proteína Ácida Fibrilar de la Glía , Inmunoglobulina G , Humanos , Proteína Ácida Fibrilar de la Glía/líquido cefalorraquídeo , Proteína Ácida Fibrilar de la Glía/inmunología , Femenino , Masculino , Acuaporina 4/inmunología , Persona de Mediana Edad , Astrocitos/inmunología , Astrocitos/metabolismo , Astrocitos/patología , Estudios Retrospectivos , Adulto , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/sangre , Anciano , Inmunoglobulina G/líquido cefalorraquídeo , Inmunoglobulina G/sangre , Proteínas de Neurofilamentos/líquido cefalorraquídeo , Proteínas de Neurofilamentos/sangre , Autoanticuerpos/líquido cefalorraquídeo , Autoanticuerpos/sangre , Adulto Joven , AdolescenteRESUMEN
The therapeutic potential of suppressing polypyrimidine tract-binding protein 1 (Ptbp1) messenger RNA by viral transduction in a post-stroke dementia mouse model has not yet been examined. In this study, 3 days after cerebral ischemia, we injected a viral vector cocktail containing adeno-associated virus (AAV)-pGFAP-mCherry and AAV-pGFAP-CasRx (control vector) or a cocktail of AAV-pGFAP-mCherry and AAV-pGFAP-CasRx-SgRNA-(Ptbp1) (1:5, 1.0 × 1011 viral genomes) into post-stroke mice via the tail vein. We observed new mCherry/NeuN double-positive neuron-like cells in the hippocampus 56 days after cerebral ischemia. A portion of mCherry/GFAP double-positive astrocyte-like glia could have been converted into new mCherry/NeuN double-positive neuron-like cells with morphological changes. The new neuronal cells integrated into the dentate gyrus and recognition memory was significantly ameliorated. These results demonstrated that the in vivo conversion of hippocampal astrocyte-like glia into functional new neurons by the suppression of Ptbp1 might be a therapeutic strategy for post-stroke dementia.
Asunto(s)
Astrocitos , Isquemia Encefálica , Modelos Animales de Enfermedad , Ribonucleoproteínas Nucleares Heterogéneas , Hipocampo , Neurogénesis , Proteína de Unión al Tracto de Polipirimidina , Animales , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Astrocitos/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Ratones , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Masculino , Neuronas/metabolismo , Memoria , Ratones Endogámicos C57BL , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificaciónRESUMEN
Multiple sclerosis (MS) is a debilitating demyelinating disease characterized by remyelination failure attributed to inadequate oligodendrocyte precursor cells (OPCs) differentiation and aberrant astrogliosis. A comprehensive cell atlas reanalysis of clinical specimens brings to light heightened clusterin (CLU) expression in a specific astrocyte subtype links to active lesions in MS patients. Our investigation reveals elevated astrocytic CLU levels in both active lesions of patient tissues and female murine MS models. CLU administration stimulates primary astrocyte proliferation while concurrently impeding astrocyte-mediated clearance of myelin debris. Intriguingly, CLU overload directly impedes OPC differentiation and induces OPCs and OLs apoptosis. Mechanistically, CLU suppresses PI3K-AKT signaling in primary OPCs via very low-density lipoprotein receptor. Pharmacological activation of AKT rescues the damage inflicted by excess CLU on OPCs and ameliorates demyelination in the corpus callosum. Furthermore, conditional knockout of CLU emerges as a promising intervention, showcasing improved remyelination processes and reduced severity in murine MS models.
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Astrocitos , Clusterina , Enfermedades Desmielinizantes , Modelos Animales de Enfermedad , Remielinización , Animales , Femenino , Humanos , Ratones , Apoptosis/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Clusterina/metabolismo , Clusterina/genética , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Vaina de Mielina/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Células Precursoras de Oligodendrocitos/efectos de los fármacos , Oligodendroglía/metabolismo , Oligodendroglía/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Remielinización/efectos de los fármacos , Transducción de SeñalRESUMEN
Purpose: To investigate the neuroplasticity hypothesis of depression by measuring brain-derived neurotrophic factor (BDNF) levels in plasma astrocyte-derived extracellular vesicles (ADEVs) and to evaluate their potential as biomarkers for depression compared with plasma BDNF levels. Patients and Methods: Thirty-five patients with major depressive disorder (MDD) and 35 matched healthy controls (HCs) were enrolled. Plasma ADEVs were isolated using a combination of ultracentrifugation and immunoaffinity capture. Isolated ADEVs were validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. BDNF levels were quantified in both ADEVs and plasma. ALG-2-interacting protein X (Alix) and cluster of differentiation 81 (CD81) levels, two established extracellular vesicle markers, were measured in ADEVs. Results: After false discovery rate correction, patients with MDD exhibited higher CD81 levels (P FDR = 0.040) and lower BDNF levels (P FDR = 0.043) in ADEVs than HCs at baseline. BDNF levels in ADEVs normalized to CD81 (P FDR = 0.002) and Alix (P FDR = 0.040) remained consistent with this finding. Following four weeks of selective serotonin reuptake inhibitor treatment (n=10), CD81 levels in ADEVs decreased (P FDR = 0.046), while BDNF levels normalized to CD81 increased (P FDR = 0.022). BDNF levels in ADEVs were more stable than in plasma. Exploratory analysis revealed no correlation between BDNF levels in ADEVs and plasma (ρ=0.117, P = 0.334). Conclusion: This study provides human in vivo evidence supporting the neuroplasticity hypothesis of depression by demonstrating altered BDNF levels in ADEVs. ADEVs may be more suitable for developing biomarkers of depression than plasma-derived biomarkers.
