Pharmacological PINK1 activation ameliorates Pathology in Parkinson's Disease models.

PINK1 loss-of-function mutations and exposure to mitochondrial toxins are causative for Parkinson's disease (PD) and Parkinsonism, respectively. We demonstrate that pathological α-synuclein deposition, the hallmark pathology of idiopathic PD, induces mitochondrial dysfunction, and impairs mitophagy as evidenced by the accumulation of the PINK1 substrate pS65-Ubiquitin (pUb). We discovered MTK458, a brain penetrant small molecule that binds to PINK1 and stabilizes its active complex, resulting in increased rates of mitophagy. Treatment with MTK458 mediates clearance of accumulated pUb and α-synuclein pathology in α-synuclein pathology models in vitro and in vivo. Our findings from preclinical PD models suggest that pharmacological activation of PINK1 warrants further clinical evaluation as a therapeutic strategy for disease modification in PD.

Pharmacological PINK1 activation ameliorates Pathology in Parkinson's Disease models.

PINK1 loss-of-function mutations and exposure to mitochondrial toxins are causative for Parkinson's disease (PD) and Parkinsonism, respectively. We demonstrate that pathological α-synuclein deposition, the hallmark pathology of idiopathic PD, induces mitochondrial dysfunction and impairs mitophagy, driving accumulation of the PINK1 substrate pS65-Ubiquitin (pUb) in primary neurons and in vivo. We synthesized MTK458, a brain penetrant small molecule that binds to PINK1 and stabilizes an active heterocomplex, thereby increasing mitophagy. MTK458 mediates clearance of α-synuclein pathology in PFF seeding models in vitro and in vivo and reduces pUb. We developed an ultrasensitive assay to quantify pUb levels in plasma and observed an increase in pUb in PD subjects that correlates with disease progression, paralleling our observations in PD models. Our combined findings from preclinical PD models and patient biofluids suggest that pharmacological activation of PINK1 is worthy of further study as a therapeutic strategy for disease modification in PD. Highlights: Discovery of a plasma Parkinson's Disease biomarker candidate, pS65-Ubiquitin (pUb)Plasma pUb levels correlate with disease status and progression in PD patients.Identification of a potent, brain penetrant PINK1 activator, MTK458MTK458 selectively activates PINK1 by stimulating dimerization and stabilization of the PINK1/TOM complexMTK458 drives clearance of α-synuclein pathology and normalizes pUb in in vivo Parkinson's models.

Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6.

Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression-the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3' UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene.

Patient-derived lymphoblastoid cell lines harboring mitochondrial DNA mutations as tool for small molecule drug discovery.

OBJECTIVE: Mitochondrial diseases are a group of devastating disorders for which there is no transformative cure. The majority of therapies for mitochondrial disease-approved, previously tested, or currently in development-are small molecules. The implementation of better cell-based models of mitochondrial disease can accelerate and improve the accuracy of small molecule drug discovery. The objective of this study is to evaluate the use of patient-derived lymphoblastoid cell lines for small molecule research in mitochondrial disease. RESULTS: Five lymphoblastoid cell lines derived from mitochondrial disease patients harboring point mutations in mtND1, mtND4, or mtATP6 were characterized in two high throughput assays assessing mitochondrial function. In a pilot "clinical trial in a dish" experiment, the efficacy of idebenone-an approved therapy for mitochondrial disease-on the lymphoblastoid cell lines was tested. Idebenone increased the basal respiration of all lymphoblastoid cell lines except those harboring the 8993T>G point mutation in mtATP6. Our results posit lymphoblastoid cell lines as a strong model for mitochondrial disease research with small molecules and have implications for the clinical efficacy of idebenone.

2-Hydroxyglutarate Inhibits ATP Synthase and mTOR Signaling.

We discovered recently that the central metabolite α-ketoglutarate (α-KG) extends the lifespan of C. elegans through inhibition of ATP synthase and TOR signaling. Here we find, unexpectedly, that (R)-2-hydroxyglutarate ((R)-2HG), an oncometabolite that interferes with various α-KG-mediated processes, similarly extends worm lifespan. (R)-2HG accumulates in human cancers carrying neomorphic mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes. We show that, like α-KG, both (R)-2HG and (S)-2HG bind and inhibit ATP synthase and inhibit mTOR signaling. These effects are mirrored in IDH1 mutant cells, suggesting a growth-suppressive function of (R)-2HG. Consistently, inhibition of ATP synthase by 2-HG or α-KG in glioblastoma cells is sufficient for growth arrest and tumor cell killing under conditions of glucose limitation, e.g., when ketone bodies (instead of glucose) are supplied for energy. These findings inform therapeutic strategies and open avenues for investigating the roles of 2-HG and metabolites in biology and disease.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ß is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit ß and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation.

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5' and 3', respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q(10). Here we show that the Q(9) content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q(9) content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis.

Target identification using drug affinity responsive target stability (DARTS).

Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule-protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein-metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.