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1.
Nat Commun ; 12(1): 5722, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588430

RESUMO

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization.


Assuntos
Neurônios/classificação , Medula Espinal/citologia , Transcriptoma , Animais , Atlas como Assunto , Núcleo Celular/genética , Conjuntos de Dados como Assunto , Camundongos , Neurônios/citologia , RNA-Seq , Análise de Célula Única , Análise Espacial , Medula Espinal/crescimento & desenvolvimento
2.
Cells ; 10(9)2021 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-34571884

RESUMO

In species that regenerate the injured spinal cord, the ependymal region is a source of new cells and a prominent coordinator of regeneration. In mammals, cells at the ependymal region proliferate in normal conditions and react after injury, but in humans, the central canal is lost in the majority of individuals from early childhood. It is replaced by a structure that does not proliferate after damage and is formed by large accumulations of ependymal cells, strong astrogliosis and perivascular pseudo-rosettes. We inform here of two additional mammals that lose the central canal during their lifetime: the Naked Mole-Rat (NMR, Heterocephalus glaber) and the mutant hyh (hydrocephalus with hop gait) mice. The morphological study of their spinal cords shows that the tissue substituting the central canal is not similar to that found in humans. In both NMR and hyh mice, the central canal is replaced by tissue reminiscent of normal lamina X and may include small groups of ependymal cells in the midline, partially resembling specific domains of the former canal. However, no features of the adult human ependymal remnant are found, suggesting that this structure is a specific human trait. In order to shed some more light on the mechanism of human central canal closure, we provide new data suggesting that canal patency is lost by delamination of the ependymal epithelium, in a process that includes apical polarity loss and the expression of signaling mediators involved in epithelial to mesenchymal transitions.


Assuntos
Epêndima/citologia , Medula Espinal/citologia , Adolescente , Adulto , Animais , Biomarcadores/metabolismo , Proliferação de Células , Epêndima/metabolismo , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos Mutantes , Pessoa de Meia-Idade , Ratos-Toupeira , Pan troglodytes , Mutação Puntual , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Especificidade da Espécie , Canal Medular/citologia , Canal Medular/metabolismo , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Adulto Jovem
3.
Nat Commun ; 12(1): 4857, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381039

RESUMO

Physical exercise stimulates adult neurogenesis, yet the underlying mechanisms remain poorly understood. A fundamental component of the innate neuroregenerative capacity of zebrafish is the proliferative and neurogenic ability of the neural stem/progenitor cells. Here, we show that in the intact spinal cord, this plasticity response can be activated by physical exercise by demonstrating that the cholinergic neurotransmission from spinal locomotor neurons activates spinal neural stem/progenitor cells, leading to neurogenesis in the adult zebrafish. We also show that GABA acts in a non-synaptic fashion to maintain neural stem/progenitor cell quiescence in the spinal cord and that training-induced activation of neurogenesis requires a reduction of GABAA receptors. Furthermore, both pharmacological stimulation of cholinergic receptors, as well as interference with GABAergic signaling, promote functional recovery after spinal cord injury. Our findings provide a model for locomotor networks' activity-dependent neurogenesis during homeostasis and regeneration in the adult zebrafish spinal cord.


Assuntos
Locomoção , Neuroglia/metabolismo , Neurônios/metabolismo , Medula Espinal/crescimento & desenvolvimento , Animais , Interneurônios/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Condicionamento Físico Animal , Receptores Colinérgicos/metabolismo , Receptores de GABA-A/metabolismo , Recuperação de Função Fisiológica , Medula Espinal/citologia , Medula Espinal/fisiologia , Transmissão Sináptica , Peixe-Zebra , Ácido gama-Aminobutírico/metabolismo
4.
Cells ; 10(7)2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34359882

