RESUMO
Aging is a major risk factor for both genetic and sporadic neurodegenerative disorders. However, it is unclear how aging interacts with genetic predispositions to promote neurodegeneration. Here, we investigate how partial loss of function of TBK1, a major genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comorbidity, leads to age-dependent neurodegeneration. We show that TBK1 is an endogenous inhibitor of RIPK1 and the embryonic lethality of Tbk1-/- mice is dependent on RIPK1 kinase activity. In aging human brains, another endogenous RIPK1 inhibitor, TAK1, exhibits a marked decrease in expression. We show that in Tbk1+/- mice, the reduced myeloid TAK1 expression promotes all the key hallmarks of ALS/FTD, including neuroinflammation, TDP-43 aggregation, axonal degeneration, neuronal loss, and behavior deficits, which are blocked upon inhibition of RIPK1. Thus, aging facilitates RIPK1 activation by reducing TAK1 expression, which cooperates with genetic risk factors to promote the onset of ALS/FTD.
Assuntos
Apoptose , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Adulto , Idoso , Envelhecimento , Animais , Apoptose/efeitos dos fármacos , Axônios/metabolismo , Comportamento Animal , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Humanos , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Knockout , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Medula Espinal/metabolismo , Estaurosporina/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The receptor-interacting protein kinase 1 (RIPK1) is recognized as a master upstream regulator that controls cell survival and inflammatory signaling as well as multiple cell death pathways, including apoptosis and necroptosis. The activation of RIPK1 kinase is extensively modulated by ubiquitination and phosphorylation, which are mediated by multiple factors that also control the activation of the NF-κB pathway. We discuss current findings regarding the genetic modulation of RIPK1 that controls its activation and interaction with downstream mediators, such as caspase-8 and RIPK3, to promote apoptosis and necroptosis. We also address genetic autoinflammatory human conditions that involve abnormal activation of RIPK1. Leveraging these new genetic and mechanistic insights, we postulate how an improved understanding of RIPK1 biology may support the development of therapeutics that target RIPK1 for the treatment of human inflammatory and neurodegenerative diseases.
Assuntos
Necroptose , Proteínas Quinases , Apoptose/genética , Humanos , Necroptose/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/genéticaRESUMO
Macrophage migration inhibitory factor (MIF) is a multifaced protein that plays important roles in multiple inflammatory conditions. However, the role of MIF in endothelial cell (EC) death under inflammatory condition remains largely unknown. Here we show that MIF actively promotes receptor-interacting protein kinase 1 (RIPK1)-mediated cell death under oxygen-glucose deprivation condition. MIF expression is induced by surgical trauma in peripheral myeloid cells both in perioperative humans and mice. We demonstrate that MIF-loaded myeloid cells induced by peripheral surgery adhere to the brain ECs after distal middle cerebral artery occlusion (dMCAO) and exacerbate the blood-brain barrier (BBB) disruption. Genetic depletion of myeloid-derived MIF in perioperative ischemic stroke (PIS) mice with MCAO following a surgical insult leads to significant reduction in ECs apoptosis and necroptosis and the associated BBB disruption. The adoptive transfer of peripheral blood mononuclear cells (PBMC) from surgical MIFΔLyz2 mice to wild-type (WT) MCAO mice also shows reduced ECs apoptosis and necroptosis compared to the transfer of PBMC from surgical MIFfââl/fââl mice to MCAO recipients. The genetic inhibition of RIPK1 also attenuates BBB disruption and ECs death compared to that of WT mice in PIS. The administration of MIF inhibitor (ISO-1) and RIPK1 inhibitor (Nec-1s) can both reduce the brain EC death and neurological deficits following PIS. We conclude that myeloid-derived MIF promotes ECs apoptosis and necroptosis through RIPK1 kinase-dependent pathway. The above findings may provide insights into the mechanism as how peripheral inflammation promotes the pathology in central nervous system.