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Astrocitos , Biomarcadores , Factor Neurotrófico Derivado del Encéfalo , Trastorno Depresivo Mayor , Vesículas Extracelulares , Plasticidad Neuronal , Humanos , Factor Neurotrófico Derivado del Encéfalo/sangre , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Masculino , Femenino , Plasticidad Neuronal/fisiología , Adulto , Persona de Mediana Edad , Trastorno Depresivo Mayor/sangre , Trastorno Depresivo Mayor/metabolismo , Biomarcadores/sangre , Astrocitos/metabolismo , Tetraspanina 28/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Estudios de Casos y Controles , Proteínas de Unión al Calcio , Proteínas de Ciclo Celular , Complejos de Clasificación Endosomal Requeridos para el TransporteRESUMEN
Vitamin D deficiency is a global problem. Vitamin D, the vitamin D receptor, and its enzymes are found throughout neuronal, ependymal, and glial cells in the brain and are implicated in certain processes and mechanisms in the brain. To investigate the processes affected by vitamin D deficiency in adults, we studied vitamin D deficient, control, and supplemented diets over 6 weeks in male and female C57Bl/6 mice. The effect of the vitamin D diets on proliferation in the neurogenic niches, changes in glial cells, as well as on memory, locomotion, and anxiety-like behavior, was investigated. Six weeks on a deficient diet was adequate time to reach deficiency. However, vitamin D deficiency and supplementation did not affect proliferation, neurogenesis, or astrocyte changes, and this was reflected on behavioral measures. Supplementation only affected microglia in the dentate gyrus of female mice. Indicating that vitamin D deficiency and supplementation do not affect these processes over a 6-week period.
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Cognición , Suplementos Dietéticos , Ratones Endogámicos C57BL , Neurogénesis , Deficiencia de Vitamina D , Vitamina D , Animales , Deficiencia de Vitamina D/complicaciones , Femenino , Masculino , Vitamina D/farmacología , Ratones , Proliferación Celular , Conducta Animal , Astrocitos/metabolismo , Giro Dentado , Ansiedad , Encéfalo/metabolismo , MemoriaRESUMEN
The generation of new neurons at the hippocampal neurogenic niche, known as adult hippocampal neurogenesis (AHN), and its impairment, have been implicated in Alzheimer's disease (AD). MicroRNA-132 (miR-132), the most consistently downregulated microRNA (miRNA) in AD, was recently identified as a potent regulator of AHN, exerting multilayered proneurogenic effects in adult neural stem cells (NSCs) and their progeny. Supplementing miR-132 in AD mouse brain restores AHN and relevant memory deficits, yet the exact mechanisms involved are still unknown. Here, we identify NACC2 as a novel miR-132 target implicated in both AHN and AD. miR-132 deficiency in mouse hippocampus induces Nacc2 expression and inflammatory signaling in adult NSCs. We show that miR-132-dependent regulation of NACC2 is involved in the initial stages of human NSC differentiation towards astrocytes and neurons. Later, NACC2 function in astrocytic maturation becomes uncoupled from miR-132. We demonstrate that NACC2 is present in reactive astrocytes surrounding amyloid plaques in mouse and human AD hippocampus, and that there is an anticorrelation between miR-132 and NACC2 levels in AD and upon induction of inflammation. Unraveling the molecular mechanisms by which miR-132 regulates neurogenesis and cellular reactivity in AD, will provide valuable insights towards its possible application as a therapeutic target.