RESUMO

Scaffolds of recombinant spider silk protein (spidroin) and hyaluronic acid (HA) hydrogel hold promise in combination with cell therapy for spinal cord injury. However, little is known concerning the human immune response to these biomaterials and grafted human neural stem/progenitor cells (hNPCs). Here, we analyzed short- and long-term in vitro activation of immune cells in human peripheral blood mononuclear cells (hPBMCs) cultured with/without recombinant spidroins, HA hydrogels, and/or allogeneic hNPCs to assess potential host-donor interactions. Viability, proliferation and phenotype of hPBMCs were analyzed using NucleoCounter and flow cytometry. hPBMC viability was confirmed after exposure to the different biomaterials. Short-term (15 h) co-cultures of hPBMCs with spidroins, but not with HA hydrogel, resulted in a significant increase in the proportion of activated CD69+ CD4+ T cells, CD8+ T cells, B cells and NK cells, which likely was caused by residual endotoxins from the Escherichia coli expression system. The observed spidroin-induced hPBMC activation was not altered by hNPCs. It is resource-effective to evaluate human compatibility of novel biomaterials early in development of the production process to, when necessary, make alterations to minimize rejection risk. Here, we present a method to evaluate biomaterials and hPBMC compatibility in conjunction with allogeneic human cells.


Assuntos
Fibroínas/farmacologia , Ácido Hialurônico/farmacologia , Hidrogéis/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Aborto Legal , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Encapsulamento de Células/métodos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Feminino , Feto , Fibroínas/química , Humanos , Ácido Hialurônico/química , Hidrogéis/química , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/imunologia , Gravidez , Primeiro Trimestre da Gravidez , Cultura Primária de Células , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Medula Espinal/citologia , Medula Espinal/imunologia , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/patologia
5.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360788

RESUMO

Neurogenesis timing is an essential developmental mechanism for neuronal diversity and organization throughout the central nervous system. In the mouse spinal cord, growing evidence is beginning to reveal that neurogenesis timing acts in tandem with spatial molecular controls to diversify molecularly and functionally distinct post-mitotic interneuron subpopulations. Particularly, in some cases, this temporal ordering of interneuron differentiation has been shown to instruct specific sensorimotor circuit wirings. In zebrafish, in vivo preparations have revealed that sequential neurogenesis waves of interneurons and motor neurons form speed-dependent locomotor circuits throughout the spinal cord and brainstem. In the present review, we discuss temporal principals of interneuron diversity taken from both mouse and zebrafish systems highlighting how each can lend illuminating insights to the other. Moving forward, it is important to combine the collective knowledge from different systems to eventually understand how temporally regulated subpopulation function differentially across speed- and/or state-dependent sensorimotor movement tasks.


Assuntos
Diferenciação Celular , Interneurônios/metabolismo , Neurogênese , Medula Espinal/embriologia , Peixe-Zebra/embriologia , Animais , Humanos , Interneurônios/citologia , Camundongos , Medula Espinal/citologia
6.
Int J Mol Sci ; 22(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299053

RESUMO

Chronic spinal cord injury (SCI) is a catastrophic condition associated with significant neurological deficit and social and financial burdens. It is currently being managed symptomatically with no real therapeutic strategies available. In recent years, a number of innovative regenerative strategies have emerged and have been continuously investigated in clinical trials. In addition, several more are coming down the translational pipeline. Among ongoing and completed trials are those reporting the use of mesenchymal stem cells, neural stem/progenitor cells, induced pluripotent stem cells, olfactory ensheathing cells, and Schwann cells. The advancements in stem cell technology, combined with the powerful neuroimaging modalities, can now accelerate the pathway of promising novel therapeutic strategies from bench to bedside. Various combinations of different molecular therapies have been combined with supportive scaffolds to facilitate favorable cell-material interactions. In this review, we summarized some of the most recent insights into the preclinical and clinical studies using stem cells and other supportive drugs to unlock the microenvironment in chronic SCI to treat patients with this condition. Successful future therapies will require these stem cells and other synergistic approaches to address the persistent barriers to regeneration, including glial scarring, loss of structural framework, and immunorejection.


