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1.
J Neurosci ; 44(25)2024 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-38658168

RESUMEN

Hexanucleotide repeat expansions within the gene C9ORF72 are the most common cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This disease-causing expansion leads to a reduction in C9ORF72 expression levels in patients, suggesting loss of C9ORF72 function could contribute to disease. To further understand the consequences of C9ORF72 deficiency in vivo, we generated a c9orf72 mutant zebrafish line. Analysis of the adult female spinal cords revealed no appreciable neurodegenerative pathology such as loss of motor neurons or increased levels of neuroinflammation. However, detailed examination of adult female c9orf72-/- retinas showed prominent neurodegenerative features, including a decrease in retinal thickness, gliosis, and an overall reduction in neurons of all subtypes. Analysis of rod and cone cells within the photoreceptor layer showed a disturbance in their outer segment structure and rhodopsin mislocalization from rod outer segments to their cell bodies and synaptic terminals. Thus, C9ORF72 may play a previously unappreciated role in retinal homeostasis and suggests C9ORF72 deficiency can induce tissue specific neuronal loss.


Asunto(s)
Proteína C9orf72 , Retina , Pez Cebra , Animales , Femenino , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Retina/metabolismo , Retina/patología , Animales Modificados Genéticamente , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/deficiencia , Proteínas/genética , Proteínas/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Médula Espinal/metabolismo , Médula Espinal/patología
2.
PLoS Genet ; 17(4): e1009515, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33914736

RESUMEN

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1ß rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.


Asunto(s)
ARN Guía de Kinetoplastida/genética , Regeneración/genética , Regeneración de la Medula Espinal/genética , Factor de Crecimiento Transformador beta1/genética , Proteínas de Pez Cebra/genética , Animales , Axones/metabolismo , Axones/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Modelos Animales de Enfermedad , Macrófagos/metabolismo , Osteonectina/genética , Recuperación de la Función/genética , Médula Espinal/crecimiento & desarrollo , Médula Espinal/patología , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Regeneración de la Medula Espinal/fisiología , Factor de Crecimiento Transformador beta3/genética , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo
3.
Development ; 143(9): 1464-74, 2016 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-26965370

RESUMEN

In adult zebrafish, relatively quiescent progenitor cells show lesion-induced generation of motor neurons. Developmental motor neuron generation from the spinal motor neuron progenitor domain (pMN) sharply declines at 48 hours post-fertilisation (hpf). After that, mostly oligodendrocytes are generated from the same domain. We demonstrate here that within 48 h of a spinal lesion or specific genetic ablation of motor neurons at 72 hpf, the pMN domain reverts to motor neuron generation at the expense of oligodendrogenesis. By contrast, generation of dorsal Pax2-positive interneurons was not altered. Larval motor neuron regeneration can be boosted by dopaminergic drugs, similar to adult regeneration. We use larval lesions to show that pharmacological suppression of the cellular response of the innate immune system inhibits motor neuron regeneration. Hence, we have established a rapid larval regeneration paradigm. Either mechanical lesions or motor neuron ablation is sufficient to reveal a high degree of developmental flexibility of pMN progenitor cells. In addition, we show an important influence of the immune system on motor neuron regeneration from these progenitor cells.


Asunto(s)
Larva/citología , Neuronas Motoras/citología , Regeneración Nerviosa/fisiología , Células-Madre Neurales/citología , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/citología , Pez Cebra/crecimiento & desarrollo , Animales , Dexametasona/farmacología , Inmunidad Innata/efectos de los fármacos , Inmunosupresores/farmacología , Larva/genética , Macrófagos/inmunología , Metronidazol/farmacología , Microglía/metabolismo , Regeneración Nerviosa/efectos de los fármacos , Oligodendroglía/citología , Factor de Transcripción PAX2/metabolismo , Proteínas de Pez Cebra/metabolismo
4.
Methods Mol Biol ; 2636: 263-277, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36881306

RESUMEN

Larval zebrafish show axonal regrowth over a complex spinal injury site and recovery of function within days after injury. Here we describe a simple protocol to disrupt gene function in this model using acute injections of highly active synthetic gRNAs to rapidly detect loss-of-function phenotypes without the need for breeding.


