RESUMO
Astrocytes are intimately linked with brain blood vessels, an essential relationship for neuronal function. However, astroglial factors driving these physical and functional associations during postnatal brain development have yet to be identified. By characterizing structural and transcriptional changes in mouse cortical astrocytes during the first two postnatal weeks, we find that high-mobility group box 1 (Hmgb1), normally upregulated with injury and involved in adult cerebrovascular repair, is highly expressed in astrocytes at birth and then decreases rapidly. Astrocyte-selective ablation of Hmgb1 at birth affects astrocyte morphology and endfoot placement, alters distribution of endfoot proteins connexin43 and aquaporin-4, induces transcriptional changes in astrocytes related to cytoskeleton remodeling, and profoundly disrupts endothelial ultrastructure. While lack of astroglial Hmgb1 does not affect the blood-brain barrier or angiogenesis postnatally, it impairs neurovascular coupling and behavior in adult mice. These findings identify astroglial Hmgb1 as an important player in postnatal gliovascular maturation.
Assuntos
Astrócitos , Barreira Hematoencefálica , Proteína HMGB1 , Animais , Camundongos , Aquaporina 4 , Encéfalo , Morfogênese , Proteína HMGB1/metabolismoRESUMO
One of the functions of small extracellular vesicles (sEVs) which has received the most attention is their capacity to deliver RNA into the cytoplasm of target cells. These studies have often been performed by transfecting RNAs into sEV-producing cells, to later purify and study sEV delivery of RNA. Transfection complexes and other delivery vehicles accumulate in late endosomes where sEV are formed and over 50% of transfection complexes or delivery vehicles administered to cells are released again to the extracellular space by exocytosis. This raises the possibility that transfection complexes could alter sEVs and contaminate sEV preparations. We found that widely used transfection reagents including RNAiMax and INTERFERin accumulated in late endosomes. These transfection complexes had a size similar to sEV and were purified by ultracentrifugation like sEV. Focusing on the lipid-based transfection reagent RNAiMax, we found that preparations of sEV from transfected cells contained lipids from transfection complexes and transfected siRNA was predominantly in particles with the density of transfection complexes, rather than sEV. This suggests that transfection complexes, such as lipid-based RNAiMax, may frequently contaminate sEV preparations and could account for some reports of sEV-mediated delivery of nucleic acids. Transfection of cells also impaired the capacity of sEVs to deliver stably-expressed siRNAs, suggesting that transfection of cells may alter sEVs and prevent the study of their endogenous capacity to deliver RNA to target cells.
Assuntos
Vesículas Extracelulares , Lipídeos , RNA Interferente Pequeno , Transfecção , UltracentrifugaçãoRESUMO
Many of the genes whose mutation causes Amyotrophic Lateral Sclerosis (ALS) are RNA-binding proteins which localize to stress granules, while others impact the assembly, stability, and elimination of stress granules. This has led to the hypothesis that alterations in the dynamics of stress granules and RNA biology cause ALS. Genetic mutations in Superoxide Dismutase 1 (SOD1) also cause ALS. Evidence demonstrates that SOD1 harboring ALS-linked mutations is recruited to stress granules, induces changes in alternative splicing, and could be an RNA-binding protein. Whether SOD1 inclusions contain RNA in disease models and whether SOD1 directly binds RNA remains uncertain. We applied methods including cross-linking immunoprecipitation and in vitro gel shift assays to detect binding of SOD1 to RNA in vitro, in cells with and without stress granules, and in mice expressing human SOD1 G93A. We find that SOD1 localizes to RNA-rich structures including stress granules, and SOD1 inclusions in mice contain mRNA. However, we find no evidence that SOD1 directly binds RNA. This suggests that SOD1 may impact stress granules, alternative splicing and RNA biology without binding directly to RNA.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Mutação/fisiologia , RNA/metabolismo , Superóxido Dismutase/metabolismo , Animais , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/genética , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , RNA/análise , RNA/genética , Superóxido Dismutase/análise , Superóxido Dismutase/genéticaRESUMO
A small percentage of the short interfering RNA (siRNA) delivered via passive lipid nanoparticles and other delivery vehicles reaches the cytoplasm of cells. The high doses of siRNA and delivery vehicle that are thus required to achieve therapeutic outcomes can lead to toxicity. Here, we show that the integration of siRNA sequences into a Dicer-independent RNA stem-loop based on pre-miR-451 microRNA-which is highly enriched in small extracellular vesicles secreted by many cell types-reduces the expression of the genes targeted by the siRNA in the liver, intestine and kidney glomeruli of mice at siRNA doses that are at least tenfold lower than the siRNA doses typically delivered via lipid nanoparticles. Small extracellular vesicles that efficiently package siRNA can significantly reduce its therapeutic dose.