Assuntos
Células-Tronco Neurais/citologia , Traumatismos da Medula Espinal/terapia , Medula Espinal/citologia , Transplante de Células-Tronco/métodos , Animais , Humanos
7.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209958

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins-and of secreted metabolites-involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome-lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte-MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Astrócitos/citologia , Metabolômica/métodos , Proteômica/métodos , Superóxido Dismutase/genética , Esclerose Amiotrófica Lateral/genética , Animais , Astrócitos/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Glutationa/metabolismo , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Cultura Primária de Células , Mapas de Interação de Proteínas , Transdução de Sinais , Medula Espinal/citologia , Medula Espinal/metabolismo
8.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208258

RESUMO

Platinum-based chemotherapeutics still play an essential role in cancer treatment. Despite their high effectiveness, severe side effects such as chemotherapy-induced neuropathy (CIPN) occur frequently. The pathophysiology of CIPN by platinum-based chemotherapeutics is not fully understood yet, but primarily the disturbance of dorsal root ganglion cells is discussed. However, there is increasing evidence of central nervous system involvement with activation of spinal cord astrocytes after treatment with chemotherapeutics. We investigated the influence of cis- or oxaliplatin on the functionality of cultured rat spinal cord astrocytes by using immunocytochemistry and patch-clamp electrophysiology. Cis- or oxaliplatin activated spinal astrocytes and led to downregulation of the excitatory amino acid transporter 1 (EAAT1) expression. Furthermore, the expression and function of potassium channel Kir4.1 were modulated. Pre-exposure to a specific Kir4.1 blocker in control astrocytes led to a reduced immune reactivity (IR) of EAAT1 and a nearly complete block of the current density. When spinal astrocytes were pre-exposed to antibiotic minocycline, all effects of cis- or oxaliplatin were abolished. Taken together, the modulation of Kir4.1 and EAAT1 proteins in astrocytes could be linked to the direct impact of cis- or oxaliplatin, identifying spinal astrocytes as a potential target in the prevention and treatment of chemotherapy-induced neuropathy.


Assuntos
Astrócitos/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Platina/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Medula Espinal/citologia , Animais , Antibacterianos/farmacologia , Astrócitos/efeitos dos fármacos , Separação Celular , Células Cultivadas , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Minociclina/farmacologia , Ratos Wistar
9.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299185

RESUMO

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.


Assuntos
Mutação , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neurônios/patologia , Proteína FUS de Ligação a RNA/metabolismo , Medula Espinal/citologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Proteína FUS de Ligação a RNA/genética , Medula Espinal/embriologia , Medula Espinal/metabolismo , Medula Espinal/patologia
10.
Nat Commun ; 12(1): 3251, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059686

RESUMO

ALS is characterized by progressive inability to execute movements. Motor neurons innervating fast-twitch muscle-fibers preferentially degenerate. The reason for this differential vulnerability and its consequences on motor output is not known. Here, we uncover that fast motor neurons receive stronger inhibitory synaptic inputs than slow motor neurons, and disease progression in the SOD1G93A mouse model leads to specific loss of inhibitory synapses onto fast motor neurons. Inhibitory V1 interneurons show similar innervation pattern and loss of synapses. Moreover, from postnatal day 63, there is a loss of V1 interneurons in the SOD1G93A mouse. The V1 interneuron degeneration appears before motor neuron death and is paralleled by the development of a specific locomotor deficit affecting speed and limb coordination. This distinct ALS-induced locomotor deficit is phenocopied in wild-type mice but not in SOD1G93A mice after appearing of the locomotor phenotype when V1 spinal interneurons are silenced. Our study identifies a potential source of non-autonomous motor neuronal vulnerability in ALS and links ALS-induced changes in locomotor phenotype to inhibitory V1-interneurons.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Interneurônios/patologia , Locomoção/fisiologia , Neurônios Motores/patologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fibras Musculares de Contração Rápida/fisiologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Medula Espinal/citologia , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética
11.
Science ; 373(6553)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34083447

