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Mol Neurodegener ; 15(1): 19, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-32143659


The two main pathological hallmarks of Parkinson's disease are loss of dopamine neurons in the substantia nigra pars compacta and proteinaceous amyloid fibrils composed mostly of α-synuclein, called Lewy pathology. Levodopa to enhance dopaminergic transmission remains one of the most effective treatment for alleviating the motor symptoms of Parkinson's disease (Olanow, Mov Disord 34:812-815, 2019). In addition, deep brain stimulation (Bronstein et al., Arch Neurol 68:165, 2011) to modulate basal ganglia circuit activity successfully alleviates some motor symptoms. MRI guided focused ultrasound in the subthalamic nucleus is a promising therapeutic strategy as well (Martinez-Fernandez et al., Lancet Neurol 17:54-63, 2018). However, to date, there exists no treatment that stops the progression of this disease. The findings that α-synuclein can be released from neurons and inherited through interconnected neural networks opened the door for discovering novel treatment strategies to prevent the formation and spread of Lewy pathology with the goal of halting PD in its tracks. This hypothesis is based on discoveries that pathologic aggregates of α-synuclein induce the endogenous α-synuclein protein to adopt a similar pathologic conformation, and is thus self-propagating. Phase I clinical trials are currently ongoing to test treatments such as immunotherapy to prevent the neuron to neuron spread of extracellular aggregates. Although tremendous progress has been made in understanding how Lewy pathology forms and spreads throughout the brain, cell intrinsic factors also play a critical role in the formation of pathologic α-synuclein, such as mechanisms that increase endogenous α-synuclein levels, selective expression profiles in distinct neuron subtypes, mutations and altered function of proteins involved in α-synuclein synthesis and degradation, and oxidative stress. Strategies that prevent the formation of pathologic α-synuclein should consider extracellular release and propagation, as well as neuron intrinsic mechanisms.

Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Progresión de la Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Agregado de Proteínas/fisiología
J Genet ; 97(5): 1315-1325, 2018 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30555080


Nodal-related protein (ndr2) is amember of the transforming growth factor type ß superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene.

Sistemas CRISPR-Cas , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Ribonucleoproteínas/genética , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Holoprosencefalia/genética , Péptidos y Proteínas de Señalización Intracelular/química , Modelos Moleculares , Fenotipo , Dominios Proteicos , Ribonucleoproteínas/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química