Assuntos
Lesões Encefálicas , Fatores Inibidores da Migração de Macrófagos , Proteína Serina-Treonina Quinases de Interação com Receptores , Animais , Humanos , Camundongos , Apoptose , Morte Celular , Células Endoteliais/metabolismo , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Leucócitos Mononucleares/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Inflamação/enzimologia , Microglia/enzimologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Ciclo Celular/genética , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Mutantes , Microglia/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Análise de Célula Única , Superóxido Dismutase-1/genética , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Dynamic synapses facilitate activity-dependent remodeling of neural circuits, thereby providing the structural substrate for adaptive behaviors. However, the mechanisms governing dynamic synapses in adult brain are still largely unknown. Here, we demonstrate that in the cortex of adult amyloid precursor protein knockout (APP-KO) mice, spine formation and elimination were both reduced while overall spine density remained unaltered. When housed under environmental enrichment, APP-KO mice failed to respond with an increase in spine density. Spine morphology was also altered in the absence of APP The underlying mechanism of these spine abnormalities in APP-KO mice was ascribed to an impairment in D-serine homeostasis. Extracellular D-serine concentration was significantly reduced in APP-KO mice, coupled with an increase of total D-serine. Strikingly, chronic treatment with exogenous D-serine normalized D-serine homeostasis and restored the deficits of spine dynamics, adaptive plasticity, and morphology in APP-KO mice. The cognitive deficit observed in APP-KO mice was also rescued by D-serine treatment. These data suggest that APP regulates homeostasis of D-serine, thereby maintaining the constitutive and adaptive plasticity of dendritic spines in adult brain.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Espinhas Dendríticas/metabolismo , Plasticidade Neuronal , Serina/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Transtornos Cognitivos/metabolismo , Feminino , Homeostase , Camundongos KnockoutRESUMO
Dysfunction of microglia is known to play an important role in Alzheimer's disease (AD). Here, we investigated the role of RIPK1 in microglia mediating the pathogenesis of AD. RIPK1 is highly expressed by microglial cells in human AD brains. Using the amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mouse model, we found that inhibition of RIPK1, using both pharmacological and genetic means, reduced amyloid burden, the levels of inflammatory cytokines, and memory deficits. Furthermore, inhibition of RIPK1 promoted microglial degradation of Aß in vitro. We characterized the transcriptional profiles of adult microglia from APP/PS1 mice and identified a role for RIPK1 in regulating the microglial expression of CH25H and Cst7, a marker for disease-associated microglia (DAM), which encodes an endosomal/lysosomal cathepsin inhibitor named Cystatin F. We present evidence that RIPK1-mediated induction of Cst7 leads to an impairment in the lysosomal pathway. These data suggest that RIPK1 may mediate a critical checkpoint in the transition to the DAM state. Together, our study highlights a non-cell death mechanism by which the activation of RIPK1 mediates the induction of a DAM phenotype, including an inflammatory response and a reduction in phagocytic activity, and connects RIPK1-mediated transcription in microglia to the etiology of AD. Our results support that RIPK1 is an important therapeutic target for the treatment of AD.
Assuntos
Doença de Alzheimer/patologia , Biomarcadores/metabolismo , Microglia/patologia , Presenilina-1/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Fenótipo , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
To successfully treat Alzheimer's disease (AD), pathophysiological events in preclinical stages need to be identified. Preclinical AD refers to the stages that exhibit amyloid deposition in the brain but have normal cognitive function, which are replicated in young adult APPswe/PS1deltaE9 (deltaE9) mice. By long-term in vivo two-photon microscopy, we demonstrate impaired adaptive spine plasticity in these transgenic mice illustrated by their failure to increase dendritic spine density and form novel neural connections when housed in enriched environment (EE). Decrease of amyloid plaques by reducing BACE1 activity restores the gain of spine density upon EE in deltaE9 mice, but not the remodeling of neural networks. On the other hand, anti-inflammatory treatment with pioglitazone or interleukin 1 receptor antagonist in deltaE9 mice successfully rescues the impairments in increasing spine density and remodeling of neural networks during EE. Our data suggest that neuroinflammation disrupts experience-dependent structural plasticity of dendritic spines in preclinical stages of AD.
