Phenotyping of the thrashing forces exerted by partially immobilized C. elegans using elastomeric micropillar arrays.

As a simple model organism, C. elegans plays an important role in gaining insight into the relationship between bodily thrashing forces and biological effects, such as disease and aging, or physical stimuli, like touch and light. Due to their similar length scale, microfluidic chips have been extensively explored for use in various biological studies involving C. elegans. However, a formidable challenge still exists due to the complexity of integrating external stimuli (chemical, mechanical or optical) with free-moving worms and subsequent imaging on the chip. In this report, we use a microfluidic device to partially immobilize a worm, which allows for measurements of the relative changes in the thrashing force under different assay conditions. Using a device adapted to the natural escape-like coiling response of a worm to immobilization, we have quantified the relative changes in the thrashing force during different developmental stages (L1, L3, L4, and young adult) and in response to various glucose concentrations and drug treatment. Our findings showed a loss of thrashing force following the introduction of glucose into a wild type worm culture that could be reversed upon treatment with the type 2 diabetes drug metformin. A morphological study of the actin filament structures in the body wall muscles provided supporting evidence for the force measurement data. Finally, we demonstrated the multiplexing capabilities of our device through recording the thrashing activities of eight worms simultaneously. The multiplexing capabilities and facile imaging available using our device open the door for high-throughput neuromuscular studies using C. elegans.

Evaluating LRRK2 genetic variants with unclear pathogenicity.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been known to be a major genetic component affecting Parkinson's disease (PD). However, the pathogenicity of many of the LRRK2 variants is unclear because they have been detected in single patients or also in patients and controls. Here, we selected 5 exonic variants (L1165P, T1410M, M1646T, L2063X, and Y2189C) from each of the protein domain of LRRK2 and analysed their possible association with pathogenicity using in vitro functional assays. Point mutations representing each of these variants were incorporated into the LRRK2 gene, and functional aspects such as the percentage of cell survival upon application of stress and kinase activity were measured. Our results showed that all 5 variants had a significantly negative effect on the survival of cells, in both presence and absence of stress, as compared to the wild-type. In addition, there was also a slight increase in kinase activity in most of the variants in comparison to the wild-type. A negative correlation between cell survival and kinase activity was observed. These data suggest that most of the variants despite being located in different domains of LRRK2 appear to exert a potential pathogenic effect possibly through an increased kinase activity, supporting a gain of function mechanism.

Mutant PINK1 upregulates tyrosine hydroxylase and dopamine levels, leading to vulnerability of dopaminergic neurons.

PINK1 mutations cause autosomal recessive forms of Parkinson disease (PD). Previous studies suggest that the neuroprotective function of wild-type (WT) PINK1 is related to mitochondrial homeostasis. PINK1 can also localize to the cytosol; however, the cytosolic function of PINK1 has not been fully elucidated. In this study we demonstrate that the extramitochondrial PINK1 can regulate tyrosine hydroxylase (TH) expression and dopamine (DA) content in dopaminergic neurons in a PINK1 kinase activity-dependent manner. We demonstrate that overexpression of full-length (FL) WT PINK1 can downregulate TH expression and DA content in dopaminergic neurons. In contrast, overexpression of PD-linked G309D, A339T, and E231G PINK1 mutations upregulates TH and DA levels in dopaminergic neurons and increases their vulnerability to oxidative stress. Furthermore transfection of FL WT PINK1 or PINK1 fragments with the PINK1 kinase domain can inhibit TH expression, whereas kinase-dead (KD) FL PINK1 or KD PINK1 fragments upregulate TH level. Our findings highlight a potential novel function of extramitochondrial PINK1 in dopaminergic neurons. Deregulation of these functions of PINK1 may contribute to PINK1 mutation-induced dopaminergic neuron degeneration. However, deleterious effects caused by PINK1 mutations may be alleviated by iron-chelating agents and antioxidant agents with DA quinone-conjugating capacity.