Parkinson’s disease (PD) is the second most common neurodegenerative disease in the world, but currently there is no cure for PD. Mutations in PINK1 and parkin cause a recessive form of early onset familial PD. PINK1 acts upstream of parkin in a common genetic pathway to regulate mitochondrial morphology and quality, which underscores that mitochondrial dysfunction contributes to PD progression. Mitochondrial morphology is maintained by a dynamic balance between the opposing actions of mitochondrial fission and fusion. PINK1 functions as a mitochondria-localized Serine/Threonine kinase and Parkin as a cytosolic E3 ligase to target the fusion GTPase Mfn for degradation, thus inhibiting mitochondrial fusion. Moreover, PINK1 and Parkin maintain mitochondrial quality by promoting autophagy of dysfunctional mitochondria, a process named “mitophagy”. Discovery of the PINK1-parkin pathway provides an entry point to isolate other genes and compounds that suppress the pathological phenotypes due to lack of PINK1/parkin, which hold great therapeutic potential for development of drugs to treat PD and/or alleviate its symptoms. In this context, we previously found that expression of the pro-fission GTPase Drp1 or inhibition of Mfn suppresses tissue damage and mitochondrial dysfunction in PINK1/parkin mutant Drosophila. In my dissertation research, I combined our Drosophila PINK1/parkin loss-of-function PD model with mammalian cell-based mitochondrial morphology and mitophagy assays, and found that expression of the evolutionarily conserved gene clueless also suppresses PINK1/parkin null mutant phenotypes in Drosophila, and that Drp1 is a critical target of clueless (Drosophila homolog)/cluH (human homolog). Overexpression of drp1 rescues adult lethality, tissue damage, cell death and mitochondrial dysfunction in clueless null mutant Drosophila. In addition, loss of clueless/cluH results in mitochondrial elongation, while clueless/cluH overexpression results in mitochondrial fragmentation in Drosophila and mammalian cells. Clueless/CluH binds Drp1 and promotes its recruitment to mitochondria, providing a mechanistic basis for the above phenotypes. Furthermore, I demonstrated that regulation of Drp1 by cluH is mediated by direct protein interactions between CluH and Drp1 receptors MiD49 and Mff in mammalian cells, and that cluH positively regulates MiD49 and Mff protein levels through posttranscriptional mechanism. Finally, loss of cluH in HeLa cells results in impaired mitophagy due to decreased mitochondrial recruitment of Drp1, and expression of the constitutive mitochondria-localized Drp1S637A rescues the mitophagy defects in cluH KO cells. Together these observations provide insights into how clueless/cluH regulates mitochondrial morphology and quality, and compensates for loss of PINK1/parkin through promoting mitochondrial fission.

As life expectancy prolongs, the incidence of neurodegenerative diseases continues to rise. The pathogenesis of neurodegenerative diseases is poorly understood and there is no effective long-term therapy. This thesis focus on two neurodegenerative disease: Parkinson’s disease (PD), the second most common neurodegenerative disease and inclusion body myopathy, Paget disease with frontotemporal dementia (IBMPFD) caused by Valosin-containing protein (VCP) mutants. Recent studies accentuate the key role of mitochondrial dysfunction in disease-onset. Mitochondrial fusion/fission is critical for mitochondrial integrity with Mitofusins (Mfn) control the initial step of fusion. We found that Mitofusins are key targets in both Parkinson’s disease and in disease by VCP mutants.

Mutations in PTEN-induced putative kinase 1 (PINK1) and parkin cause autosomal recessive PD. Genetic studies indicate PINK1 and parkin function in a common pathway and negatively regulate Mfn. In chapter two, we identify VCP as a suppressor for PINK1 and parkin mutants and its suppression is achieved through Mfn degradation. VCP missense mutations cause IBMPFD and other neurodegenerative disorders. The pathological mechanism of IBMPFD is not clear and there is no cure. we generated an in vivo IBMPFD model in Drosophila, which recapitulates disease pathologies in human patients; we demonstrated common VCP disease mutants act as hyperactive alleles and found that VCP inhibitors reverse the disease pathology in in vivo disease models as well as in patients’ fibroblasts. Our results strongly suggest VCP inhibitors have therapeutic value.

Mitofusins are key substrates of ubiquitin E3 ligase Parkin. Yet the exact mechanism of the ubiquitination is not well-understood. In chapter three, we identified critical lysine sites for Mitofusin ubiquitination by Parkin in Drosophila as well as in mammalian cells. Flies harboring mutated lysine sites display severe tissue defects. Parkin-mediated mitophagy plays a major role in mitochondrial quality control. Lysine mutants delay Parkin-mediated degradation of Mitofusins and mitophagy. Our results uncover the important regulatory role of Mitofusins in mitochondrial quality control and tissue health.

