LRRK2 functions in synaptic vesicle endocytosis through a kinase-dependent mechanism.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, but the precise physiological function of the protein remains ill-defined. Recently, our group proposed a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses in the Drosophila melanogaster neuromuscular junctions.Flies harbor only one Lrrk gene, which might encompass the functions of both mammalian LRRK1 and LRRK2. We therefore studied the role of LRRK2 in mammalian synaptic function and provide evidence that knockout or pharmacological inhibition of LRRK2 results in defects in synaptic vesicle endocytosis, altered synaptic morphology and impairments in neurotransmission. In addition, our data indicate that mammalian endophilin A1 (EndoA1,also known as SH3GL2) is phosphorylated by LRRK2 in vitro at T73 and S75, two residues in the BAR domain. Hence, our results indicate that LRRK2 kinase activity has an important role in the regulation of clathrin-mediated endocytosis of synaptic vesicles and subsequent neurotransmission at the synapse.

Mutations in the intellectual disability gene Ube2a cause neuronal dysfunction and impair parkin-dependent mitophagy.

The prevalence of intellectual disability is around 3%; however, the etiology of the disease remains unclear in most cases. We identified a series of patients with X-linked intellectual disability presenting mutations in the Rad6a (Ube2a) gene, which encodes for an E2 ubiquitin-conjugating enzyme. Drosophila deficient for dRad6 display defective synaptic function as a consequence of mitochondrial failure. Similarly, mouse mRad6a (Ube2a) knockout and patient-derived hRad6a (Ube2a) mutant cells show defective mitochondria. Using in vitro and in vivo ubiquitination assays, we show that RAD6A acts as an E2 ubiquitin-conjugating enzyme that, in combination with an E3 ubiquitin ligase such as Parkin, ubiquitinates mitochondrial proteins to facilitate the clearance of dysfunctional mitochondria in cells. Hence, we identify RAD6A as a regulator of Parkin-dependent mitophagy and establish a critical role for RAD6A in maintaining neuronal function.

LRRK2 controls an EndoA phosphorylation cycle in synaptic endocytosis.

LRRK2 is a kinase mutated in Parkinson's disease, but how the protein affects synaptic function remains enigmatic. We identified LRRK2 as a critical regulator of EndophilinA. Using genetic and biochemical studies involving Lrrk loss-of-function mutants and Parkinson-related LRRK2(G2019S) gain-of-kinase function, we show that LRRK2 affects synaptic endocytosis by phosphorylating EndoA at S75, a residue in the BAR domain. We show that LRRK2-mediated EndoA phosphorylation has profound effects on EndoA-dependent membrane tubulation and membrane association in vitro and in vivo and on synaptic vesicle endocytosis at Drosophila neuromuscular junctions in vivo. Our work uncovers a regulatory mechanism that indicates that reduced LRRK2 kinase activity facilitates EndoA membrane association, while increased kinase activity inhibits membrane association. Consequently, both too much and too little LRRK2-dependent EndoA phosphorylation impedes synaptic endocytosis, and we propose a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses.

Old yellow enzymes, highly homologous FMN oxidoreductases with modulating roles in oxidative stress and programmed cell death in yeast.

In a genetic screen to identify modifiers of Bax-dependent lethality in yeast, the C terminus of OYE2 was isolated based on its capacity to restore sensitivity to a Bax-resistant yeast mutant strain. Overexpression of full-length OYE2 suppresses Bax lethality in yeast, lowers endogenous reactive oxygen species (ROS), increases resistance to H(2)O(2)-induced programmed cell death (PCD), and significantly lowers ROS levels generated by organic prooxidants. Reciprocally, Delta oye2 yeast strains are sensitive to prooxidant-induced PCD. Overexpression and knock-out analysis indicate these OYE2 antioxidant activities are opposed by OYE3, a highly homologous heterodimerizing protein, which functions as a prooxidant promoting H(2)O(2)-induced PCD in wild type yeast. To exert its effect OYE3 requires the presence of OYE2. Deletion of the 12 C-terminal amino acids and catalytic inactivation of OYE2 by a Y197F mutation enhance significantly survival upon H(2)O(2)-induced PCD in wild type cells, but accelerate PCD in Delta oye3 cells, implicating the oye2p-oye3p heterodimer for promoting cell death upon oxidative stress. Unexpectedly, a strain with a double knock-out of these genes (Delta oye2 oye3) is highly resistant to H(2)O(2)-induced PCD, exhibits increased respiratory capacity, and undergoes less cell death during the adaptive response in chronological aging. Simultaneous deletion of OYE2 and other antioxidant genes hyperinduces endogenous levels of ROS, promoting H(2)O(2)-induced cell death: in Delta oye2 glr1 yeast high levels of oxidized glutathione elicited gross morphological aberrations involving the actin cytoskeleton and defects in organelle partitioning. Altering the ratio of reduced to oxidized glutathione by exogenous addition of GSH fully reversed these alterations. Based on this work, OYE proteins are firmly placed in the signaling network connecting ROS generation, PCD modulation, and cytoskeletal dynamics in yeast.