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Enfermedad de Alzheimer , Astrocitos , Hipocampo , MicroARNs , Células-Madre Neurales , Neurogénesis , MicroARNs/genética , MicroARNs/metabolismo , Neurogénesis/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Humanos , Células-Madre Neurales/metabolismo , Ratones , Hipocampo/metabolismo , Hipocampo/patología , Astrocitos/metabolismo , Neuronas/metabolismo , Diferenciación Celular , Regulación de la Expresión GénicaRESUMEN
Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion.
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Astrocitos , Reprogramación Celular , Corteza Cerebral , Dependovirus , Vectores Genéticos , Neuronas , Animales , Astrocitos/metabolismo , Neuronas/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Ratones , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Dependovirus/genética , Reprogramación Celular/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones Endogámicos C57BL , Masculino , Retroviridae/genéticaRESUMEN
Inflammation with expression of interleukin 6 (IL-6) in the central nervous system (CNS) occurs in several neurodegenerative/neuroinflammatory conditions and may cause neurochemical changes to endogenous neuroprotective systems. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two neuropeptides with well-established protective and anti-inflammatory properties. Yet, whether PACAP and VIP levels are altered in mice with CNS-restricted, astrocyte-targeted production of IL-6 (GFAP-IL6) remains unknown. In this study, PACAP/VIP levels were assessed in the brain of GFAP-IL6 mice. In addition, we utilised bi-genic GFAP-IL6 mice carrying the human sgp130-Fc transgene (termed GFAP-IL6/sgp130Fc mice) to determine whether trans-signalling inhibition rescued PACAP/VIP changes in the CNS. Transcripts and protein levels of PACAP and VIP, as well as their receptors PAC1, VPAC1 and VPAC2, were significantly increased in the cerebrum and cerebellum of GFAP-IL6 mice vs. wild type (WT) littermates. These results were paralleled by a robust activation of the JAK/STAT3, NF-κB and ERK1/2MAPK pathways in GFAP-IL6 mice. In contrast, co-expression of sgp130Fc in GFAP-IL6/sgp130Fc mice reduced VIP expression and activation of STAT3 and NF-κB pathways, but it failed to rescue PACAP, PACAP/VIP receptors and Erk1/2MAPK phosphorylation. We conclude that forced expression of IL-6 in astrocytes induces the activation of the PACAP/VIP neuropeptide system in the brain, which is only partly modulated upon IL-6 trans-signalling inhibition. Increased expression of PACAP/VIP neuropeptides and receptors may represent a homeostatic response of the CNS to an uncontrolled IL-6 synthesis and its neuroinflammatory consequences.
Asunto(s)
Encéfalo , Interleucina-6 , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Transducción de Señal , Péptido Intestinal Vasoactivo , Animales , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Interleucina-6/metabolismo , Interleucina-6/genética , Ratones , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/genética , Encéfalo/metabolismo , Astrocitos/metabolismo , Humanos , Ratones Transgénicos , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Sistema Nervioso Central/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Masculino , Ratones Endogámicos C57BLRESUMEN
Astrogliosis is a process by which astrocytes, when exposed to inflammation, exhibit hypertrophy, motility, and elevated expression of reactivity markers such as Glial Fibrillar Acidic Protein, Vimentin, and Connexin43. Since 1999, our laboratory in Chile has been studying molecular signaling pathways associated with "gliosis" and has reported that reactive astrocytes upregulate Syndecan 4 and αVß3 Integrin, which are receptors for the neuronal glycoprotein Thy-1. Thy-1 engagement stimulates adhesion and migration of reactive astrocytes and induces neurons to retract neurites, thus hindering neuronal network repair. Reportedly, we have used DITNC1 astrocytes and neuron-like CAD cells to study signaling mechanisms activated by the Syndecan 4-αVß3 Integrin/Thy-1 interaction. Importantly, the sole overexpression of ß3 Integrin in non-reactive astrocytes turns them into reactive cells. In vitro, extensive passaging is a simile for "aging", and aged fibroblasts have shown ß3 Integrin upregulation. However, it is not known if astrocytes upregulate ß3 Integrin after successive cell passages. Here, we hypothesized that astrocytes undergoing long-term passaging increase ß3 Integrin expression levels and behave as reactive astrocytes without needing pro-inflammatory stimuli. We used DITNC1 cells with different passage numbers to study reactivity markers using immunoblots, immunofluorescence, and astrocyte adhesion/migration assays. We also evaluated ß3 Integrin levels by immunoblot and flow cytometry, as well as the neurotoxic effects of reactive astrocytes. Serial cell passaging mimicked the effects of inflammatory stimuli, inducing astrocyte reactivity. Indeed, in response to Thy-1, ß3 Integrin levels, as well as cell adhesion and migration, gradually increased with multiple passages. Importantly, these long-lived astrocytes expressed and secreted factors that inhibited neurite outgrowth and caused neuronal death, just like reactive astrocytes in culture. Therefore, we describe two DITNC1 cell types: a non-reactive type that can be activated with Tumor Necrosis Factor (TNF) and another one that exhibits reactive astrocyte features even in the absence of TNF treatment. Our results emphasize the importance of passage numbers in cell behavior. Likewise, we compare the pro-inflammatory stimulus versus long-term in-plate passaging of cell cultures and introduce them as astrocyte models to study the reactivity process.
