RESUMO
SARS-CoV-2 infects less than 1% of cells in the human body, yet it can cause severe damage in a variety of organs. Thus, deciphering the non-cell-autonomous effects of SARS-CoV-2 infection is imperative for understanding the cellular and molecular disruption it elicits. Neurological and cognitive defects are among the least understood symptoms of COVID-19 patients, with olfactory dysfunction being their most common sensory deficit. Here, we show that both in humans and hamsters, SARS-CoV-2 infection causes widespread downregulation of olfactory receptors (ORs) and of their signaling components. This non-cell-autonomous effect is preceded by a dramatic reorganization of the neuronal nuclear architecture, which results in dissipation of genomic compartments harboring OR genes. Our data provide a potential mechanism by which SARS-CoV-2 infection alters the cellular morphology and the transcriptome of cells it cannot infect, offering insight to its systemic effects in olfaction and beyond.
Assuntos
Anosmia , COVID-19 , Animais , Cricetinae , Regulação para Baixo , Humanos , Receptores Odorantes , SARS-CoV-2 , OlfatoRESUMO
Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAPTg;Gfap+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAPTg;Gfap+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;Gfap+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap+ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;Gfap+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAPTg;Gfap+/R236H mice.
Assuntos
Doença de Alexander , Doença de Alexander/genética , Doença de Alexander/metabolismo , Doença de Alexander/patologia , Animais , Astrócitos/metabolismo , Modelos Animais de Doenças , Gliose/metabolismo , Gliose/patologia , Humanos , Camundongos , Camundongos Transgênicos , Mutação , ProteômicaRESUMO
Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with acute and postacute cognitive and neuropsychiatric symptoms including impaired memory, concentration, attention, sleep and affect. Mechanisms underlying these brain symptoms remain understudied. Here we report that SARS-CoV-2-infected hamsters exhibit a lack of viral neuroinvasion despite aberrant blood-brain barrier permeability. Hamsters and patients deceased from coronavirus disease 2019 (COVID-19) also exhibit microglial activation and expression of interleukin (IL)-1ß and IL-6, especially within the hippocampus and the medulla oblongata, when compared with non-COVID control hamsters and humans who died from other infections, cardiovascular disease, uraemia or trauma. In the hippocampal dentate gyrus of both COVID-19 hamsters and humans, we observed fewer neuroblasts and immature neurons. Protracted inflammation, blood-brain barrier disruption and microglia activation may result in altered neurotransmission, neurogenesis and neuronal damage, explaining neuropsychiatric presentations of COVID-19. The involvement of the hippocampus may explain learning, memory and executive dysfunctions in COVID-19 patients.
Assuntos
COVID-19 , Humanos , Citocinas , SARS-CoV-2 , Hipocampo , Neurogênese/fisiologiaRESUMO
Post-zygotically acquired genetic variants, or somatic variants, that arise during cortical development have emerged as important causes of focal epilepsies, particularly those due to malformations of cortical development. Pathogenic somatic variants have been identified in many genes within the PI3K-AKT-mTOR-signalling pathway in individuals with hemimegalencephaly and focal cortical dysplasia (type II), and more recently in SLC35A2 in individuals with focal cortical dysplasia (type I) or non-dysplastic epileptic cortex. Given the expanding role of somatic variants across different brain malformations, we sought to delineate the landscape of somatic variants in a large cohort of patients who underwent epilepsy surgery with hemimegalencephaly or focal cortical dysplasia. We evaluated samples from 123 children with hemimegalencephaly (n = 16), focal cortical dysplasia type I and related phenotypes (n = 48), focal cortical dysplasia type II (n = 44), or focal cortical dysplasia type III (n = 15). We performed high-depth exome sequencing in brain tissue-derived DNA from each case and identified somatic single nucleotide, indel and large copy number variants. In 75% of individuals with hemimegalencephaly and 29% with focal cortical dysplasia type II, we identified pathogenic variants in PI3K-AKT-mTOR pathway genes. Four of 48 cases with focal cortical dysplasia type I (8%) had a likely pathogenic variant in SLC35A2. While no other gene had multiple disease-causing somatic variants across the focal cortical dysplasia type I cohort, four individuals in this group had a single pathogenic or likely pathogenic somatic variant in CASK, KRAS, NF1 and NIPBL, genes previously associated with neurodevelopmental disorders. No rare pathogenic or likely pathogenic somatic variants in any neurological disease genes like those identified in the focal cortical dysplasia type I cohort were found in 63 neurologically normal controls (P = 0.017), suggesting a role for these novel variants. We also identified a somatic loss-of-function variant in the known epilepsy gene, PCDH19, present in a small number of alleles in the dysplastic tissue from a female patient with focal cortical dysplasia IIIa with hippocampal sclerosis. In contrast to focal cortical dysplasia type II, neither focal cortical dysplasia type I nor III had somatic variants in genes that converge on a unifying biological pathway, suggesting greater genetic heterogeneity compared to type II. Importantly, we demonstrate that focal cortical dysplasia types I, II and III are associated with somatic gene variants across a broad range of genes, many associated with epilepsy in clinical syndromes caused by germline variants, as well as including some not previously associated with radiographically evident cortical brain malformations.
