Discovery and functional prioritization of Parkinson's disease candidate genes from large-scale whole exome sequencing.

BACKGROUND: Whole-exome sequencing (WES) has been successful in identifying genes that cause familial Parkinson's disease (PD). However, until now this approach has not been deployed to study large cohorts of unrelated participants. To discover rare PD susceptibility variants, we performed WES in 1148 unrelated cases and 503 control participants. Candidate genes were subsequently validated for functions relevant to PD based on parallel RNA-interference (RNAi) screens in human cell culture and Drosophila and C. elegans models. RESULTS: Assuming autosomal recessive inheritance, we identify 27 genes that have homozygous or compound heterozygous loss-of-function variants in PD cases. Definitive replication and confirmation of these findings were hindered by potential heterogeneity and by the rarity of the implicated alleles. We therefore looked for potential genetic interactions with established PD mechanisms. Following RNAi-mediated knockdown, 15 of the genes modulated mitochondrial dynamics in human neuronal cultures and four candidates enhanced α-synuclein-induced neurodegeneration in Drosophila. Based on complementary analyses in independent human datasets, five functionally validated genes-GPATCH2L, UHRF1BP1L, PTPRH, ARSB, and VPS13C-also showed evidence consistent with genetic replication. CONCLUSIONS: By integrating human genetic and functional evidence, we identify several PD susceptibility gene candidates for further investigation. Our approach highlights a powerful experimental strategy with broad applicability for future studies of disorders with complex genetic etiologies.

Identification of an evolutionary conserved structural loop that is required for the enzymatic and biological function of tryptophan 2,3-dioxygenase.

The enzyme TDO (tryptophan 2,3-dioxygenase; TDO-2 in Caenorhabditis elegans) is a potential therapeutic target to cancer but is also thought to regulate proteotoxic events seen in the progression of neurodegenerative diseases. To better understand its function and develop specific compounds that target TDO we need to understand the structure of this molecule. In C. elegans we compared multiple different CRISPR/Cas9-induced tdo-2 deletion mutants and identified a motif of three amino acids (PLD) that is required for the enzymatic conversion of tryptophan to N-formylkynurenine. Loss of TDO-2's enzymatic activity in PDL deletion mutants was accompanied by an increase in motility during aging and a prolonged lifespan, which is in line with the previously observed phenotypes induced by a knockdown of the full enzyme. Comparison of sequence structures suggests that blocking this motif might interfere with haem binding, which is essential for the enzyme's activity. The fact that these three residues are situated in an evolutionary conserved structural loop of the enzyme suggests that the findings can be translated to humans. The identification of this specific loop region in TDO-2-essential for its catalytic function-will aid in the design of novel inhibitors to treat diseases in which the TDO enzyme is overexpressed or hyperactive.

Proteome-wide Changes in Protein Turnover Rates in C. elegans Models of Longevity and Age-Related Disease.

The balance between protein synthesis and protein breakdown is a major determinant of protein homeostasis, and loss of protein homeostasis is one of the hallmarks of aging. Here we describe pulsed SILAC-based experiments to estimate proteome-wide turnover rates of individual proteins. We applied this method to determine protein turnover rates in Caenorhabditis elegans models of longevity and Parkinson's disease, using both developing and adult animals. Whereas protein turnover in developing, long-lived daf-2(e1370) worms is about 30% slower than in controls, the opposite was observed in day 5 adult worms, in which protein turnover in the daf-2(e1370) mutant is twice as fast as in controls. In the Parkinson's model, protein turnover is reduced proportionally over the entire proteome, suggesting that the protein homeostasis network has a strong ability to adapt. The findings shed light on the relationship between protein turnover and healthy aging.

A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans.

Mitochondrial dysfunction is at the core of many diseases ranging from inherited metabolic diseases to common conditions that are associated with aging. Although associations between aging and mitochondrial function have been identified using mammalian models, much of the mechanistic insight has emerged from Caenorhabditis elegans. Mitochondrial respiration is recognized as an indicator of mitochondrial health. The Seahorse XF96 respirometer represents the state-of-the-art platform for assessing respiration in cells, and we adapted the technique for applications involving C. elegans. Here we provide a detailed protocol to optimize and measure respiration in C. elegans with the XF96 respirometer, including the interpretation of parameters and results. The protocol takes ∼2 d to complete, excluding the time spent culturing C. elegans, and it includes (i) the preparation of C. elegans samples, (ii) selection and loading of compounds to be injected, (iii) preparation and execution of a run with the XF96 respirometer and (iv) postexperimental data analysis, including normalization. In addition, we compare our XF96 application with other existing techniques, including the eight-well Seahorse XFp. The main benefits of the XF96 include the limited number of worms required and the high throughput capacity due to the 96-well format.

LRRK2 transport is regulated by its novel interacting partner Rab32.

Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson's disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2.

Genetic screens in Caenorhabditis elegans models for neurodegenerative diseases.

Caenorhabditis elegans comprises unique features that make it an attractive model organism in diverse fields of biology. Genetic screens are powerful to identify genes and C. elegans can be customized to forward or reverse genetic screens and to establish gene function. These genetic screens can be applied to "humanized" models of C. elegans for neurodegenerative diseases, enabling for example the identification of genes involved in protein aggregation, one of the hallmarks of these diseases. In this review, we will describe the genetic screens employed in C. elegans and how these can be used to understand molecular processes involved in neurodegenerative and other human diseases. This article is part of a Special Issue entitled: From Genome to Function.

Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation.

Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer's diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-ß and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra L-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases.