GAK and PRKCD kinases regulate basal mitophagy.

The removal of mitochondria in a programmed or stress-induced manner is essential for maintaining cellular homeostasis. To date, much research has focused upon stress-induced mitophagy that is largely regulated by the E3 ligase PRKN, with limited insight into the mechanisms regulating basal "housekeeping" mitophagy levels in different model organisms. Using iron chelation as an inducer of PRKN-independent mitophagy, we recently screened an siRNA library of lipid-binding proteins and determined that two kinases, GAK and PRKCD, act as positive regulators of PRKN-independent mitophagy. We demonstrate that PRKCD is localized to mitochondria and regulates recruitment of ULK1-ATG13 upon induction of mitophagy. GAK activity, by contrast, modifies the mitochondrial network and lysosomal morphology that compromise efficient transport of mitochondria for degradation. Impairment of either kinase in vivo blocks basal mitophagy, demonstrating the biological relevance of our findings.Abbreviations: CCCP: carbonyl cyanide-m-chlorophenyl hydrazone; DFP: deferiprone; GAK: cyclin G associated kinase; HIF1A: hypoxia inducible factor 1 subunit alpha; PRKC/PKC: protein kinase C; PRKCD: protein kinase C delta; PRKN: parkin RBR E3 ubiquitin protein ligase.

GAK and PRKCD are positive regulators of PRKN-independent mitophagy.

The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive. Here, we have screened for regulators of PRKN-independent mitophagy using an siRNA library targeting 197 proteins containing lipid interacting domains. We identify Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCD) as regulators of PRKN-independent mitophagy, with both being dispensable for PRKN-dependent mitophagy and starvation-induced autophagy. We demonstrate that the kinase activity of both GAK and PRKCD are required for efficient mitophagy in vitro, that PRKCD is present on mitochondria, and that PRKCD facilitates recruitment of ULK1/ATG13 to early autophagic structures. Importantly, we demonstrate in vivo relevance for both kinases in the regulation of basal mitophagy. Knockdown of GAK homologue (gakh-1) in C. elegans or knockout of PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy regulation.

Phenotypic Characterization of Larval Zebrafish (Danio rerio) with Partial Knockdown of the cacna1a Gene.

The CACNA1A gene encodes the pore-forming α1 subunit of voltage-gated P/Q type Ca2+ channels (Cav2.1). Mutations in this gene, among others, have been described in patients and rodents suffering from absence seizures and episodic ataxia type 2 with/without concomitant seizures. In this study, we aimed for the first time to assess phenotypic and behavioral alterations in larval zebrafish with partial cacna1aa knockdown, placing special emphasis on changes in epileptiform-like electrographic discharges in larval brains. Whole-mount in situ hybridization analysis revealed expression of cacna1aa in the optic tectum and medulla oblongata of larval zebrafish at 4 and 5 days post-fertilization. Next, microinjection of two antisense morpholino oligomers (individually or in combination) targeting all splice variants of cacna1aa into fertilized zebrafish eggs resulted in dose-dependent mortality and decreased or absent touch response. Over 90% knockdown of cacna1aa on protein level induced epileptiform-like discharges in the optic tectum of larval zebrafish brains. Incubation of morphants with antiseizure drugs (sodium valproate, ethosuximide, lamotrigine, topiramate) significantly decreased the number and, in some cases, cumulative duration of epileptiform-like discharges. In this context, sodium valproate seemed to be the least effective. Carbamazepine did not affect the number and duration of epileptiform-like discharges. Altogether, our data indicate that cacna1aa loss-of-function zebrafish may be considered a new model of absence epilepsy and may prove useful both for the investigation of Cacna1a-mediated epileptogenesis and for in vivo drug screening.

HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.