Assuntos
Epilepsia , Hemimegalencefalia , Malformações do Desenvolvimento Cortical , Caderinas , Proteínas de Ciclo Celular , Feminino , Humanos , Malformações do Desenvolvimento Cortical do Grupo I , Mutação , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Protocaderinas , Serina-Treonina Quinases TORRESUMO
Many patients with SARS-CoV-2 infection develop neurological signs and symptoms; although, to date, little evidence exists that primary infection of the brain is a significant contributing factor. We present the clinical, neuropathological and molecular findings of 41 consecutive patients with SARS-CoV-2 infections who died and underwent autopsy in our medical centre. The mean age was 74 years (38-97 years), 27 patients (66%) were male and 34 (83%) were of Hispanic/Latinx ethnicity. Twenty-four patients (59%) were admitted to the intensive care unit. Hospital-associated complications were common, including eight patients (20%) with deep vein thrombosis/pulmonary embolism, seven (17%) with acute kidney injury requiring dialysis and 10 (24%) with positive blood cultures during admission. Eight (20%) patients died within 24 h of hospital admission, while 11 (27%) died more than 4 weeks after hospital admission. Neuropathological examination of 20-30 areas from each brain revealed hypoxic/ischaemic changes in all brains, both global and focal; large and small infarcts, many of which appeared haemorrhagic; and microglial activation with microglial nodules accompanied by neuronophagia, most prominently in the brainstem. We observed sparse T lymphocyte accumulation in either perivascular regions or in the brain parenchyma. Many brains contained atherosclerosis of large arteries and arteriolosclerosis, although none showed evidence of vasculitis. Eighteen patients (44%) exhibited pathologies of neurodegenerative diseases, which was not unexpected given the age range of our patients. We examined multiple fresh frozen and fixed tissues from 28 brains for the presence of viral RNA and protein, using quantitative reverse-transcriptase PCR, RNAscope® and immunocytochemistry with primers, probes and antibodies directed against the spike and nucleocapsid regions. The PCR analysis revealed low to very low, but detectable, viral RNA levels in the majority of brains, although they were far lower than those in the nasal epithelia. RNAscope® and immunocytochemistry failed to detect viral RNA or protein in brains. Our findings indicate that the levels of detectable virus in coronavirus disease 2019 brains are very low and do not correlate with the histopathological alterations. These findings suggest that microglial activation, microglial nodules and neuronophagia, observed in the majority of brains, do not result from direct viral infection of brain parenchyma, but more likely from systemic inflammation, perhaps with synergistic contribution from hypoxia/ischaemia. Further studies are needed to define whether these pathologies, if present in patients who survive coronavirus disease 2019, might contribute to chronic neurological problems.
