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In recent years, considerable effort has been devoted to the development of a fish model for Parkinson’s disease (PD) to examine the pathological mechanisms of neurodegeneration. To effectively evaluate PD pathology, the ability to accurately and reliably count dopaminergic neurons is important. However, there is currently no such standardized method. Due to the relatively small number of dopaminergic neurons in fish, stereological estimation would not be suitable. In addition, serial sectioning requires proficiency to not lose any sections, and it permits double counting due to the large size of some of the dopaminergic neurons. In this study, we report an optimized protocol for staining dopaminergic neurons in zebrafish and provide a reliable counting method. Finally, using our optimized protocol, we confirmed that administration of 6-hydroxydopamine (a neurotoxin) or the deletion of the PINK1 gene (one of the causative genes of familiar PD) in zebrafish caused significant reduction in the number of dopaminergic and noradrenergic neurons. In summary, this method will serve as an important tool for the appropriate evaluation and establishment of fish PD models.

Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson’s disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD., Author Summary Parkinson’s disease (PD) is a neurodegenerative disease characterized by intraneuronal accumulation of alpha-synuclein (α-syn) called Lewy bodies and Lewy neurites. Recent genetic studies have revealed that mutations in glucocerebrosidase (GBA), a causative gene of Gaucher disease (GD), are a strong risk for PD. However, its pathological mechanisms leading to PD remain largely unknown. Here, we generated GBA nonsense mutant (GBA -/-) medaka which survive long enough for pathological analysis of disease progression. These mutant medaka display not only the phenotypes resembling human neuronopathic GD but also axonal accumulation of α-syn accompanied by impairment of the autophagy-lysosome pathway. Furthermore, the present study demonstrates this α-syn accumulation has negligible contribution to the pathogenesis of neuronopathic GD in medaka. GBA -/- medaka represent a valuable model for exploring the pathological mechanisms of PD with GBA mutations as well as neuronopathic GD, and our findings have important implications for the association of GBA mutations with PD.