RESUMO

The meninges are a membranous structure enveloping the central nervous system (CNS) that host a rich repertoire of immune cells mediating CNS immune surveillance. Here, we report that the mouse meninges contain a pool of monocytes and neutrophils supplied not from the blood but by adjacent skull and vertebral bone marrow. Under pathological conditions, including spinal cord injury and neuroinflammation, CNS-infiltrating myeloid cells can originate from brain borders and display transcriptional signatures distinct from their blood-derived counterparts. Thus, CNS borders are populated by myeloid cells from adjacent bone marrow niches, strategically placed to supply innate immune cells under homeostatic and pathological conditions. These findings call for a reinterpretation of immune-cell infiltration into the CNS during injury and autoimmunity and may inform future therapeutic approaches that harness meningeal immune cells.


Assuntos
Células da Medula Óssea/fisiologia , Doenças do Sistema Nervoso Central/imunologia , Sistema Nervoso Central/imunologia , Meninges/imunologia , Células Mieloides/fisiologia , Crânio/anatomia & histologia , Coluna Vertebral/anatomia & histologia , Animais , Medula Óssea/fisiologia , Encéfalo/citologia , Encéfalo/imunologia , Encéfalo/fisiologia , Movimento Celular , Sistema Nervoso Central/citologia , Doenças do Sistema Nervoso Central/patologia , Dura-Máter/citologia , Dura-Máter/imunologia , Dura-Máter/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Homeostase , Meninges/citologia , Meninges/fisiologia , Camundongos , Monócitos/fisiologia , Neutrófilos/fisiologia , Medula Espinal/citologia , Medula Espinal/imunologia , Medula Espinal/fisiologia , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/patologia
12.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070932

RESUMO

The neuronal networks that generate locomotion are well understood in swimming animals such as the lamprey, zebrafish and tadpole. The networks controlling locomotion in tetrapods remain, however, still enigmatic with an intricate motor pattern required for the control of the entire limb during the support, lift off, and flexion phase, and most demandingly when the limb makes contact with ground again. It is clear that the inhibition that occurs between bursts in each step cycle is produced by V2b and V1 interneurons, and that a deletion of these interneurons leads to synchronous flexor-extensor bursting. The ability to generate rhythmic bursting is distributed over all segments comprising part of the central pattern generator network (CPG). It is unclear how the rhythmic bursting is generated; however, Shox2, V2a and HB9 interneurons do contribute. To deduce a possible organization of the locomotor CPG, simulations have been elaborated. The motor pattern has been simulated in considerable detail with a network composed of unit burst generators; one for each group of close synergistic muscle groups at each joint. This unit burst generator model can reproduce the complex burst pattern with a constant flexion phase and a shortened extensor phase as the speed increases. Moreover, the unit burst generator model is versatile and can generate both forward and backward locomotion.


Assuntos
Geradores de Padrão Central/fisiologia , Interneurônios/fisiologia , Locomoção/fisiologia , Atividade Motora/fisiologia , Redes Neurais de Computação , Medula Espinal/fisiologia , Animais , Gatos , Geradores de Padrão Central/citologia , Simulação por Computador , Extremidades/inervação , Extremidades/fisiologia , Humanos , Interneurônios/citologia , Lampreias/fisiologia , Larva/fisiologia , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Roedores/fisiologia , Medula Espinal/citologia , Peixe-Zebra/fisiologia
13.
Elife ; 102021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33942723

RESUMO

A major barrier to intraspinal regeneration after dorsal root (DR) injury is the DR entry zone (DREZ), the CNS/PNS interface. DR axons stop regenerating at the DREZ, even if regenerative capacity is increased by a nerve conditioning lesion. This potent blockade has long been attributed to myelin-associated inhibitors and (CSPGs), but incomplete lesions and conflicting reports have prevented conclusive agreement. Here, we evaluated DR regeneration in mice using novel strategies to facilitate complete lesions and analyses, selective tracing of proprioceptive and mechanoreceptive axons, and the first simultaneous targeting of Nogo/Reticulon-4, MAG, OMgp, CSPGs, and GDNF. Co-eliminating myelin inhibitors and CSPGs elicited regeneration of only a few conditioning-lesioned DR axons across the DREZ. Their absence, however, markedly and synergistically enhanced regeneration of GDNF-stimulated axons, highlighting the importance of sufficiently elevating intrinsic growth capacity. We also conclude that myelin inhibitors and CSPGs are not the primary mechanism stopping axons at the DREZ.