Asunto(s)
Traumatismos de la Médula Espinal , Pez Cebra , Animales , Pez Cebra/genética , Fenotipo , Traumatismos de la Médula Espinal/genética , Axones , Larva/genética
5.
Dev Cell ; 56(11): 1617-1630.e6, 2021 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-34033756

RESUMEN

Central nervous system injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. Activation of the innate immune system promotes regenerative neurogenesis, but it is fundamentally unknown whether this is indirect through the activation of known developmental signaling pathways or whether immune cells directly signal to progenitor cells using mechanisms that are unique to regeneration. Using single-cell RNA-seq of progenitor cells and macrophages, as well as cell-type-specific manipulations, we provide evidence for a direct signaling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, TNFa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This establishes the principle that macrophages directly communicate to spinal progenitor cells via non-developmental signals after injury, providing potential targets for future interventions in the regeneration-deficient spinal cord of mammals.


Asunto(s)
Histona Desacetilasa 1/genética , Neurogénesis/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Regeneración/genética , Médula Espinal/crecimiento & desarrollo , Proteínas de Pez Cebra/genética , Animales , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/genética , Macrófagos/citología , Macrófagos/metabolismo , RNA-Seq , Transducción de Señal/genética , Análisis de la Célula Individual , Médula Espinal/metabolismo , Células Madre/citología , Células Madre/metabolismo , Factor de Transcripción AP-1/genética , Pez Cebra/genética
6.
Nat Commun ; 9(1): 4670, 2018 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-30405119

RESUMEN

Spinal cord injury leads to a massive response of innate immune cells in non-regenerating mammals, but also in successfully regenerating zebrafish. However, the role of the immune response in successful regeneration is poorly defined. Here we show that inhibiting inflammation reduces and promoting it accelerates axonal regeneration in spinal-lesioned zebrafish larvae. Mutant analyses show that peripheral macrophages, but not neutrophils or microglia, are necessary for repair. Macrophage-less irf8 mutants show prolonged inflammation with elevated levels of Tnf-α and Il-1ß. Inhibiting Tnf-α does not rescue axonal growth in irf8 mutants, but impairs it in wildtype animals, indicating a pro-regenerative role of Tnf-α. In contrast, decreasing Il-1ß levels or number of Il-1ß+ neutrophils rescue functional regeneration in irf8 mutants. However, during early regeneration, interference with Il-1ß function impairs regeneration in irf8 and wildtype animals. Hence, inflammation is dynamically controlled by macrophages to promote functional spinal cord regeneration in zebrafish.


Asunto(s)
Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Regeneración Nerviosa , Médula Espinal/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Pez Cebra/metabolismo , Animales , Axones/metabolismo , Colágeno Tipo XII/metabolismo , Microglía/metabolismo , Microglía/patología , Mutación/genética , Neutrófilos/metabolismo , Médula Espinal/patología , Pez Cebra/inmunología
7.
Nat Commun ; 8(1): 126, 2017 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-28743881

RESUMEN

The inhibitory extracellular matrix in a spinal lesion site is a major impediment to axonal regeneration in mammals. In contrast, the extracellular matrix in zebrafish allows substantial axon re-growth, leading to recovery of movement. However, little is known about regulation and composition of the growth-promoting extracellular matrix. Here we demonstrate that activity of the Wnt/ß-catenin pathway in fibroblast-like cells in the lesion site is pivotal for axon re-growth and functional recovery. Wnt/ß-catenin signaling induces expression of col12a1a/b and deposition of Collagen XII, which is necessary for axons to actively navigate the non-neural lesion site environment. Overexpression of col12a1a rescues the effects of Wnt/ß-catenin pathway inhibition and is sufficient to accelerate regeneration. We demonstrate that in a vertebrate of high regenerative capacity, Wnt/ß-catenin signaling controls the composition of the lesion site extracellular matrix and we identify Collagen XII as a promoter of axonal regeneration. These findings imply that the Wnt/ß-catenin pathway and Collagen XII may be targets for extracellular matrix manipulations in non-regenerating species.Following spinal injury in zebrafish, non-neural cells establish an extracellular matrix to promote axon re-growth but how this is regulated is unclear. Here, the authors show that Wnt/ß-catenin signaling in fibroblast-like cells at a lesion activates axon re-growth via deposition of Collagen XII.


Asunto(s)
Colágeno Tipo XII/metabolismo , Regeneración de la Medula Espinal , Vía de Señalización Wnt , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Colágeno Tipo XII/genética , Larva/genética , Larva/metabolismo , Larva/fisiología , Microscopía Confocal , Recuperación de la Función , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Imagen de Lapso de Tiempo/métodos , Pez Cebra/genética , Pez Cebra/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , beta Catenina/metabolismo
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