Assuntos
Infarto Encefálico/patologia , Encéfalo/patologia , COVID-19/patologia , Hipóxia-Isquemia Encefálica/patologia , Hemorragias Intracranianas/patologia , Injúria Renal Aguda/complicações , Injúria Renal Aguda/fisiopatologia , Injúria Renal Aguda/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/complicações , Encéfalo/metabolismo , Infarto Encefálico/complicações , COVID-19/complicações , COVID-19/fisiopatologia , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Feminino , Humanos , Hipóxia-Isquemia Encefálica/complicações , Inflamação , Unidades de Terapia Intensiva , Hemorragias Intracranianas/complicações , Masculino , Microglia/patologia , Pessoa de Meia-Idade , Neurônios/patologia , Fagocitose , Fosfoproteínas/metabolismo , Embolia Pulmonar/complicações , Embolia Pulmonar/fisiopatologia , RNA Viral/metabolismo , Diálise Renal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Taxa de Sobrevida , Linfócitos T/patologia , Trombose Venosa/complicações , Trombose Venosa/fisiopatologiaRESUMO
Orbital schwannomas are typically slow-growing benign tumors that can cause gradual loss of vision, proptosis, and limitation of ocular motility. The authors present an atypical case of a rapidly growing orbital apex schwannoma in a patient with preexisting vision loss secondary to presumed sarcoidal optic neuritis. Contrary to the slowly progressive nature of a typical orbital schwannoma, the lesion was observed to enlarge from radiologically undiscernible to 3.5 cm over 4 years.
Assuntos
Neurilemoma/diagnóstico , Órbita/patologia , Neoplasias Orbitárias/diagnóstico , Biópsia , Progressão da Doença , Feminino , Humanos , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.
Assuntos
Doença de Alexander , Agregados Proteicos , Proteoma/análise , Animais , Astrócitos , Ciclina D2/análise , RNA Helicases DEAD-box/análise , Proteínas de Ligação ao GTP/análise , Humanos , Imuno-Histoquímica , Camundongos , Proteínas de Neoplasias/análise , Neuropeptídeos/análise , Agregação Patológica de Proteínas , RNA Helicases/análise , Receptores de Quinase C Ativada , Receptores de Superfície Celular/análiseRESUMO
Although microglia have long been considered as brain resident immune cells, increasing evidence suggests that they also have physiological roles in the development of the normal CNS. In this study, we found large numbers of activated microglia in the forebrain subventricular zone (SVZ) of the rat from P1 to P10. Pharmacological suppression of the activation, which produces a decrease in levels of a number of proinflammatory cytokines (i.e., IL-1ß, IL-6, TNF-α, and IFN-γ) significantly inhibited neurogenesis and oligodendrogenesis in the SVZ. In vitro neurosphere assays reproduced the enhancement of neurogenesis and oligodendrogenesis by activated microglia and showed that the cytokines revealed the effects complementarily. These results suggest that activated microglia accumulate in the early postnatal SVZ and that they enhance neurogenesis and oligodendrogenesis via released cytokines.
Assuntos
Ventrículos Cerebrais/fisiologia , Microglia/fisiologia , Neurogênese/fisiologia , Oligodendroglia/fisiologia , Animais , Animais Recém-Nascidos , Proliferação de Células , Células Cultivadas , Ventrículos Cerebrais/citologia , Feminino , Masculino , Ratos , Ratos WistarRESUMO
To examine the diversity of astrocytes in the human brain, we immunostained surgical specimens of temporal cortex and hippocampus and autopsy brains for CD44, a plasma membrane protein and extracellular matrix receptor. CD44 antibodies outline the details of astrocyte morphology to a degree not possible with glial fibrillary acidic protein (GFAP) antibodies. CD44+ astrocytes could be subdivided into two groups. First, CD44+ astrocytes with long processes were consistently found in the subpial area ("interlaminar" astrocytes), the deep isocortical layers, and the hippocampus. Many of these processes ended on blood vessels. Some were also found adjacent to large blood vessels, from which they extended long processes. We observed these CD44+, long-process astrocytes in every brain we examined, from fetal to adult. These astrocytes generally displayed high immunostaining for GFAP, S100ß, and CD44, but low immunostaining for glutamine synthetase, excitatory amino-acid transporter 1 (EAAT1), and EAAT2. Aquaporin 4 (AQP4) appeared distributed all over the cell bodies and processes of the CD44+ astrocytes, while, in contrast, AQP4 localized to perivascular end feet in the CD44- protoplasmic astrocytes. Second, there were CD44+ astrocytes without long processes in the cortex. These were not present during gestation or at birth, and in adult brains varied substantially in number, shape, and immunohistochemical phenotype. Many of these displayed a "mixed" morphological and immunocytochemical phenotype between protoplasmic and fibrous astrocytes. We conclude that the diversity of astrocyte populations in the isocortex and archicortex in the human brain reflects both intrinsic and acquired phenotypes, the latter perhaps representing a shift from CD44- "protoplasmic" to CD44+ "fibrous"-like astrocytes.