Assuntos
Axônios/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Bainha de Mielina/metabolismo , Medula Espinal/citologia , Raízes Nervosas Espinhais/patologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
14.
Neurochem Res ; 46(8): 1970-1980, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33973102

RESUMO

Reportedly, TWIK-related spinal cord K+ (TRESK) deficiency in spinal cord neurons positively correlates with the mechanism underlying neuropathic pain (NP). However, the precise effects of TRESK on neurons of the spinal cord remain elusive. In the present study, we investigated the impact of TRESK silencing on spinal cord neurons to further elucidate the downstream mechanisms of TRESK. Herein, neurons of the dorsal spinal cord were cultured as a cell model for investigations. Apoptosis, oxidative stress, and DNA damage-related proteins were evaluated. Additionally, flow cytometry, microarray profiling, real-time polymerase chain reaction (PCR), western blotting, fluorescence in situ hybridization (FISH), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were performed. In cultured neurons, the downregulation of TRESK mRNA expression induced apoptosis of dorsal spinal cord neurons. Using real-time PCR and western blotting, the upregulation of LncRNA Gm11874 (Gm11874) and ATP5i, screened from the gene chip, was confirmed. On silencing TRESK, expression levels of γ-H2AX, poly [ADP-ribose] polymerase 1 (PARP-1), FoxO1, FoxO3, MitoSOX, malondialdehyde (MDA), and 8-hydroxy-2' -deoxyguanosine (8-OHdG), which are known indices of oxidative stress and DNA damage, were significantly elevated. Moreover, ATP induced oxidative stress, DNA damage, and apoptosis were reduced by ATP5i siRNA. Finally, Gm11874 and ATP5i were co-expressed in spinal cord neurons in a FISH experiment, and the expression of ATP5i was positively regulated by Gm11874. These results implied that ATP5i induced oxidative stress and DNA damage, resulting in neuronal apoptosis, and Gm11874 was confirmed to act upstream of ATP5i. Our study revealed that TRESK silencing upregulated Gm11874 to induce apoptosis of spinal cord neurons, which resulted in ATP5i promoting oxidative stress and DNA damage. These findings could highlight the TRESK-mediated NP mechanism.


Assuntos
Apoptose/fisiologia , Dano ao DNA/fisiologia , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Canais de Potássio/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Camundongos , RNA Interferente Pequeno/farmacologia , Medula Espinal/citologia , Medula Espinal/metabolismo , Regulação para Cima/fisiologia
15.
Nat Commun ; 12(1): 2471, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931636

RESUMO

In vertebrates, motor control relies on cholinergic neurons in the spinal cord that have been extensively studied over the past hundred years, yet the full heterogeneity of these neurons and their different functional roles in the adult remain to be defined. Here, we develop a targeted single nuclear RNA sequencing approach and use it to identify an array of cholinergic interneurons, visceral and skeletal motor neurons. Our data expose markers for distinguishing these classes of cholinergic neurons and their rich diversity. Specifically, visceral motor neurons, which provide autonomic control, can be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons is also reflected in our analysis with alpha, gamma, and a third subtype, possibly corresponding to the elusive beta motor neurons, clearly distinguished. In combination, our data provide a comprehensive transcriptomic description of this important population of neurons that control many aspects of physiology and movement and encompass the cellular substrates for debilitating degenerative disorders.


