Complex I deficiency and dopaminergic neuronal cell loss in parkin-deficient zebrafish (Danio rerio)

Abstract

Currently, only symptomatic therapy is available for Parkinson's disease. The zebrafish is a vertebrate animal model ideally suited for high throughput compound screening to identify disease-modifying compounds for Parkinson's disease. We have developed a zebrafish model for Parkin deficiency, the most commonly mutated gene in early onset Parkinson's disease. The zebrafish Parkin protein is 62% identical to its human counterpart with 78% identity in functionally relevant regions. The parkin gene is expressed throughout zebrafish development and ubiquitously in adult zebrafish tissue. Abrogation of Parkin activity leads to a significant decrease in the number of ascending dopaminergic neurons in the posterior tuberculum (homologous to the substantia nigra in humans), an effect enhanced by exposure to MPP+. Both light microscopic analysis and staining with the pan-neuronal marker HuC confirmed that this loss of dopaminergic neurons is not due to general impairment of brain development. Neither serotonergic nor motor neurons were affected, further emphasizing that the effect of parkin knockdown appears to be specific for dopaminergic neurons. Notably, parkin knockdown zebrafish embryos also develop specific reduction in the activity of the mitochondrial respiratory chain complex I, making this the first vertebrate model to share both important pathogenic mechanisms (i.e. complex I deficiency) and the pathological hallmark (i.e. dopaminergic cell loss) with human parkin-mutant patients. The zebrafish model is thus ideally suited for future drug screens and other studies investigating the functional mechanisms underlying neuronal cell death in early onset Parkinson's Disease. Additional electron microscopy studies revealed electron dense material in the t-tubules within the muscle tissue of parkin knockdown zebrafish. T-tubules are rich in L-type calcium channels, therefore our work might also provide a tentative link between genetically determined early onset Parkinson's disease and recent studies attributing an important role to these L-type calcium channels in late onset sporadic Parkinson's disease.

DJ-1 binds to mitochondrial complex I and maintains its activity

“…Reduced activity level and reduced amount of mitochondrial complex Ｉ have been reported in PD patients [17][18][19][20][21] and in animal models [22,23]. These findings indicate that the function of mitochondrial complex I is related to the onset of PD.…”

α3Na+/K+-ATPase Deficiency Causes Brain Ventricle Dilation and Abrupt Embryonic Motility in Zebrafish

“…Atp1a3a co-localized with the DA neuron marker, TH (Fig. 6A), a marker previously used to identify DA neurons in zebrafish (39). Confocal images highlight the classical arrangement of TH-positive cells in the diencephalon, a region that shows widespread expression of Atp1a3a mRNA (Fig.…”

Mitophagy and neurodegeneration

“…Gross morphological analysis has been performed on mitochondria in some zebrafish models of PD, 116,117 but these did not show any abnormalities. It is possible that analysis in morphants is too early to observe gross morphological defects in mitochondria, but that these arise over a longer time course as a result of compromised mitochondrial function.…”

“…116 The gene structure of zebrafish park2 is identical to that of human PARK2, consisting of 12 exons. In 2009, Flinn et al 116 developed a zebrafish park2 knockdown model by MO, which produced very interesting results ( Table 3). The MO resulted in a 51 amino acid deletion disrupting the in-between ring domain required for ubiquitination of some proteins.…”

“…Other domains, including the ubiquitin ligase domain and two RING domains, remained intact and so the protein may have some residual enzymatic function. 116 It was shown that by 3 dpf, zebrafish park2 morphants display a reduction in dopaminergic neurons of ~20% in the diencephalon. 116 These zebrafish also display an ~45% reduction in the activity of mitochondrial respiratory chain complex I; this corroborates well with the view that complex I function is specifically lost in human PARK2-related PD and sporadic PD.…”