Assuntos
Doença de Alzheimer/imunologia , Espinhas Dendríticas/imunologia , Neuroimunomodulação/imunologia , Plasticidade Neuronal/imunologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/patologia , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroimunomodulação/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Pioglitazona , Células Piramidais/efeitos dos fármacos , Células Piramidais/imunologia , Células Piramidais/patologia , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Receptores Tipo I de Interleucina-1/metabolismo , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/imunologia , Córtex Somatossensorial/patologia , Tiazolidinedionas/farmacologiaRESUMO
Synaptic failure is an immediate cause of cognitive decline and memory dysfunction in Alzheimer's disease. Dendritic spines are specialized structures on neuronal processes, on which excitatory synaptic contacts take place and the loss of dendritic spines directly correlates with the loss of synaptic function. Dendritic spines are readily accessible for both in vitro and in vivo experiments and have, therefore, been studied in great detail in Alzheimer's disease mouse models. To date, a large number of different mechanisms have been proposed to cause dendritic spine dysfunction and loss in Alzheimer's disease. For instance, amyloid beta fibrils, diffusible oligomers or the intracellular accumulation of amyloid beta have been found to alter the function and structure of dendritic spines by distinct mechanisms. Furthermore, tau hyperphosphorylation and microglia activation, which are thought to be consequences of amyloidosis in Alzheimer's disease, may also contribute to spine loss. Lastly, genetic and therapeutic interventions employed to model the disease and elucidate its pathogenetic mechanisms in experimental animals may cause alterations of dendritic spines on their own. However, to date none of these mechanisms have been translated into successful therapeutic approaches for the human disease. Here, we critically review the most intensely studied mechanisms of spine loss in Alzheimer's disease as well as the possible pitfalls inherent in the animal models of such a complex neurodegenerative disorder.
Assuntos
Doença de Alzheimer/patologia , Espinhas Dendríticas/patologia , Doença de Alzheimer/fisiopatologia , Amiloide/metabolismo , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Espinhas Dendríticas/fisiologia , HumanosRESUMO
Alzheimer's disease (AD) is thought to be caused by accumulation of amyloid-ß protein (Aß), which is a cleavage product of amyloid precursor protein (APP). Transgenic mice overexpressing APP have been used to recapitulate amyloid-ß pathology. Among them, APP23 and APPswe/PS1deltaE9 (deltaE9) mice are extensively studied. APP23 mice express APP with Swedish mutation and develop amyloid plaques late in their life, while cognitive deficits are observed in young age. In contrast, deltaE9 mice with mutant APP and mutant presenilin-1 develop amyloid plaques early but show typical cognitive deficits in old age. To unveil the reasons for different progressions of cognitive decline in these commonly used mouse models, we analyzed the number and turnover of dendritic spines as important structural correlates for learning and memory. Chronic in vivo two-photon imaging in apical tufts of layer V pyramidal neurons revealed a decreased spine density in 4-5-month-old APP23 mice. In age-matched deltaE9 mice, in contrast, spine loss was only observed on cortical dendrites that were in close proximity to amyloid plaques. In both cases, the reduced spine density was caused by decreased spine formation. Interestingly, the patterns of alterations in spine morphology differed between these two transgenic mouse models. Moreover, in APP23 mice, APP was found to accumulate intracellularly and its content was inversely correlated with the absolute spine density and the relative number of mushroom spines. Collectively, our results suggest that different pathological mechanisms, namely an intracellular accumulation of APP or extracellular amyloid plaques, may lead to spine abnormalities in young adult APP23 and deltaE9 mice, respectively. These distinct features, which may represent very different mechanisms of synaptic failure in AD, have to be taken into consideration when translating results from animal studies to the human disease.
Assuntos
Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Espinhas Dendríticas/patologia , Mutação/genética , Neurônios/patologia , Presenilina-1/metabolismo , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Presenilina-1/genética , Estatísticas não ParamétricasRESUMO
Graphite-based dual-ion batteries are with potential higher energy density, making them a unique candidate in energy storage systems. However, anion insertion into graphite in aqueous environment remains a significant challenge. Herein, we report that the reversible insertion of Al-Cl superhalide into expanded graphite (EG) delivers an ultrahigh specific capacity of ~171â mAh g-1 from an aqueous deep eutectic solvent (DES) gel electrolyte of 50â m ChCl+5â m AlCl3 . High-resolution transmission electron microscopy (HRTEM), Raman spectra and X-ray diffraction (XRD) show that the EG generates turbostratic structure during Al-Cl superhalide (de)insertion instead of presenting typical graphite intercalation compounds (GIC), thus attributing to the high capacity during Al-Cl superhalide insertion.
