RESUMO
Disability in multiple sclerosis (MS) is driven in part by the failure of remyelination and progressive neurodegeneration. Microglia, and specifically triggering receptor expressed on myeloid cells 2 (TREM2), a factor highly expressed in microglia, have been shown to play an important role in remyelination. Here, using a focal demyelination model in the brain, we demonstrate that demyelination is persistent in TREM2 knockout mice, lasting more than 6 weeks after lysolecithin injection and resulting in substantial neurodegeneration. We also find that TREM2 knockout mice exhibit an altered glial response following demyelination. TREM2 knockout microglia demonstrate defects in migration and phagocytosis of myelin debris. In addition, human monocyte-derived macrophages from subjects with a TREM2 mutation prevalent in human disease also show a defect in myelin debris phagocytosis. Together, we highlight the central role of TREM2 signaling in remyelination and neuroprotection. These findings provide insights into how chronic demyelination might lead to axonal damage and could help identify novel neuroprotective therapeutic targets for MS.
Assuntos
Esclerose Múltipla , Remielinização , Animais , Camundongos , Humanos , Microglia/fisiologia , Neuroproteção , Esclerose Múltipla/tratamento farmacológico , Bainha de Mielina , Camundongos Knockout , Camundongos Endogâmicos C57BL , Glicoproteínas de Membrana/genética , Receptores Imunológicos/genéticaRESUMO
Gene therapy constitutes one of the most promising mode of disease treatments. Two key properties for therapeutic delivery vectors are its transduction efficiency (how well the vector delivers therapeutic cargo to desired target cells) and specificity (how well it avoids off-target delivery into unintended cells within the body). Here we developed an integrated bioinformatics and experimental pipeline that enables multiplex measurement of transduction efficiency and specificity, particularly by measuring how libraries of delivery vectors transduce libraries of diverse cell types. We demonstrated that pairing high-throughput measurement of AAV identity with high-resolution single-cell RNA transcriptomic sequencing maps how natural and engineered AAV variants transduce individual cells within human cerebral and ocular organoids. We further demonstrate that efficient AAV transduction observed in organoids is recapitulated in vivo in non-human primates. This library-on-library technology will be important for determining the safety and efficacy of therapeutic delivery vectors.
Assuntos
Dependovirus , Vetores Genéticos , Animais , Bioensaio , Dependovirus/metabolismo , Vetores Genéticos/genética , RNA/metabolismo , Transdução Genética , Tropismo ViralRESUMO
Oligodendrocyte dysfunction has been implicated in the pathogenesis of neurodegenerative diseases, so understanding oligodendrocyte activation states would shed light on disease processes. We identify three distinct activation states of oligodendrocytes from single-cell RNA sequencing (RNA-seq) of mouse models of Alzheimer's disease (AD) and multiple sclerosis (MS): DA1 (disease-associated1, associated with immunogenic genes), DA2 (disease-associated2, associated with genes influencing survival), and IFN (associated with interferon response genes). Spatial analysis of disease-associated oligodendrocytes (DAOs) in the cuprizone model reveals that DA1 and DA2 are established outside of the lesion area during demyelination and that DA1 repopulates the lesion during remyelination. Independent meta-analysis of human single-nucleus RNA-seq datasets reveals that the transcriptional responses of MS oligodendrocytes share features with mouse models. In contrast, the oligodendrocyte activation signature observed in human AD is largely distinct from those observed in mice. This catalog of oligodendrocyte activation states (http://research-pub.gene.com/OligoLandscape/) will be important to understand disease progression and develop therapeutic interventions.
Assuntos
Doenças Desmielinizantes , Esclerose Múltipla , Doenças Neurodegenerativas , Animais , Cuprizona/uso terapêutico , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , OligodendrogliaRESUMO
As the primary phagocytic cells of the central nervous system, microglia exquisitely regulate their lysosomal activity to facilitate brain development and homeostasis. However, mechanisms that coordinate lysosomal activity with microglia development, chemotaxis, and function remain unclear. Here, we show that embryonic macrophages require the lysosomal guanosine triphosphatase (GTPase) RagA and the GTPase-activating protein Folliculin to colonize the brain in zebrafish. We demonstrate that embryonic macrophages in rraga mutants show increased expression of lysosomal genes but display significant down-regulation of immune- and chemotaxis-related genes. Furthermore, we find that RagA and Folliculin repress the key lysosomal transcription factor Tfeb and its homologs Tfe3a and Tfe3b in the macrophage lineage. Using RNA sequencing, we establish that Tfeb and Tfe3 are required for activation of lysosomal target genes under conditions of stress but not for basal expression of lysosomal pathways. Collectively, our data define a lysosomal regulatory circuit essential for macrophage development and function in vivo.