Assuntos
Astrócitos/fisiologia , Córtex Cerebral/fisiologia , Heterogeneidade Genética , Hipocampo/fisiologia , Plasticidade Neuronal/fisiologia , Fenótipo , Adolescente , Adulto , Idoso , Encéfalo/citologia , Encéfalo/fisiologia , Córtex Cerebral/citologia , Criança , Pré-Escolar , Feminino , Hipocampo/citologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Alexander disease (AxD) is a rare neurodegenerative disorder characterized pathologically by the presence of eosinophilic inclusions known as Rosenthal fibers (RFs) within astrocytes, and is caused by dominant mutations in the coding region of the gene encoding glial fibrillary acidic protein (GFAP). GFAP is the major astrocytic intermediate filament, and in AxD patient brain tissue GFAP is a major component of RFs. TAR DNA binding protein of 43 kDa (TDP-43) is the major pathological protein in almost all cases of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and â¼50% of frontotemporal lobar degeneration (FTLD), designated as FTLD-TDP. In ALS and FTLD-TDP, TDP-43 becomes insoluble, ubiquitinated, and pathologically phosphorylated and accumulates in cytoplasmic inclusions in both neurons and glia of affected brain and spinal cord regions. Previously, TDP-43 was detected in RFs of human pilocytic astrocytomas; however, involvement of TDP-43 in AxD has not been determined. Here we show that TDP-43 is present in RFs in AxD patient brains, and that insoluble phosphorylated full-length and high molecular weight TDP-43 accumulates in white matter of such brains. Phosphorylated TDP-43 also accumulates in the detergent-insoluble fraction from affected brain regions of Gfap(R236H/+) knock-in mice, which harbor a GFAP mutation homologous to one that causes AxD in humans, and TDP-43 colocalizes with astrocytic RF pathology in Gfap(R236H/+) mice and transgenic mice overexpressing human wild-type GFAP. These findings suggest common pathogenic mechanisms in ALS, FTLD, and AxD, and this is the first report of TDP-43 involvement in a neurological disorder primarily affecting astrocytes.
Assuntos
Doença de Alzheimer/patologia , Astrócitos/patologia , Proteinopatias TDP-43/patologia , Adolescente , Adulto , Idoso , Envelhecimento/fisiologia , Animais , Western Blotting , Criança , Citoplasma/metabolismo , Proteínas de Ligação a DNA , Feminino , Imunofluorescência , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/fisiologia , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Lactente , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Fosforilação , Adulto JovemRESUMO
Major gaps in our understanding of the leukodystrophies result from their rarity and the lack of tissue for the interdisciplinary studies required to extend our knowledge of the pathophysiology of the diseases. This study details the natural evolution of changes in the CNS of the shaking pup (shp), a model of the classical form of the X-linked disorder Pelizaeus-Merzbacher disease, in particular in glia, myelin, and axons, which is likely representative of what occurs over time in the human disease. The mutation in the proteolipid protein gene, PLP1, leads to a delay in differentiation, increased cell death, and a marked distension of the rough endoplasmic reticulum in oligodendrocytes. However, over time, more oligodendrocytes differentiate and survive in the spinal cord leading to an almost total recovery of myelination, In contrast, the brain remains persistently hypomyelinated. These data suggest that shp oligodendrocytes may be more functional than previously realized and that their early recruitment could have therapeutic value.