RESUMO
Defects in the prelamin A processing enzyme caused by loss-of-function mutations in the ZMPSTE24 gene are responsible for a spectrum of progeroid disorders characterized by the accumulation of farnesylated prelamin A. Here we report that defective prelamin A processing triggers nuclear RIPK1-dependent signalling that leads to necroptosis and inflammation. We show that accumulated prelamin A recruits RIPK1 to the nucleus to facilitate its activation upon tumour necrosis factor stimulation in ZMPSTE24-deficient cells. Kinase-activated RIPK1 then promotes RIPK3-mediated MLKL activation in the nucleus, leading to nuclear envelope disruption and necroptosis. This signalling relies on prelamin A farnesylation, which anchors prelamin A to nuclear envelope to serve as a nucleation platform for necroptosis. Genetic inactivation of necroptosis ameliorates the progeroid phenotypes in Zmpste24-/- mice. Our findings identify an unconventional nuclear necroptosis pathway resulting from ZMPSTE24 deficiency with pathogenic consequences in progeroid disorder and suggest RIPK1 as a feasible target for prelamin A-associated progeroid disorders.
Assuntos
Lamina Tipo A , Necroptose , Animais , Camundongos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Mutação , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating fatal neurodegenerative disease with no cure. Receptor-interacting protein kinase 1 (RIPK1) has been proposed to mediate pathogenesis of ALS. Primidone has been identified as an old drug that can also inhibit RIPK1 kinase. We conducted a drug-repurposing biomarker study of primidone as a RIPK1 inhibitor using SOD1G93A mice and ALS patients. SOD1G93A mice treated with primidone showed significant delay of symptomatic onset and improved motor performance. One-hundred-sixty-two ALS participants dosed daily with primidone (62.5 mg) completed 24-week follow-up. A significant reduction was showed in serum levels of RIPK1 and IL-8, which were significantly higher in ALS patients than that of healthy controls (P < 0.0001). Serum RIPK1 levels were correlated positively with the severity of bulbar symptoms (P < 0.05). Our study suggests that serum levels of RIPK1 and IL-8 in peripheral can be used as clinical biomarkers for the activation of RIPK1 in central nervous system in human ALS patients. Repurposing primidone may provide a promising therapeutic strategy for ALS. The effect of primidone for the treatment of other inflammatory diseases may also be considered, since the activation of RIPK1 has been implicated in mediating a variety of inflammatory diseases including COVID-19-associated cytokine release syndrome (CRS). (ChiCTR2200060149).
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Biomarcadores , Interleucina-8/genética , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Doenças Neurodegenerativas/metabolismo , Primidona/metabolismo , Primidona/farmacologia , Primidona/uso terapêutico , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/farmacologia , Superóxido Dismutase/metabolismo , Superóxido Dismutase/farmacologia , Superóxido Dismutase/uso terapêutico , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/farmacologiaRESUMO
Microglial dysfunction is a major contributor to the pathogenesis of multiple neurodegenerative diseases. The neurotoxicity of microglia associated with oxidative stress largely depends on NF-κB pathway activation, which promotes the production and release of microglial proinflammatory cytokines and chemokines. In this review, we discuss the current literature on the essential role of the NF-κB pathway on microglial activation that exacerbates neurodegeneration, with a particular focus on RIPK1 kinase activity-dependent microglial dysfunction. As upregulated RIPK1 kinase activity is associated with reactive oxygen species (ROS) accumulation in neurodegenerative diseases, we also discuss the current knowledge about the mechanistic links between RIPK1 activation and ROS generation. Given RIPK1 kinase activity and oxidative stress are closely regulated with each other in a vicious cycle, future studies are required to be conducted to fully understand how RIPK1 and ROS collude together to disturb microglial homeostasis that drives neurodegenerative pathogenesis.
