Anna Pilsl

BACKGROUND: Mutations in the gene encoding the E3 ubiquitin ligase parkin (PARK2) are responsible for the majority of autosomal recessive parkinsonism. Similarly to other knockout mouse models of PD-associated genes, parkin knockout mice do not show a substantial neuropathological or behavioral phenotype, while loss of parkin in Drosophila melanogaster leads to a severe phenotype, including reduced lifespan, apoptotic flight muscle degeneration and male sterility. In order to study the function of parkin in more detail and to address possible differences in its role in different species, we chose Danio rerio as a different vertebrate model system. METHODOLOGY/PRINCIPAL FINDINGS: We first cloned zebrafish parkin to compare its biochemical and functional aspects with that of human parkin. By using an antisense knockdown strategy we generated a zebrafish model of parkin deficiency (knockdown efficiency between 50% and 60%) and found that the transient knockdown of parkin does not cause morphological or behavioral alterations. Specifically, we did not observe a loss of dopaminergic neurons in parkin-deficient zebrafish. In addition, we established transgenic zebrafish lines stably expressing parkin by using a Gal4/UAS-based bidirectional expression system. While parkin-deficient zebrafish are more vulnerable to proteotoxicity, increased parkin expression protected transgenic zebrafish from cell death induced by proteotoxic stress. CONCLUSIONS/SIGNIFICANCE: Similarly to human parkin, zebrafish parkin is a stress-responsive protein which protects cells from stress-induced cell death. Our transgenic zebrafish model is a novel tool to characterize the protective capacity of parkin in vivo.

Loss-of-function mutations in the Parkin gene (PARK2) are responsible for the majority of autosomal recessive Parkinson disease. A growing body of evidence indicates that misfolding and aggregation of Parkin is a major mechanism of Parkin inactivation, accounting for the loss-of-function phenotype of various pathogenic Parkin mutants. Remarkably, wild-type Parkin is also prone to misfolding under certain cellular conditions, suggesting a more general role of Parkin in the pathogenesis of Parkinson disease. We now show that misfolding of Parkin can lead to two phenotypes: the formation of detergent-insoluble, aggregated Parkin, or destabilization of Parkin resulting in an accelerated proteasomal degradation. By combining two pathogenic Parkin mutations, we could demonstrate that destabilization of Parkin is dominant over the formation of detergent-insoluble Parkin aggregates. Furthermore, a comparative analysis with HHARI, an E3 ubiquitin ligase with an RBR domain highly homologous to that of Parkin, revealed that folding of Parkin is specifically dependent on the integrity of the C-terminal domain, but not on the presence of a putative PDZ-binding motif at the extreme C terminus.

PACRG can functionally substitute for the LUBAC adaptor subunit SHARPIN to stimulate NF-κB signaling.