Assuntos
Neurônios Colinérgicos/citologia , Interneurônios/citologia , Neurônios Motores/citologia , Análise de Célula Única/métodos , Núcleo Solitário/metabolismo , Medula Espinal/metabolismo , Transcriptoma/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Feminino , Hibridização In Situ , Interneurônios/metabolismo , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , RNA-Seq , Medula Espinal/citologia , Medula Espinal/fisiologia
16.
Mol Syst Biol ; 17(4): e9945, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33890404

RESUMO

Positive feedback driven by transcriptional regulation has long been considered a key mechanism underlying cell lineage segregation during embryogenesis. Using the developing spinal cord as a paradigm, we found that canonical, transcription-driven feedback cannot explain robust lineage segregation of motor neuron subtypes marked by two cardinal factors, Hoxa5 and Hoxc8. We propose a feedback mechanism involving elementary microRNA-mRNA reaction circuits that differ from known feedback loop-like structures. Strikingly, we show that a wide range of biologically plausible post-transcriptional regulatory parameters are sufficient to generate bistable switches, a hallmark of positive feedback. Through mathematical analysis, we explain intuitively the hidden source of this feedback. Using embryonic stem cell differentiation and mouse genetics, we corroborate that microRNA-mRNA circuits govern tissue boundaries and hysteresis upon motor neuron differentiation with respect to transient morphogen signals. Our findings reveal a previously underappreciated feedback mechanism that may have widespread functions in cell fate decisions and tissue patterning.


Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Retroalimentação Fisiológica , MicroRNAs/genética , Neurônios Motores/metabolismo , Medula Espinal/citologia , Animais , Sequência de Bases , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/metabolismo , Cinética , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq , Transdução de Sinais , Análise de Célula Única , Fatores de Transcrição/metabolismo , Transcrição Genética , Tretinoína/metabolismo
17.
J Neurochem ; 158(2): 482-499, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33905537

RESUMO

Nucleocytosolic transport, a membrane process, is impaired in motor neurons in amyotrophic lateral sclerosis (ALS). This study analyzes the nuclear lipidome in motor neurons in ALS and examines molecular pathways linked to the major lipid alterations. Nuclei were obtained from the frozen anterior horn of the lumbar spinal cord of ALS patients and age-matched controls. Lipidomic profiles of this subcellular fraction were obtained using liquid chromatography and mass spectrometry. We validated the mechanisms behind presumable lipidomic changes by exploring ALS surrogate models including human motor neurons (derived from ALS lines and controls) subjected to oxidative stress, the hSOD-G93A transgenic mice, and samples from an independent cohort of ALS patients. Among the differential lipid species, we noted 41 potential identities, mostly belonging to phospholipids (particularly ether phospholipids, as plasmalogens), as well as diacylglycerols and triacylglycerides. Decreased expression of alkyldihydroxyacetonephosphate synthase (AGPS)-a critical peroxisomal enzyme in plasmalogen synthesis-is found in motor neuron disease models; this occurs in parallel with an increase in the expression of sterol carrier protein 2 (SCP2) mRNA in ALS and Scp2 levels in G93A transgenic mice. Further, we identified diminished expression of diacylglycerol-related enzymes, such as phospholipase C ßI (PLCßI) and protein kinase CßII (PKCßII), linked to diacylglycerol metabolism. Finally, lipid droplets were recognized in the nuclei, supporting the identification of triacylglycerides as differential lipids. Our results point to the potentially pathogenic role of altered composition of nuclear membrane lipids and lipids in the nucleoplasm in the anterior horn of the spinal cord in ALS. Overall, these data support the usefulness of subcellular lipidomics applied to neurodegenerative diseases.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Núcleo Celular/genética , Lipidômica , Idoso , Animais , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Citosol/metabolismo , Diglicerídeos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios Motores/metabolismo , Estresse Oxidativo , Projetos Piloto , Medula Espinal/citologia , Medula Espinal/metabolismo , Frações Subcelulares/metabolismo , Superóxido Dismutase-1
18.
Cells ; 10(3)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804060