Asunto(s)
Astrocitos , Adhesión Celular , Movimiento Celular , Gliosis , Astrocitos/metabolismo , Gliosis/metabolismo , Gliosis/patología , Animales , Antígenos Thy-1/metabolismo , Integrina alfaVbeta3/metabolismo , Inflamación/metabolismo , Inflamación/patología , Sindecano-4/metabolismo , Sindecano-4/genética , Ratones , Línea Celular , Humanos , Células Cultivadas , Transducción de SeñalRESUMEN
Even though several highly effective treatments have been developed for multiple sclerosis (MS), the underlying pathological mechanisms and drivers of the disease have not been fully elucidated. In recent years, there has been a growing interest in studying neuroinflammation in the context of glial cell involvement as there is increasing evidence of their central role in disease progression. Although glial cell communication and proper function underlies brain homeostasis and maintenance, their multiple effects in an MS brain remain complex and controversial. In this review, we aim to provide an overview of the contribution of glial cells, oligodendrocytes, astrocytes, and microglia in the pathology of MS during both the activation and orchestration of inflammatory mechanisms, as well as of their synergistic effects during the repair and restoration of function. Additionally, we discuss how the understanding of glial cell involvement in MS may provide new therapeutic targets either to limit disease progression or to facilitate repair.
Asunto(s)
Esclerosis Múltiple , Neuroglía , Enfermedades Neuroinflamatorias , Humanos , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Neuroglía/metabolismo , Neuroglía/patología , Animales , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Microglía/metabolismo , Microglía/patología , Astrocitos/metabolismo , Astrocitos/patología , Oligodendroglía/metabolismo , Oligodendroglía/patología , Encéfalo/metabolismo , Encéfalo/patologíaRESUMEN
Depression is a prevalent and debilitating mental disorder that affects millions worldwide. Current treatments, such as antidepressants targeting the serotonergic system, have limitations, including delayed onset of action and high rates of treatment resistance, necessitating novel therapeutic strategies. Ginsenoside Rc (G-Rc) has shown potential anti-inflammatory and neuroprotective effects, but its antidepressant properties remain unexplored. This study investigated the antidepressant effects of G-Rc in an L-alpha-aminoadipic acid (L-AAA)-induced mouse model of depression, which mimics the astrocytic pathology and neuroinflammation observed in major depressive disorder. Mice were administered G-Rc, vehicle, or imipramine orally after L-AAA injection into the prefrontal cortex. G-Rc significantly reduced the immobility time in forced swimming and tail suspension tests compared to vehicle treatment, with more pronounced effects than imipramine. It also attenuated the expression of pro-inflammatory cytokines (TNF-α, IL-6, TGF-ß, lipocalin-2) and alleviated astrocytic degeneration, as indicated by increased GFAP and decreased IBA-1 levels. Additionally, G-Rc modulated apoptosis-related proteins, decreasing caspase-3 and increasing Bcl-2 levels compared to the L-AAA-treated group. These findings suggest that G-Rc exerts antidepressant effects by regulating neuroinflammation, astrocyte-microglia crosstalk, and apoptotic pathways in the prefrontal cortex, highlighting its potential as a novel therapeutic agent for depression.