RESUMO
The cells and tissue in the human body are orderly and directionally arranged, and constructing an ideal biomimetic extracellular matrix is still a major problem to be solved in tissue engineering. In the field of the bioresorbable vascular grafts, the long-term functional prognosis requires that cells first migrate and grow along the physiological arrangement direction of the vessel itself. Moreover, the graft is required to promote the formation of neointima and the development of the vessel walls while ensuring that the whole repair process does not form a thrombus. In this study, poly (l-lactide-co-ε-caprolactone) (PLCL) shell layers and polyethylene oxide (PEO) core layers with different microstructures and loaded with sodium tanshinone IIA sulfonate (STS) were prepared by coaxial electrospinning. The mechanical properties proved that the fiber membranes had good mechanical support, higher than that of the human aorta, as well as great suture retention strengths. The hydrophilicity of the oriented-fiber membranes was greatly improved compared with that of the random-fiber membranes. Furthermore, we investigated the biocompatibility and hemocompatibility of different functional fiber membranes, and the results showed that the oriented-fiber membranes containing sodium tanshinone IIA sulfonate had an excellent antiplatelet adhesion effect compared to other fiber membranes. Cytological analysis confirmed that the functional fiber membranes were non-cytotoxic and had significant cell proliferation capacities. The oriented-fiber membranes induced cell growth along the orientation direction. Degradation tests showed that the pH variation range had little change, the material mass was gradually reduced, and the fiber morphology was slowly destroyed. Thus, results indicated the degradation rate of the oriented-fiber graft likely is suitable for the process of new tissue regeneration, while the random-fiber graft with a low degradation rate may cause the material to reside in the tissue for too long, which would impede new tissue reconstitution. In summary, the oriented-functional-fiber membranes possessing core-shell structures with sodium tanshinone IIA sulfonate/polyethylene oxide loading could be used as tissue engineering materials for applications such as vascular grafts with good prospects, and their clinical application potential will be further explored in future research.
RESUMO
RIPK1 is a master regulator of multiple cell death pathways, including apoptosis and necroptosis, and inflammation. Importantly, activation of RIPK1 has also been shown to promote the transcriptional induction of proinflammatory cytokines in cells undergoing necroptosis, in animal models of amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD), and in human ALS and AD. Rare human genetic carriers of non-cleavable RIPK1 variants (D324V and D324H) exhibit distinct symptoms of recurrent fevers and increased transcription of proinflammatory cytokines. Multiple RIPK1 inhibitors have been advanced into human clinical trials as new therapeutics for human inflammatory and neurodegenerative diseases, such as ALS and AD. However, it is unclear whether and how RIPK1 kinase activity directly mediates inflammation independent of cell death as the nuclear function of RIPK1 has not yet been explored. Here we show that nuclear RIPK1 is physically associated with the BAF complex. Upon RIPK1 activation, the RIPK1/BAF complex is recruited by specific transcription factors to active enhancers and promoters marked by H3K4me1 and H3K27ac. Activated nuclear RIPK1 mediates the phosphorylation of SMARCC2, a key component of the BAF complex, to promote chromatin remodeling and the transcription of specific proinflammatory genes. Increased nuclear RIPK1 activation and RIPK1/BAF-mediated chromatin-remodeling activity were found in cells expressing non-cleavable RIPK1, and increased enrichment of activated RIPK1 on active enhancers and promoters was found in an animal model and human pathological samples of ALS. Our results suggest that RIPK1 kinase serves as a transcriptional coregulator in nucleus that can transmit extracellular stimuli to the BAF complex to modulate chromatin accessibility and directly regulate the transcription of specific genes involved in mediating inflammatory responses.
Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/genética , Animais , Apoptose , Cromatina , Montagem e Desmontagem da Cromatina , Citocinas/metabolismo , Inflamação/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Benzodiazepines are widely administered drugs to treat anxiety and insomnia. In addition to tolerance development and abuse liability, their chronic use may cause cognitive impairment and increase the risk for dementia. However, the mechanism by which benzodiazepines might contribute to persistent cognitive decline remains unknown. Here we report that diazepam, a widely prescribed benzodiazepine, impairs the structural plasticity of dendritic spines, causing cognitive impairment in mice. Diazepam induces these deficits via the mitochondrial 18 kDa translocator protein (TSPO), rather than classical γ-aminobutyric acid type A receptors, which alters microglial morphology, and phagocytosis of synaptic material. Collectively, our findings demonstrate a mechanism by which TSPO ligands alter synaptic plasticity and, as a consequence, cause cognitive impairment.
Assuntos
Diazepam , Microglia , Receptores de GABA/metabolismo , Animais , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Cognição , Diazepam/farmacologia , Camundongos , Microglia/metabolismo , Proteínas MitocondriaisRESUMO
Loss-of-function mutations in NEK1 gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS. Here we show that NEK1 regulates retromer-mediated endosomal trafficking by phosphorylating VPS26B. NEK1 deficiency disrupts endosomal trafficking of plasma membrane proteins and cerebral proteome homeostasis to promote mitochondrial and lysosomal dysfunction and aggregation of α-synuclein. The metabolic and proteomic defects of NEK1 deficiency disrupts the integrity of blood-brain barrier (BBB) by promoting lysosomal degradation of A20, a key modulator of RIPK1, thus sensitizing cerebrovascular endothelial cells to RIPK1-dependent apoptosis and necroptosis. Genetic inactivation of RIPK1 or metabolic rescue with ketogenic diet can prevent postnatal lethality and BBB damage in NEK1 deficient mice. Inhibition of RIPK1 reduces neuroinflammation and aggregation of α-synuclein in the brains of NEK1 deficient mice. Our study identifies a molecular mechanism by which retromer trafficking and metabolism regulates cerebrovascular integrity, cerebral proteome homeostasis and RIPK1-mediated neuroinflammation.