RESUMO

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, the role of each central nervous system (CNS)-resident cell type during inflammation, neurodegeneration, and remission has been frequently addressed. Although protocols for the isolation of different individual CNS-resident cell types exist, none can harvest all of them within a single experiment. In addition, isolation of individual cells is more demanding in adult mice and even more so from the inflamed CNS. Here, we present a protocol for the simultaneous purification of viable single-cell suspensions of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) from adult mice-applicable in healthy mice as well as in EAE. After dissociation of the brain and spinal cord from adult mice, microglia, oligodendrocytes, astrocytes and, neurons were isolated via magnetic-activated cell sorting (MACS). Validations comprised flow cytometry, immunocytochemistry, as well as functional analyses (immunoassay and Sholl analysis). The purity of each cell isolation averaged 90%. All cells displayed cell-type-specific morphologies and expressed specific surface markers. In conclusion, this new protocol for the simultaneous isolation of all major CNS-resident cell types from one CNS offers a sophisticated and comprehensive way to investigate complex cellular networks ex vivo and simultaneously reduce mice numbers to be sacrificed.


Assuntos
Encéfalo/citologia , Separação Celular , Microglia/citologia , Oligodendroglia/citologia , Medula Espinal/citologia , Animais , Encéfalo/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Inflamação/metabolismo , Camundongos , Microglia/metabolismo , Esclerose Múltipla/metabolismo , Oligodendroglia/metabolismo , Medula Espinal/metabolismo
19.
Science ; 372(6540): 385-393, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33888637

RESUMO

Motor and sensory functions of the spinal cord are mediated by populations of cardinal neurons arising from separate progenitor lineages. However, each cardinal class is composed of multiple neuronal types with distinct molecular, anatomical, and physiological features, and there is not a unifying logic that systematically accounts for this diversity. We reasoned that the expansion of new neuronal types occurred in a stepwise manner analogous to animal speciation, and we explored this by defining transcriptomic relationships using a top-down approach. We uncovered orderly genetic tiers that sequentially divide groups of neurons by their motor-sensory, local-long range, and excitatory-inhibitory features. The genetic signatures defining neuronal projections were tied to neuronal birth date and conserved across cardinal classes. Thus, the intersection of cardinal class with projection markers provides a unifying taxonomic solution for systematically identifying distinct functional subsets.


Assuntos
Vias Neurais , Neurônios/fisiologia , Medula Espinal/citologia , Transcriptoma , Animais , Medula Cervical/citologia , Feminino , Masculino , Camundongos , Neurônios Motores/fisiologia , Propriocepção , RNA-Seq , Células Receptoras Sensoriais/fisiologia , Análise de Célula Única , Análise Espacial , Medula Espinal/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Cell Biochem Funct ; 39(5): 679-687, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33904209

RESUMO

The resumption of blood supply in spinal cord (SC) after injury is a prerequisite of its recovery. To expose the mechanisms of damaged SC revascularization we have used an organotypic SC/aortic fragments (AF) co-culture where, as we showed previously, damaged SC tissue induces AF cell sprouting but repels them away. Supplementation of culture medium with exogenous VEGF-A165 redirects the migrating aortic endothelial cells towards SC tissue. This effect and the pattern of sFlt1 expression (a soluble form of VEGFR1) suggest that the low level of SC-secreted VEGF and the presence of sFlt1 in SC slices together prevent the migration of aortic CD31+ cells to the SC in the absence of exogenous VEGF. VEGF-A165 supplementation sequesters this inhibitory activity of sFlt1 by direct binding thus allowing CD31+ cell migration in to SC tissue. Proteome analysis has shown that migration/proliferation of CD31+ and αSMA+ aortic cells in neuronal culture medium used in our SC/AF model (which obstruct sprouting by itself) was resumed by combined action of several pro- (aFGF, bFGF, Osteopontin, TF, IGFBP2, SDF1) and anti-angiogenic (Endostatin/Collagen18) factors. The mutual influence of AF and SC tissues is a key factor balancing these factors and thus driving endothelial sprouting in SC injury zone.


Assuntos
Aorta/citologia , Técnicas de Cocultura , Células Endoteliais/metabolismo , Medula Espinal/citologia , Animais , Aorta/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Medula Espinal/metabolismo
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