Assuntos
Modelos Animais de Doenças , Progressão da Doença , Doença de Pelizaeus-Merzbacher/fisiopatologia , Animais , Astrócitos/patologia , Astrócitos/fisiologia , Axônios/patologia , Axônios/fisiologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Morte Celular/fisiologia , Cães , Feminino , Masculino , Mutação , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/patologia , Bainha de Mielina/fisiologia , Oligodendroglia/patologia , Oligodendroglia/fisiologia , Doença de Pelizaeus-Merzbacher/genética , Doença de Pelizaeus-Merzbacher/patologia , Medula Espinal/patologia , Medula Espinal/fisiopatologiaRESUMO
Astrocytes and microglia are commonly involved in a wide variety of CNS pathologies. However, they are typically involved in a secondary response in which many cell types are affected simultaneously and therefore it is difficult to know their contributions to the pathology. Here, we show that pathological astrocytes in a mouse model of Alexander disease (AxD; GFAP (Tg);Gfap (+/R236H)) cause a pronounced immune response. We have studied the inflammatory response in the hippocampus and spinal cord of these mice and have found marked microglial activation, which follows that of astrocytes in a spatial pathological progression, as shown by increased levels of Iba1 and microglial cell (Iba1+) density. RNA sequencing and subsequent gene ontology (GO) analysis revealed that a majority of the most upregulated genes in GFAP (Tg);Gfap (+/R236H) mice are directly associated with immune function and that cytokine and chemokine GO attributes represent nearly a third of the total immune attributes. Cytokine and chemokine analysis showed CXCL10 and CCL2 to be the most and earliest increased molecules, showing concentrations as high as EAE or stroke models. CXCL10 was localized exclusively to astrocytes while CCL2 was also present in microglia. Despite the high levels of CXCL10 and CCL2, T cell infiltration was mild and no B cells were found. Thus, mutations in GFAP are sufficient to trigger a profound inflammatory response. The cellular stress caused by the accumulation of GFAP likely leads to the production of inflammatory molecules and microglial activation. Examination of human AxD CNS tissues also revealed microglial activation and T cell infiltrates. Therefore, the inflammatory environment may play an important role in producing the neuronal dysfunction and seizures of AxD.
Assuntos
Doença de Alexander/imunologia , Doença de Alexander/patologia , Astrócitos/imunologia , Astrócitos/patologia , Animais , Pré-Escolar , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/imunologia , Hipocampo/patologia , Humanos , Lactente , Masculino , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Neuroimunomodulação/fisiologia , Medula Espinal/imunologia , Medula Espinal/patologiaRESUMO
Mechanisms that regulate oligodendrocyte (OL) precursor migration and differentiation are important in normal development and in demyelinating/remyelinating conditions. We previously found that the tetraspanin CD82 is far more highly expressed in O4(+) OL precursors of the adult rat brain than those of the neonatal brain. CD82 has been physically linked to cMet, the hepatocyte growth factor (HGF) receptor, in tumor cells, and this interaction decreases downstream signaling. We show here that CD82 inhibits the HGF activation of cMet in neonatal and adult rat OL precursors. CD82 expression is sufficient to allow precursor differentiation into mature OLs even in the presence of HGF. In contrast, CD82 downregulation in adult O4(+)/CD82(+) cells inhibits their differentiation, decreases their accumulation of myelin proteins, and causes a reversion to less mature stages. CD82 expression in neonatal O4(+)/CD82(-) cells also blocks Rac1 activation, suggesting a possible regulatory effect on cytoskeletal organization and mobility. Thus, CD82 is a negative regulator of HGF/cMet during OL development and overcomes HGF inhibitory regulation of OL precursor maturation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Proteína Kangai-1/farmacologia , Oligodendroglia/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , Células-Tronco/efeitos dos fármacos , Adulto , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Contagem de Células , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Humanos , Proteína Kangai-1/metabolismo , Oligodendroglia/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Células-Tronco/classificaçãoRESUMO
Alexander Disease (AxD) is a primary disorder of astrocytes, caused by heterozygous mutations in GFAP, which encodes the major astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP). Astrocytes in AxD display hypertrophy, massive increases in GFAP, and the accumulation of Rosenthal fibers, cytoplasmic protein inclusions containing GFAP, and small heat shock proteins. To study the effects of GFAP mutations on astrocyte morphology and physiology, we have examined hippocampal astrocytes in three mouse models of AxD, a transgenic line (GFAP(Tg)) in which the normal human GFAP is expressed in several copies, a knock-in line (Gfap(+/R236H)) in which one of the Gfap genes bears an R236H mutation, and a mouse derived from the mating of these two lines (GFAP(Tg); Gfap(+/R236H)). We report changes in astrocyte phenotype in all lines, with the most severe in the GFAP(Tg);Gfap(+/R236H), resulting in the conversion of protoplasmic astrocytes to cells that have lost their bushy-like morphology because of a reduction of distal fine processes, and become multinucleated and hypertrophic. Astrocytes activate the mTOR cascade, acquire CD44, and lose GLT-1. The altered astrocytes display a microheterogeneity in phenotypes, even neighboring cells. Astrocytes also show diminished glutamate transporter current, are significantly depolarized, and not coupled to adjacent astrocytes. Thus, the accumulation of GFAP in the AxD mouse astrocytes initiates a conversion of normal, protoplasmic astrocytes to astrocytes that display severely "reactive" characteristics, many of which may be detrimental to neighboring neurons and oligodendrocytes.
