Small molecule inhibitors of α-synuclein oligomers identified by targeting early dopamine-mediated motor impairment in C. elegans

Abstract

Background Parkinson’s disease is a disabling neurodegenerative movement disorder characterized by dopaminergic neuron loss induced by α-synuclein oligomers. There is an urgent need for disease-modifying therapies for Parkinson’s disease, but drug discovery is challenged by lack of in vivo models that recapitulate early stages of neurodegeneration. Invertebrate organisms, such as the nematode worm Caenorhabditis elegans, provide in vivo models of human disease processes that can be instrumental for initial pharmacological studies. Methods To identify early motor impairment of animals expressing α-synuclein in dopaminergic neurons, we first used a custom-built tracking microscope that captures locomotion of single C. elegans with high spatial and temporal resolution. Next, we devised a method for semi-automated and blinded quantification of motor impairment for a population of simultaneously recorded animals with multi-worm tracking and custom image processing. We then used genetic and pharmacological methods to define the features of early motor dysfunction of α-synuclein-expressing C. elegans. Finally, we applied the C. elegans model to a drug repurposing screen by combining it with an artificial intelligence platform and cell culture system to identify small molecules that inhibit α-synuclein oligomers. Screen hits were validated using in vitro and in vivo mammalian models. Results We found a previously undescribed motor phenotype in transgenic α-synuclein C. elegans that correlates with mutant or wild-type α-synuclein protein levels and results from dopaminergic neuron dysfunction, but precedes neuronal loss. Together with artificial intelligence-driven in silico and in vitro screening, this C. elegans model identified five compounds that reduced motor dysfunction induced by α-synuclein. Three of these compounds also decreased α-synuclein oligomers in mammalian neurons, including rifabutin which has not been previously investigated for Parkinson’s disease. We found that treatment with rifabutin reduced nigrostriatal dopaminergic neurodegeneration due to α-synuclein in a rat model. Conclusions We identified a C. elegans locomotor abnormality due to dopaminergic neuron dysfunction that models early α-synuclein-mediated neurodegeneration. Our innovative approach applying this in vivo model to a multi-step drug repurposing screen, with artificial intelligence-driven in silico and in vitro methods, resulted in the discovery of at least one drug that may be repurposed as a disease-modifying therapy for Parkinson’s disease.

Using a Caenorhabditis elegans Parkinson’s Disease Model to Assess Disease Progression and Therapy Efficiency

“…By utilizing the nematode model for PD that over-expresses the human disease-causing α-synuclein fused to a fluorescent reporter in the body wall muscles, strain NL5901 [ 27 ], it is possible to directly monitor α-synuclein aggregation throughout life. This nematode strain mimics the α-synuclein aggregation found in Lewy bodies and idiopathic PD [ 3 , 47 ]; however, it should be noted that this strain does not recapitulate the progressive loss of dopaminergic neurons [ 48 ], which is beyond the scope of this work. The open-source ImageJ software [ 49 ] was employed to quantify changes in the aggregation of the α-synuclein throughout the lifespan of the nematode in parallel to a behavioral assay as a robust and representative readout of neuromuscular health.…”

Discovery of a Gut Bacterial Metabolic Pathway that Drives α-Synuclein Aggregation and Neurodegeneration

“…54 In particular, we selected transgenic C. elegans models of PD that have been used to elucidate dopamine’s role in the formation of toxic α-syn oligomers and in neurodegeneration. 55, 56…”

“…54 In particular, we selected transgenic C. elegans models of PD that have been used to elucidate dopamine's role in the formation of toxic α-syn oligomers and in neurodegeneration. 55,56 First, we used C. elegans strain UA287 in which the six frontal dopaminergic neurons in this animal both express human α-syn A53T mutant and fluoresce by virtue of GFP, with expression of both proteins controlled by dopamine transporter promoter P dat-1 . 55 Neurodegeneration in these nematodes is evidenced by decline of the fluorescent signal (indicating neuron death) as well as by changes in the morphology of neuritic processes and soma (indicating decline in neuronal activity).…”

Alpha-Synuclein Targeting Therapeutics for Parkinson's Disease and Related Synucleinopathies

“…Combinations of angiotensin receptor II blockers and dihydropyridine calcium channel blockers, angiotensin converting enzyme inhibitors and diuretics were found to be inversely associated with time to PD diagnosis and thus be of interest for having disease modifying potential in PD ( 257 ). Recently, following an AI-based screen, several compounds were systematically evaluated in cell and animal models of asyn overexpression and revealed rifabutin as a potential drug to reduce nigrostriatal degeneration ( 258 ).…”