RESUMO
The gene GPNMB is known to play roles in phagocytosis and tissue repair, and is upregulated in microglia in many mouse models of neurodegenerative disease as well as in human patients. Nearby genomic variants are associated with both elevated Parkinson's disease (PD) risk and higher expression of this gene, suggesting that inhibiting GPNMB activity might be protective in Parkinson's disease. We tested this hypothesis in three different mouse models of neurological diseases: a remyelination model and two models of alpha-synuclein pathology. We found that Gpnmb deletion had no effect on histological, cellular, behavioral, neurochemical or gene expression phenotypes in any of these models. These data suggest that Gpnmb does not play a major role in the development of pathology or functional defects in these models and that further work is necessary to study its role in the development or progression of Parkinson's disease.
Assuntos
Proteínas do Olho/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Doença de Parkinson/metabolismo , Remielinização/genética , Substância Negra/metabolismo , Sinucleinopatias/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Doença de Parkinson/patologia , Substância Negra/patologia , Sinucleinopatias/metabolismo , Sinucleinopatias/patologiaRESUMO
In multiple sclerosis (MS) and other neurological diseases, the failure to repair demyelinated lesions contributes to axonal damage and clinical disability. Here, we provide evidence that Mertk, a gene highly expressed by microglia that alters MS risk, is required for efficient remyelination. Compared to wild-type (WT) mice, Mertk-knockout (KO) mice show impaired clearance of myelin debris and remyelination following demyelination. Using single-cell RNA sequencing, we characterize Mertk-influenced responses to cuprizone-mediated demyelination and remyelination across different cell types. Mertk-KO brains show an attenuated microglial response to demyelination but an elevated proportion of interferon (IFN)-responsive microglia. In addition, we identify a transcriptionally distinct subtype of surviving oligodendrocytes specific to demyelinated lesions. The inhibitory effect of myelin debris on remyelination is mediated in part by IFNγ, which further impedes microglial clearance of myelin debris and inhibits oligodendrocyte differentiation. Together, our work establishes a role for Mertk in microglia activation, phagocytosis, and migration during remyelination.
Assuntos
Microglia/metabolismo , Esclerose Múltipla/patologia , c-Mer Tirosina Quinase/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Cuprizona/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Esclerose Múltipla/genética , Bainha de Mielina/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fagocitose , Remielinização/efeitos dos fármacos , c-Mer Tirosina Quinase/deficiência , c-Mer Tirosina Quinase/genéticaRESUMO
Studying myelination in vitro and in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs) in vitro, although scalable, does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe an ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0-2 mice, after which they myelinate ex vivo for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.
