RESUMEN
The neuronal ceroid lipofuscinoses are a group of lysosomal storage disorders that comprise the most common, genetically heterogeneous, fatal neurodegenerative disorders of children. They are characterised by childhood onset, visual failure, epileptic seizures, psychomotor retardation and dementia. CLN3 disease, also known as Batten disease, is caused by autosomal recessive mutations in the CLN3 gene, 80-85% of which are a ~1 kb deletion. Currently no treatments exist, and after much suffering, the disease inevitably results in premature death. The aim of this study was to generate a zebrafish model of CLN3 disease using antisense morpholino injection, and characterise the pathological and functional consequences of Cln3 deficiency, thereby providing a tool for future drug discovery. The model was shown to faithfully recapitulate the pathological signs of CLN3 disease, including reduced survival, neuronal loss, retinopathy, axonopathy, loss of motor function, lysosomal storage of subunit c of mitochondrial ATP synthase, and epileptic seizures, albeit with an earlier onset and faster progression than the human disease. Our study provides proof of principle that the advantages of the zebrafish over other model systems can be utilised to further our understanding of the pathogenesis of CLN3 disease and accelerate drug discovery.
Asunto(s)
Epilepsia/complicaciones , Degeneración Nerviosa/complicaciones , Lipofuscinosis Ceroideas Neuronales/complicaciones , Pez Cebra/fisiología , Animales , Apoptosis/efectos de los fármacos , Axones/efectos de los fármacos , Axones/patología , Conducta Animal/efectos de los fármacos , Encéfalo/anomalías , Encéfalo/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia/patología , Técnicas de Silenciamiento del Gen , Gliosis/patología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Morfolinos/farmacología , Morfolinos/toxicidad , Actividad Motora/efectos de los fármacos , Miocardio/patología , Degeneración Nerviosa/patología , Lipofuscinosis Ceroideas Neuronales/patología , Subunidades de Proteína/metabolismo , ARN sin Sentido/metabolismo , Retina/efectos de los fármacos , Retina/patología , Análisis de Supervivencia , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Tripeptidyl peptidase 1 (TPP1) deficiency causes CLN2 disease, late infantile (or classic late infantile neuronal ceroid lipofuscinosis), a paediatric neurodegenerative disease of autosomal recessive inheritance. Patients suffer from blindness, ataxia, epilepsy and cognitive defects, with MRI indicating widespread brain atrophy, and profound neuron loss is evident within the retina and brain. Currently there are no effective therapies for this disease, which causes premature death in adolescence. Zebrafish have been successfully used to model a range of neurological and behavioural abnormalities. The aim of this study was to characterize the pathological and functional consequences of Tpp1 deficiency in zebrafish and to correlate these with human CLN2 disease, thereby providing a platform for drug discovery. Our data show that homozygous tpp1(sa0011) mutant (tpp1(sa0011)(-/-)) zebrafish display a severe, progressive, early onset neurodegenerative phenotype, characterized by a significantly small retina, a small head and curved body. The mutant zebrafish have significantly reduced median survival with death occurring 5 days post-fertilization. As in human patients with CLN2 disease, mutant zebrafish display storage of subunit c of mitochondrial ATP-synthase, hypertrophic lysosomes as well as localized apoptotic cell death in the retina, optic tectum and cerebellum. Further neuropathological phenotypes of these mutants provide novel insights into mechanisms of pathogenesis in CLN2 disease. Secondary neurogenesis in the retina, optic tectum and cerebellum is impaired and axon tracts within the spinal cord, optic nerve and the posterior commissure are disorganized, with the optic nerve failing to reach its target. This severe neurodegenerative phenotype eventually results in functional motor impairment, but this is preceded by a phase of hyperactivity that is consistent with seizures. Importantly, both of these locomotion phenotypes can be assayed in an automated manner suitable for high-throughput studies. Our study provides proof-of-principle that tpp1(sa0011)(-/-) mutants can utilize the advantages of zebrafish for understanding pathogenesis and drug discovery in CLN2 disease and other epilepsies.