Assuntos
Barreira Hematoencefálica/metabolismo , Glucose/metabolismo , Complexos Multiproteicos/metabolismo , Quinase 1 Relacionada a NIMA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Microglia/metabolismo , Quinase 1 Relacionada a NIMA/genética , Necroptose/genética , Fosforilação , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
In our study, a two-phase culture system was developed to acquire large amount of CD41+ and polyploidy cells. Human mobilized peripheral blood CD34+ (PB CD34+) cells were first cultured in expansion medium (Cocktail or CC100 medium) for 3,4,5 or 6 days, and then cultured in megakaryocytic differentiation medium containing TPO and SCF for additional 7, 8 or 9 days. Cell expansion, morphology, CD41+ cell percentage and DNA content were investigated to evaluate the protocol. The result showed that more CD41+ and polyploidy cells could be obtained following the two-phase culture with Cocktail medium than with CC100. Moreover, with 3 days expansion in Cocktail medium plus 7 days in differentiation medium, the initial CD 34+ cells obtained 16-fold expansion of CD41+ cells and 3-fold expansion of polyploidy cells, such obtained level being significantly higher than that of culturing cells with only one step in TPO or TPO+SCF. We conclude that with the two-phase culture system, PB CD34+ cells can expand and differentiate to more CD41+ and polyploidy cells than those cultured only in accordance to the one-stage culture protocol, so a new and highly efficient megakaryocyte differentiation model for megakaryocyte and platelet related researches is provided already.
Assuntos
Antígenos CD34/sangue , Células Sanguíneas/citologia , Diferenciação Celular/fisiologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Megacariócitos/citologia , Técnicas de Cultura de Células/métodos , Fatores Estimuladores de Colônias/fisiologia , Humanos , Células-Tronco/citologiaRESUMO
The contribution and mechanism of cerebrovascular pathology in Alzheimer's disease (AD) pathogenesis are still unclear. Here, we show that venular and capillary cerebral endothelial cells (ECs) are selectively vulnerable to necroptosis in AD. We identify reduced cerebromicrovascular expression of murine N-acetyltransferase 1 (mNat1) in two AD mouse models and hNat2, the human ortholog of mNat1 and a genetic risk factor for type-2 diabetes and insulin resistance, in human AD. mNat1 deficiency in Nat1-/- mice and two AD mouse models promotes blood-brain barrier (BBB) damage and endothelial necroptosis. Decreased mNat1 expression induces lysosomal degradation of A20, an important regulator of necroptosis, and LRP1ß, a key component of LRP1 complex that exports Aß in cerebral ECs. Selective restoration of cerebral EC expression of mNAT1 delivered by adeno-associated virus (AAV) rescues cerebromicrovascular levels of A20 and LRP1ß, inhibits endothelial necroptosis and activation, ameliorates mitochondrial fragmentation, reduces Aß deposits, and improves cognitive function in the AD mouse model.
Assuntos
Doença de Alzheimer/metabolismo , Arilamina N-Acetiltransferase/metabolismo , Isoenzimas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Arilamina N-Acetiltransferase/genética , Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cérebro/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Humanos , Isoenzimas/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necroptose/fisiologia , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Single-cell RNA sequencing (scRNA-seq) is an exciting new technology allowing the analysis of transcriptomes from individual cells, and is ideally suited to address the inherent complexity and dynamics of the central nervous system. scRNA-seq has already been applied to the study of molecular taxonomy of the brain. These works have paved the way to expanding our understanding of the nervous system and provide insights into cellular susceptibilities and molecular mechanisms in neurological and neurodegenerative diseases. We discuss recent progress and challenges in applying this technology to advance our understanding of the brain. We advocate the application of scRNA-seq in the discovery of targets and biomarkers as a new approach in developing novel therapeutics for the treatment of neurodegenerative diseases.