Assuntos
Doença de Alexander/genética , Doença de Alexander/patologia , Astrócitos/patologia , Corrente Citoplasmática/fisiologia , Modelos Animais de Doenças , Fenótipo , Doença de Alexander/metabolismo , Animais , Astrócitos/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de ÓrgãosRESUMO
Cytomegalic neurons, characterized by increased size and a hyperactive mechanistic target of rapamycin complex 1 (mTORC1), are pathognomonic for tuberous sclerosis complex (TSC). To model these neurons, we recently generated a murine Tsc1 conditional knockout model in which Tsc1 deletion in late embryonic radial glia results in neuronal hypertrophy of a subset of isocortical pyramidal neurons. In the current study, we compared the cellular pathology of these cytomegalic neurons to those of the enlarged neurons in human cortical tubers. Neurons from the mice showed unique features, such as cytoplasmic vacuoles associated with Golgi complexes and the ectopic formation of perineuronal nets (PNNs), a feature of inhibitory neurons, rarely present in excitatory cortical neurons. The membranes of these vacuoles were enriched for the plasma membrane proteins CD44, KCC2, and Na+/K+ ATPase, suggesting deficits in Golgi membrane trafficking. These aberrant features in the mouse appeared only after the onset of seizures, probably due to the prolonged seizure activity in the context of constitutive mTORC1 activation. Similar PNNs and cytoplasmic vacuoles were present in the cytomegalic neurons of human cortical tubers. Our findings reveal novel pathological features of Golgi complexes and PNNs in the cytomegalic neurons in TSC.
RESUMO
In the mammalian isocortex, CD44, a cell surface receptor for extracellular matrix molecules, is present in pial-based and fibrous astrocytes of white matter but not in protoplasmic astrocytes. In the hominid isocortex, CD44+ astrocytes comprise the subpial "interlaminar" astrocytes, sending long processes into the cortex. The hippocampus also contains similar astrocytes. We have examined all levels of the human central nervous system and found CD44+ astrocytes in every region. Astrocytes in white matter and astrocytes that interact with large blood vessels but not with capillaries in gray matter are CD44+, the latter extending long processes into the parenchyma. Motor neurons in the brainstem and spinal cord, such as oculomotor, facial, hypoglossal, and in the anterior horn of the spinal cord, are surrounded by CD44+ processes, contrasting with neurons in the cortex, basal ganglia, and thalamus. We found CD44+ processes that intercalate between ependymal cells to reach the ventricle. We also found CD44+ astrocytes in the molecular layer of the cerebellar cortex. Protoplasmic astrocytes, which do not normally contain CD44, acquire it in pathologies like hypoxia and seizures. The pervasive and inducible expression of CD44 in astrocytes is a novel finding that lays the foundations for functional studies into the significance of CD44 in health and disease.
Assuntos
Receptores de Hialuronatos , Hipóxia , Convulsões , Animais , Humanos , Astrócitos , Receptores de Hialuronatos/metabolismo , Hipóxia/metabolismo , Neocórtex , Convulsões/metabolismo , Substância BrancaRESUMO
The mechanisms underlying the selective regional vulnerability to neurodegeneration in Huntington's disease (HD) have not been fully defined. To explore the role of astrocytes in this phenomenon, we used single-nucleus and bulk RNAseq, lipidomics, HTT gene CAG repeat-length measurements, and multiplexed immunofluorescence on HD and control post-mortem brains. We identified genes that correlated with CAG repeat length, which were enriched in astrocyte genes, and lipidomic signatures that implicated poly-unsaturated fatty acids in sensitizing neurons to cell death. Because astrocytes play essential roles in lipid metabolism, we explored the heterogeneity of astrocytic states in both protoplasmic and fibrous-like (CD44+) astrocytes. Significantly, one protoplasmic astrocyte state showed high levels of metallothioneins and was correlated with the selective vulnerability of distinct striatal neuronal populations. When modeled in vitro, this state improved the viability of HD-patient-derived spiny projection neurons. Our findings uncover key roles of astrocytic states in protecting against neurodegeneration in HD.