Assuntos
Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , MicroRNAs/química , Neurônios Motores/efeitos dos fármacos , Nanopartículas/administração & dosagem , Interferência de RNA , RNA Interferente Pequeno/químicaRESUMO
Future changes in climate are widely anticipated to increase fire frequency, particularly in boreal forests where extreme warming is expected to occur. Feedbacks between vegetation and fire may modify the direct effects of warming on fire activity and shape ecological responses to changing fire frequency. We investigate these interactions using extensive field data from the Boreal Shield of Saskatchewan, Canada, a region where >40% of the forest has burned in the past 30 years. We use geospatial and field data to assess the resistance and resilience of eight common vegetation states to frequent fire by quantifying the occurrence of short-interval fires and their effect on recovery to a similar vegetation state. These empirical relationships are combined with data from published literature to parameterize a spatially explicit, state-and-transition simulation model of fire and forest succession. We use this model to ask if and how: (a) feedbacks between vegetation and wildfire may modify fire activity on the landscape, and (b) more frequent fire may affect landscape forest composition and age structure. Both field and GIS data suggest the probability of fire is low in the initial decades after fire, supporting the hypothesis that fuel accumulation may exert a negative feedback on fire frequency. Field observations of pre- and postfire composition indicate that switches in forest state are more likely in conifer stands that burn at a young age, supporting the hypothesis that resilience is lower in immature stands. Stands dominated by deciduous trees or jack pine were generally resilient to fire, while mixed conifer and well-drained spruce forests were less resilient. However, simulation modeling suggests increased fire activity may result in large changes in forest age structure and composition, despite the feedbacks between vegetation-fire likely to occur with increased fire activity.
Assuntos
Taiga , Traqueófitas/parasitologia , Incêndios Florestais , Mudança Climática , Monitoramento Ambiental , Modelos Teóricos , Saskatchewan , Especificidade da Espécie , Fatores de Tempo , Traqueófitas/classificação , Traqueófitas/crescimento & desenvolvimentoRESUMO
Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.
Assuntos
Esclerose Lateral Amiotrófica/genética , Autofagia/genética , Proteína C9orf72/genética , Proteína FUS de Ligação a RNA/genética , Proteína Sequestossoma-1/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Arginina/metabolismo , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , Embrião de Mamíferos , Células HeLa , Humanos , Metilação , Camundongos , Camundongos Knockout , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Cultura Primária de Células , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/metabolismo , Estresse Fisiológico , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
BACKGROUND: Inflammation due to remote pathogen exposure combined to hypoxia/ischemia (HI) is one of the most common causes of neonatal encephalopathy affecting at-term or near-term human newborn, which will consequently develop cerebral palsy. Within term-equivalent rat brains exposed to systemic lipopolysaccharide (LPS) plus HI, it was previously showed that neurons produce IL-1ß earlier than do glial cells, and that blocking IL-1 was neuroprotective. To further define the mechanisms whereby IL-1 exerts its neurotoxic effect, we hypothesize that IL-1ß plays a pivotal role in a direct and/or indirect mechanistic loop of neuronal self-injury through matrix metalloproteinase (MMP)-9. METHODS: An established preclinical rat model of LPS+HI-induced neonatal encephalopathy was used. In situ hybridization, ELISA, and immunolabeling techniques were employed. Selective blocking compounds allowed addressing the respective roles of IL-1 and MMP-9. RESULTS: In LPS+HI-exposed forebrains, neuronal IL-1ß was first detected in infarcted neocortical and striatal areas and later in glial cells of the adjacent white matter. Neuronal IL-1ß played a key role: (i) in the early post-HI exacerbation of neuroinflammation and (ii) in generating both core and penumbral infarcted cerebral areas. Systemically administered IL-1 receptor antagonist (IL-1Ra) reached the brain and bound to the neocortical and deep gray neuronal membranes. Then, IL-1Ra down-regulated IL-1ß mRNA and MMP-9 neuronal synthesis. Immediately post-HI, neuronal IL-1ß up-regulated cytokine-induced neutrophil chemoattractant (CINC-1), monocyte chemoattractant protein-1 (MCP-1), and inducible nitric oxide synthase. MMP-9 would disrupt the blood-brain barrier, which, combined to CINC-1 up-regulation, would play a role in polymorphonuclear cell (PMN) infiltration into the LPS+HI-exposed brain. IL-1ß blockade prevented PMN infiltration and oriented the phenotype of macrophagic/microglial cells towards anti-inflammatory and neurotrophic M2 profile. IL-1ß increased the expression of activated caspase-3 and of receptor-interacting-protein (RIP)-3 within infarcted forebrain area. Such apoptotic and necroptotic pathway activations were prevented by IL-1Ra, as well as ensuing cerebral palsy-like brain damage and motor impairment. CONCLUSIONS: This work uncovered a new paradigm of neuronal self-injury orchestrated by neuronal synthesis of IL-1ß and MMP-9. In addition, it reinforced the translational neuroprotective potential of IL-1 blockers to alleviate human perinatal brain injuries.