Together, our work demonstrates that utilizing Drosophila and human cells, we could establish reliable in vivo and in vitro models of neurodegenerative disease, unravel novel disease mechanism and identify therapy for these devastating diseases.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Poor understanding of PD pathogenesis has limited the development of effective therapies. Recently, studies on PD associated genes have linked mitochondrial dysfunction to underlying causes of PD. Mutations in the mitochondrial Ser/Thr kinase PINK1 or the Ubiquitin E3 ligase Parkin cause early onset hereditary PD. Genetic studies indicate that PINK1 and parkin function in the same pathway to regulate mitochondrial dynamics through Mitofusin (MFN). This dissertation aims to identify other components in the PINK1/Parkin pathway. In chapter two, we investigate genetic interaction between PINK1 and Omi/HtrA2 in vivo. Previously, mutations in the mitochondrial protease Omi/HtrA2 were identified in PD patients, and in vitro studies show that phosphorylation of Omi/HtrA2 is PINK1-dependent, suggesting that Omi/HtrA2 acts downstream of PINK1. However, our work suggests that Omi/HtrA2 does not function in the same genetic pathway as PINK1. Omi/HtrA2 null mutants in Drosophila do not share any of PINK1 mutant phenotypes. Furthermore, Omi/HtrA2 and PINK1 fail to modify each other's mutant phenotypes. Based on our results, we do not favor a hypothesis in which Omi/HtrA2 plays an essential role in PINK1/parkin mediated PD pathogenesis. These data are also consistent with recent human genetics studies that show no association between Omi/HtrA2 and PD. In chapter three, we identify MUL1, a mitochondrial ubiquitin E3 ligase, as a novel suppressor of PINK1 mutants. MUL1 suppresses PINK1 and parkin phenotypes including mitochondrial morphology, muscle degeneration, and dopaminergic neuronal phenotypes. MUL1 suppresses PINK1 phenotypes by reducing MFN levels through ubiquitination. Further genetic epistasis studies indicate that MUL1 acts in parallel to the PINK1/parkin pathway to regulate mitochondrial integrity. In mammalian cells, the PINK1/Parkin pathway has been shown to mediate the selective degradation of damaged mitochondria called mitophagy. We found that although MUL1 regulates MFN levels, Parkin-mediated mitophagy is not affected. In consistent with our genetic studies in Drosophila, these data support that MUL1 acting in parallel to PINK1/Parkin is conserved. Our work suggests that reduction of MFN by MUL1 is sufficient to reverse PINK1/parkin deficiency phenotypes and proposes MUL1 as a potential therapeutic target to modulate PD pathology.

In the mature mammalian central nervous system (CNS), axons do not regenerate after injury. To improve regeneration, past studies have focused on removing the cell extrinsic signals that block regeneration. However, recent studies suggest that the decline of intrinsic growth capacity of neurons might be the major reason why axons do not regenerate in the adult CNS. The Dual Leucine Zipper Kinase (DLK) pathway is a key intrinsic signal for regeneration after axonal injury. Overexpression of dlk leads to robust regeneration while loss of dlk completely abolishes regeneration after injury. In contrast to the extensive studies on DLK and its downstream signaling cascade, upstream signaling pathways that control DLK remain largely unknown. This dissertation aims to identify the upstream regulators of dlk. In chapter two, we showed that the scaffolding proteins X11Lα and X11Lβ are negative regulators of the Drosophila homolog of DLK, Wallenda (Wnd). We found that loss of X11 leads to enhanced axonal regeneration that is dependent on the DLK/Wnd pathway because inhibition of this pathway suppresses the regeneration phenotypes. Our data suggest that X11 normally prevents DLK/Wnd from promoting axonal growth. Upon injury, increase in intracellular Ca2+ triggers degradation of X11 by the Ca2+-dependent protease CalpB. The degradation of X11 in turn activates DLK/Wnd and initiates axonal regeneration. In Chapter three, we identified a small GTPase, rheb, that greatly enhances axonal regeneration and suppresses axonal degeneration in Drosophila when overexpressed. Enhanced axonal regeneration and reduced axonal degeneration caused by rheb overexpression are dependent on the DLK/Wnd pathway. Furthermore, we found that overexpression of rheb leads to an increase of DLK/Wnd protein level. Finally, we found that inhibition of the mTORC1 pathway suppresses rheb-mediated axonal overgrowth. Taken together, our results suggest that rheb regulates Wnd level by increasing protein translation of DLK/Wnd through the mTORC1 pathway. This thesis provides mechanistic insights into DLK/Wnd regulation by X11 and rheb that will help develop therapeutic strategies for regeneration of the injured CNS.