Assuntos
Astrócitos , Doença de Huntington , Neurônios , Doença de Huntington/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Astrócitos/metabolismo , Astrócitos/patologia , Humanos , Neurônios/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Masculino , Feminino , Lipidômica/métodos , Pessoa de Meia-Idade , Metalotioneína/metabolismo , Metalotioneína/genética , Encéfalo/metabolismo , Encéfalo/patologia , Metabolismo dos Lipídeos , Idoso , MultiômicaRESUMO
Frontotemporal dementia (FTD) is an incurable group of early-onset dementias that can be caused by the deposition of hyperphosphorylated tau in patient brains. However, the mechanisms leading to neurodegeneration remain largely unknown. Here, we combined single-cell analyses of FTD patient brains with a stem cell culture and transplantation model of FTD. We identified disease phenotypes in FTD neurons carrying the MAPT-N279K mutation, which were related to oxidative stress, oxidative phosphorylation, and neuroinflammation with an upregulation of the inflammation-associated protein osteopontin (OPN). Human FTD neurons survived less and elicited an increased microglial response after transplantation into the mouse forebrain, which we further characterized by single nucleus RNA sequencing of microdissected grafts. Notably, downregulation of OPN in engrafted FTD neurons resulted in improved engraftment and reduced microglial infiltration, indicating an immune-modulatory role of OPN in patient neurons, which may represent a potential therapeutic target in FTD.
Assuntos
Demência Frontotemporal , Neurônios , Osteopontina , Proteínas tau , Osteopontina/metabolismo , Osteopontina/genética , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Demência Frontotemporal/metabolismo , Humanos , Neurônios/metabolismo , Neurônios/patologia , Animais , Proteínas tau/metabolismo , Camundongos , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Microglia/metabolismo , Microglia/patologia , Mutação/genéticaRESUMO
The pathological involvement of the central nervous system in SARS-CoV2 (COVID-19) patients is established. The burden of pathology is most pronounced in the brain stem including the medulla oblongata. Hypoxic/ischemic damage is the most frequent neuropathologic abnormality. Other neuropathologic features include neuronophagia, microglial nodules, and hallmarks of neurodegenerative diseases: astrogliosis and microglial reactivity. It is still unknown if these pathologies are secondary to hypoxia versus a combination of inflammatory response combined with hypoxia. It is also unknown how astrocytes react to neuroinflammation in COVID-19, especially considering evidence supporting the neurotoxicity of certain astrocytic phenotypes. This study aims to define the link between astrocytic and microglial pathology in COVID-19 victims in the inferior olivary nucleus, which is one of the most severely affected brain regions in COVID-19, and establish whether COVID-19 pathology is driven by hypoxic damage. Here, we conducted neuropathologic assessments and multiplex-immunofluorescence studies on the medulla oblongata of 18 COVID-19, 10 pre-pandemic patients who died of acute respiratory distress syndrome (ARDS), and 7-8 control patients with no ARDS or COVID-19. The comparison of ARDS and COVID-19 allows us to identify whether the pathology in COVID-19 can be explained by hypoxia alone, which is common to both conditions. Our results showed increased olivary astrogliosis in ARDS and COVID-19. However, microglial density and microglial reactivity were increased only in COVID-19, in a region-specific manner. Also, olivary hilar astrocytes increased YKL-40 (CHI3L1) in COVID-19, but to a lesser extent than ARDS astrocytes. COVID-19 astrocytes also showed lower levels of Aquaporin-4 (AQP4), and Metallothionein-3 in subsets of COVID-19 brain regions. Cluster analysis on immunohistochemical attributes of astrocytes and microglia identified ARDS and COVID-19 clusters with correlations to clinical history and disease course. Our results indicate that olivary glial pathology and neuroinflammation in the COVID-19 cannot be explained solely by hypoxia and suggest that failure of astrocytes to upregulate the anti-inflammatory YKL-40 may contribute to the neuroinflammation. Notwithstanding the limitations of retrospective studies in establishing causality, our experimental design cannot adequately control for factors external to our design. Perturbative studies are needed to confirm the role of the above-described astrocytic phenotypes in neuroinflammation.