Assuntos
Encefalopatias , Paralisia Cerebral/complicações , Hipóxia-Isquemia Encefálica/complicações , Interleucina-1beta/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neurônios/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Encefalopatias/etiologia , Encefalopatias/metabolismo , Encefalopatias/patologia , Caspase 3/metabolismo , Paralisia Cerebral/induzido quimicamente , Paralisia Cerebral/imunologia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Metaloproteinase 9 da Matriz/genética , Atividade Motora/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos Endogâmicos Lew , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Comportamento Estereotipado/efeitos dos fármacosRESUMO
BACKGROUND: Infection-inflammation combined with hypoxia-ischemia (HI) is the most prevalent pathological scenario involved in perinatal brain damage leading to life-long neurological disabilities. Following lipopolysaccharide (LPS) and/or HI aggression, different patterns of inflammatory responses have been uncovered according to the brain differentiation stage. In fact, LPS pre-exposure has been reported to aggravate HI brain lesions in post-natal day 1 (P1) and P7 rat models that are respectively equivalent - in terms of brain development - to early and late human preterm newborns. However, little is known about the innate immune response in LPS plus HI-induced lesions of the full-term newborn forebrain and the associated neuropathological and neurobehavioral outcomes. METHODS: An original preclinical rat model has been previously documented for the innate neuroimmune response at different post-natal ages. It was used in the present study to investigate the neuroinflammatory mechanisms that underline neurological impairments after pathogen-induced inflammation and HI in term newborns. RESULTS: LPS and HI exerted a synergistic detrimental effect on rat brain. Their effect led to a peculiar pattern of parasagittal cortical-subcortical infarcts mimicking those in the human full-term newborn with subsequent severe neurodevelopmental impairments. An increased IL-1ß response in neocortical and basal gray neurons was demonstrated at 4 h after LPS + HI-exposure and preceded other neuroinflammatory responses such as microglial and astroglial cell activation. Neurological deficits were observed during the acute phase of injury followed by a recovery, then by a delayed onset of profound motor behavior impairment, reminiscent of the delayed clinical onset of motor system impairments observed in humans. Interleukin-1 receptor antagonist (IL-1ra) reduced the extent of brain lesions confirming the involvement of IL-1ß response in their pathophysiology. CONCLUSION: In rat pups at a neurodevelopmental age corresponding to full-term human newborns, a systemic pre-exposure to a pathogen component amplified HI-induced mortality and morbidities that are relevant to human pathology. Neuronal cells were the first cells to produce IL-1ß in LPS + HI-exposed full-term brains. Such IL-1ß production might be responsible for neuronal self-injuries via well-described neurotoxic mechanisms such as IL-1ß-induced nitric oxide production, or IL-1ß-dependent exacerbation of excitotoxic damage.
Assuntos
Encefalopatias/etiologia , Encefalopatias/metabolismo , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encefalopatias/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Imuno-Histoquímica , Hibridização In Situ , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Imageamento por Ressonância Magnética , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Endogâmicos LewRESUMO
The recessive lyp allele, which harbors a defective gimap5 (GTPase of immunity-associated nucleotide binding protein 5) gene, causes spontaneous apoptosis of T lymphocytes in the biobreeding diabetes-prone strain of rats. Mechanisms underlying the pro-survival function of GIMAP5 remain unclear. In this study, we show that gimap5(lyp/lyp) T cells display diminished calcium flux in response to thapsigargin or signaling via the T cell antigen receptor. This defect is manifested in mature single positive thymocytes, where the survival defect first occurs. We also show that GIMAP5 deficiency does not affect the thapsigargin-induced calcium release from the intracellular stores but impairs subsequent calcium entry across the plasma membrane. Our findings suggest that GIMAP5 is an important regulator of calcium response in T lymphocytes and impaired calcium signaling might underlie spontaneous apoptosis of gimap5(lyp/lyp) T cells.