Multimodal imaging and high-throughput image-processing for drug screening on living organisms on-chip.

A major step for the validation of medical drugs is the screening on whole organisms, which gives the systemic information that is missing when using cellular models. Caenorhabditis elegans is a soil worm that catches the interest of researchers who study systemic physiopathology (e.g., metabolic and neurodegenerative diseases) because: (1) its large genetic homology with humans supports translational analysis; (2) worms are much easier to handle and grow in large amounts compared with rodents, for which (3) the costs and (4) the ethical concerns are substantial. Here, we demonstrate how multimodal optical imaging on such an organism can provide high-content information relevant to the drug development pipeline (e.g., mode-of-action identification, dose-response analysis), especially when combined with on-chip multiplexing capability. After designing a microfluidic array to select small separated populations of C. elegans, we combine fluorescence and bright-field imaging along with high-throughput feature recognition and signal detection to enable the identification of the mode-of-action of an antibiotic. For this purpose, we use a genetically encoded fluorescence reporter of mitochondrial stress, which we studied in living specimens during their entire development. Furthermore, we demonstrate real-time, very large field-of-view capability on multiplexed motility assays for the assessment of the dose-response relation of an anesthetic.

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation.

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr-/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.

A method to identify and validate mitochondrial modulators using mammalian cells and the worm C. elegans.

Mitochondria are semi-autonomous organelles regulated by a complex network of proteins that are vital for many cellular functions. Because mitochondrial modulators can impact many aspects of cellular homeostasis, their identification and validation has proven challenging. It requires the measurement of multiple parameters in parallel to understand the exact nature of the changes induced by such compounds. We developed a platform of assays scoring for mitochondrial function in two complementary models systems, mammalian cells and C. elegans. We first optimized cell culture conditions and established the mitochondrial signature of 1,200 FDA-approved drugs in liver cells. Using cell-based and C. elegans assays, we further defined the metabolic effects of two pharmacological classes that emerged from our hit list, i.e. imidazoles and statins. We found that these two drug classes affect respiration through different and cholesterol-independent mechanisms in both models. Our screening strategy enabled us to unequivocally identify compounds that have toxic or beneficial effects on mitochondrial activity. Furthermore, the cross-species approach provided novel mechanistic insight and allowed early validation of hits that act on mitochondrial function.