Assuntos
Cerebelo/crescimento & desenvolvimento , Cerebelo/patologia , Bainha de Mielina/metabolismo , Remielinização/fisiologia , Ativinas/metabolismo , Animais , Diferenciação Celular , Meios de Cultura , Modelos Animais de Doenças , Compostos Heterocíclicos com 3 Anéis/farmacologia , Camundongos Transgênicos , Oligodendroglia/citologiaRESUMO
Cell growth is controlled by a lysosomal signalling complex containing Rag small GTPases and mammalian target of rapamycin complex 1 (mTORC1) kinase. Here, we carried out a microscopy-based genome-wide human short interfering RNA screen and discovered a lysosome-localized G protein-coupled receptor (GPCR)-like protein, GPR137B, that interacts with Rag GTPases, increases Rag localization and activity, and thereby regulates mTORC1 translocation and activity. High GPR137B expression can recruit and activate mTORC1 in the absence of amino acids. Furthermore, GPR137B also regulates the dissociation of activated Rag from lysosomes, suggesting that GPR137B controls a cycle of Rag activation and dissociation from lysosomes. GPR137B-knockout cells exhibited defective autophagy and an expanded lysosome compartment, similar to Rag-knockout cells. Like zebrafish RagA mutants, GPR137B-mutant zebrafish had upregulated TFEB target gene expression and an expanded lysosome compartment in microglia. Thus, GPR137B is a GPCR-like lysosomal regulatory protein that controls dynamic Rag and mTORC1 localization and activity as well as lysosome morphology.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Genoma Humano/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas G/genética , Animais , Autofagia/genética , Regulação da Expressão Gênica/genética , Humanos , Lisossomos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Microglia/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Myelin allows for fast and efficient axonal conduction, but much remains to be determined about the mechanisms that regulate myelin formation. To investigate the genetic basis of myelination, we carried out a genetic screen using zebrafish. Here, we show that the lysosomal G protein RagA is essential for CNS myelination. In rraga-/- mutant oligodendrocytes, target genes of the lysosomal transcription factor Tfeb are upregulated, consistent with previous evidence that RagA represses Tfeb activity. Loss of Tfeb function is sufficient to restore myelination in RagA mutants, indicating that hyperactive Tfeb represses myelination. Conversely, tfeb-/- single mutants exhibit ectopic myelin, further indicating that Tfeb represses myelination during development. In a mouse model of de- and remyelination, TFEB expression is increased in oligodendrocytes, but the protein is localized to the cytoplasm, and hence inactive, especially during remyelination. These results define essential regulators of myelination and may advance approaches to therapeutic remyelination.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Bainha de Mielina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Endossomos/metabolismo , Proteínas de Homeodomínio/genética , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Oligodendroglia/fisiologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.
Assuntos
Mesoderma/citologia , Transdução de Sinais , Proteínas Morfogenéticas Ósseas/metabolismo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Coração/crescimento & desenvolvimento , Proteínas de Homeodomínio/metabolismo , Humanos , Mesoderma/metabolismo , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/metabolismo , Análise de Célula Única , Somitos/metabolismo , Células-Tronco , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismoRESUMO
Microglia are resident macrophages of the CNS that are essential for phagocytosis of apoptotic neurons and weak synapses during development. We show that RagA and Lamtor4, two components of the Rag-Ragulator complex, are essential regulators of lysosomes in microglia. In zebrafish lacking RagA function, microglia exhibit an expanded lysosomal compartment, but they are unable to properly digest apoptotic neuronal debris. Previous biochemical studies have placed the Rag-Ragulator complex upstream of mTORC1 activation in response to cellular nutrient availability. Nonetheless, RagA and mTOR mutant zebrafish have distinct phenotypes, indicating that the Rag-Ragulator complex has functions independent of mTOR signaling. Our analysis reveals an essential role of the Rag-Ragulator complex in proper lysosome function and phagocytic flux in microglia.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Lisossomos/metabolismo , Microglia/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Análise Mutacional de DNA , Embrião não Mamífero/metabolismo , Hibridização In Situ , Microscopia Confocal , Mutagênese , Fagocitose , Fenótipo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Mutations in Kinesin proteins (Kifs) are linked to various neurological diseases, but the specific and redundant functions of the vertebrate Kifs are incompletely understood. For example, Kif5A, but not other Kinesin-1 heavy-chain family members, is implicated in Charcot-Marie-Tooth disease (CMT) and Hereditary Spastic Paraplegia (HSP), but the mechanism of its involvement in the progressive axonal degeneration characteristic of these diseases is not well understood. We report that zebrafish kif5Aa mutants exhibit hyperexcitability, peripheral polyneuropathy, and axonal degeneration reminiscent of CMT and HSP. Strikingly, although kif5 genes are thought to act largely redundantly in other contexts, and zebrafish peripheral neurons express five kif5 genes, kif5Aa mutant peripheral sensory axons lack mitochondria and degenerate. We show that this Kif5Aa-specific function is cell autonomous and is mediated by its C-terminal tail, as only Kif5Aa and chimeric motors containing the Kif5Aa C-tail can rescue deficits. Finally, concurrent loss of the kinesin-3, kif1b, or its adaptor kbp, exacerbates axonal degeneration via a nonmitochondrial cargo common to Kif5Aa. Our results shed light on Kinesin complexity and reveal determinants of specific Kif5A functions in mitochondrial transport, adaptor binding, and axonal maintenance.