Asunto(s)
Ácido 2-Aminoadípico , Antidepresivos , Astrocitos , Ginsenósidos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Ratones , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Ginsenósidos/farmacología , Masculino , Ácido 2-Aminoadípico/farmacología , Depresión/tratamiento farmacológico , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Modelos Animales de Enfermedad , Citocinas/metabolismo , Ratones Endogámicos C57BL , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Apoptosis/efectos de los fármacosRESUMEN
Agathisflavone is a flavonoid that exhibits anti-inflammatory and anti-oxidative properties. Here, we investigated the neuroprotective effects of agathisflavone on central nervous system (CNS) neurons and glia in the cerebellar slice ex vivo model of neonatal ischemia. Cerebellar slices from neonatal mice, in which glial fibrillary acidic protein (GFAP) and SOX10 drive expression of enhanced green fluorescent protein (EGFP), were used to identify astrocytes and oligodendrocytes, respectively. Agathisflavone (10 µM) was administered preventively for 60 min before inducing ischemia by oxygen and glucose deprivation (OGD) for 60 min and compared to controls maintained in normal oxygen and glucose (OGN). The density of SOX-10+ oligodendrocyte lineage cells and NG2 immunopositive oligodendrocyte progenitor cells (OPCs) were not altered in OGD, but it resulted in significant oligodendroglial cell atrophy marked by the retraction of their processes, and this was prevented by agathisflavone. OGD caused marked axonal demyelination, determined by myelin basic protein (MBP) and neurofilament (NF70) immunofluorescence, and this was blocked by agathisflavone preventative treatment. OGD also resulted in astrocyte reactivity, exhibited by increased GFAP-EGFP fluorescence and decreased expression of glutamate synthetase (GS), and this was prevented by agathisflavone pretreatment. In addition, agathisflavone protected Purkinje neurons from ischemic damage, assessed by calbindin (CB) immunofluorescence. The results demonstrate that agathisflavone protects neuronal and myelin integrity in ischemia, which is associated with the modulation of glial responses in the face of ischemic damage.
Asunto(s)
Animales Recién Nacidos , Cerebelo , Flavonoides , Fármacos Neuroprotectores , Animales , Fármacos Neuroprotectores/farmacología , Ratones , Cerebelo/metabolismo , Cerebelo/efectos de los fármacos , Flavonoides/farmacología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Glucosa/metabolismo , BiflavonoidesRESUMEN
Fructose 1,6-bisphosphatase 2 (Fbp2) is a regulatory enzyme of gluco- and glyconeogenesis which, in the course of evolution, acquired non-catalytic functions. Fbp2 promotes cell survival during calcium stress, regulates glycolysis via inhibition of Hif-1α activity, and is indispensable for the formation of long-term potentiation in hippocampus. In hippocampal astrocytes, the amount of Fbp2 protein is reduced by signals delivered in neuronal extracellular vesicles (NEVs) through an unknown mechanism. The physiological role of Fbp2 (determined by its subcellular localization/interactions) depends on its oligomeric state and thus, we asked whether the cargo of NEVs is sufficient to change also the ratio of Fbp2 dimer/tetramer and, consequently, influence astrocyte basal metabolism. We found that the NEVs cargo reduced the Fbp2 mRNA level, stimulated the enzyme degradation and affected the cellular titers of different oligomeric forms of Fbp2. This was accompanied with increased glucose uptake and lactate release by astrocytes. Our results revealed that neuronal signals delivered to astrocytes in NEVs provide the necessary balance between enzymatic and non-enzymatic functions of Fbp2, influencing not only its amount but also subcellular localization. This may allow for the metabolic adjustments and ensure protection of mitochondrial membrane potential during the neuronal activity-related increase in astrocytic [Ca2+].
Asunto(s)
Astrocitos , Vesículas Extracelulares , Fructosa-Bifosfatasa , Glucólisis , Neuronas , Astrocitos/metabolismo , Animales , Vesículas Extracelulares/metabolismo , Neuronas/metabolismo , Fructosa-Bifosfatasa/metabolismo , Fructosa-Bifosfatasa/genética , Hipocampo/metabolismo , Hipocampo/citología , Ratas , Glucosa/metabolismo , Células Cultivadas , Proteolisis , Multimerización de ProteínaRESUMEN
Astrocytes, the predominant glial cells in the central nervous system, play essential roles in maintaining brain function. Reprogramming induced pluripotent stem cells (iPSCs) to become astrocytes through overexpression of the transcription factors, NFIB and SOX9, is a rapid and efficient approach for studying human neurological diseases and identifying therapeutic targets. However, the precise differentiation path and molecular signatures of induced astrocytes remain incompletely understood. Accordingly, we performed single-cell RNA sequencing analysis on 64,736 cells to establish a comprehensive atlas of NFIB/SOX9-directed astrocyte differentiation from human iPSCs. Our dataset provides detailed information about the path of astrocyte differentiation, highlighting the stepwise molecular changes that occur throughout the differentiation process. This dataset serves as a valuable reference for dissecting uncharacterized transcriptomic features of NFIB/SOX9-induced astrocytes and investigating lineage progression during astrocyte differentiation. Moreover, these findings pave the way for future studies on neurological diseases using the NFIB/SOX9-induced astrocyte model.