Neurotoxin exposure of zebrafish larvae has been used to mimic a Parkinson's disease (PD) phenotype and to facilitate high-throughput drug screening. However, the vulnerability of zebrafish to various neurotoxins was shown to be variable. Here, we provide a direct comparison of ablative effectiveness in order to identify the optimal neurotoxin-mediated dopaminergic (DAnergic) neuronal death in larval zebrafish. Transgenic zebrafish, Tg(dat:eGFP), were exposed to different concentrations of the neurotoxins MPTP, MPP + , paraquat, 6-OHDA, and rotenone for four days, starting at three days post-fertilization. The LC 50 of each respective neurotoxin concentration was determined. Confocal live imaging on Tg(dat:eGFP) showed that MPTP, MPP + , and rotenone caused comparable DAnergic cell loss in the ventral diencephalon (vDC) region while, paraquat and 6-OHDA caused fewer losses of DAnergic cells. These results were further supported by respective gene expression analyses of dat, th, and p53. Importantly, the loss of DAnergic cells from exposure to MPTP, MPP + , and rotenone impacted larval locomotor function. MPTP induced the largest motor deficit, but this was accompanied by the most severe morphological impairment. We conclude that, of the tested neurotoxins, MPP + recapitulates a substantial degree of DAnergic ablation and slight locomotor perturbations without systemic defects indicative of a Parkinsonian phenotype.

Mitochondria are dynamic organelles that mediate the energetic supply to cells and mitigate oxidative stress through the intricate balance of fission and fusion. Mitochondrial dysfunction is a prominent feature within Parkinson disease (PD) etiologies. To date, there have been conflicting studies of neurotoxin impact on dopaminergic cell death, mitochondrial function and behavioral impairment using adult zebrafish. Here, we performed cerebroventricular microinjections (CVMIs) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on adult transgenic zebrafish that resulted in significant reductions in dopaminergic neurons within the telencephalon and olfactory bulbs (OB) of Tg(dat:eGFP) fish. Visualization of mCherry and mitochondrial gene expression analysis in Tg(dat:tom20 MLS:mCherry) fish reveal that MPTP induces mitochondrial fragmentation in dopaminergic neurons and the activation of the pink1/parkin pathway involved mitophagy. Moreover, the loss of dopaminergic neurons translated into a transient locomotor and olfactory phenotype. Taken together, these data can contribute to a better understanding of the mitochondrial impact on dopaminergic survivability.

The presenilin-associated rhomboid-like (PARL) gene was found to contribute to mitochondrial morphology and function and was linked to familial Parkinson’s disease (PD). The PARL gene product is a mitochondrial intramembrane cleaving protease that acts on a number of mitochondrial proteins involved in mitochondrial morphology, apoptosis, and mitophagy. To date, functional and genetic studies of PARL have been mainly performed in mammals. However, little is known about PARL function and its role in dopaminergic (DA) neuron development in vertebrates. The zebrafish genome comprises two PARL paralogs: parla and parlb. Here, we established a loss-of-function mutation in parla via CRISPR/Cas9-mediated mutagenesis. We examined DA neuron numbers in the adult brain and expression of genes associated with DA neuron function in larvae and adults. We show that loss of parla function results in loss of DA neurons, mainly in the olfactory bulb. Changes in the levels of tyrosine hydroxylase transcripts supported this neuronal loss. Expression of fis1, a gene involved in mitochondrial fission, was increased in parla mutants. Finally, we showed that loss of parla function translates into impaired olfaction and altered locomotion parameters. These results suggest a role for parla in the development and/or maintenance of DA neuron function in zebrafish.

Adult zebrafish have the ability to regenerate cells of the central nervous system. However, few neuronal regeneration studies in adult zebrafish addressed their ability to regenerate specific types of neurons following cell specific ablation. We show here that treatment of transgenic Tg( dat:CFP-NTR ) adult zebrafish with the prodrug metronidazole (Mtz) according to our administration regimen predominantly ablates dopamine (DA) neurons within the olfactory bulb (OB) of adult fish. Loss of DA neurons was accompanied by an impaired olfaction phenotype, as early as 1-week post-treatment, in which fish were unable to sense the presence of the repulsive stimulus cadaverine. The olfactory impairment was reversed within 45 days and coincided with the recovery of DA neuron counts in the OB. A multi-label pulse-chase analysis with BrdU and EdU over the first seventeen days-post Mtz exposure showed that newly formed DA neurons were recruited within the first nine days following exposure and led to functional and morphological recovery of the OB.

No abstract