A zebrafish model of CLN2 disease is deficient in tripeptidyl peptidase 1 and displays progressive neurodegeneration accompanied by a reduction in proliferation

Abstract

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.

Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms

“…Also known as CLN2 disease, LINCL is caused by loss-of-function mutations in the CLN2 gene encoding Tri-peptidyl peptidase 1 (TPP1), 80 and interestingly, the majority of symptoms and pathologies found in LINCL are also present in the zebrafish tpp1 mutant. 81 Thus, tpp1 homozygous mutant larvae at 72 hours post-fertilisation (hpf) exhibit much more frequent and prolonged bouts of movement than wild-type siblings. At later stages, tpp1 mutants become immobile and unresponsive to touch, making the detection of seizures more challenging.…”

Building a zebrafish toolkit for investigating the pathobiology of epilepsy and identifying new treatments for epileptic seizures

“…One of the first genetic studies of seizure susceptibility in zebrafish identified mutations which suppressed the ability of PTZ to induce acute seizures in larvae (Baraban et al, 2007). Other mutations have since been identified which promote seizure onset, including the mind bomb mutation of the Notch pathway regulator ube3a (Hortopan et al, 2010), and mutations in zebrafish orthologues of OCRL1 (Ramirez et al, 2012), the sodium channel subunit SCN1A ( Baraban et al, 2013), the neuronal ceroid lipofuscinosis (CLN2) disease gene TPP1 (Mahmood et al, 2013a), the EAAT2 glutamate transporter mutated in both epilepsy and Amyotrophic Lateral Sclerosis (McKeown et al, 2012) and the glycine receptor subunit GLRB mutated in hyperekplexia (Hirata et al, 2005). The use of antisense morpholinos to knock-down expression of specific genes provides an alternative approach to mutant analysis for elucidating gene functions in zebrafish, revealing roles for many additional genes in determining seizure susceptibility.…”

Developing ‘integrative’ zebrafish models of behavioral and metabolic disorders

“…Likewise, transgenic mouse studies show that neurodegeneration itself may contribute to obesity [253]. Given the utility of zebrafish models to study aging [254,255], obesity (Tables 2 and 3) and neurodegeneration [256,257], it is possible to expect that novel aquatic models can be useful to mimic pathogenetic interplay between metabolic and neurodegenerative disorders. With the growing population affected by obesity, AD and PD worldwide [258], this direction of zebrafish biomedical research becomes especially timely and clinically relevant.…”