Morphological hallmarks of dopaminergic neurodegeneration are associated with altered neuron function in Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) is an excellent model system to study neurodegenerative diseases, such as Parkinson's disease, as it enables analysis of both neuron morphology and function in live animals. Multiple structural changes in neurons, such as cephalic dendrite morphological abnormalities, have been considered hallmarks of neurodegeneration in this model, but their relevance to changes in neuron function are not entirely clear. We sought to test whether hallmark morphological changes associated with chemically induced dopaminergic neuron degeneration, such as dendrite blebbing, breakage, and loss, are indicative of neuronal malfunction and result in changes in behavior. We adapted an established dopaminergic neuronal function assay by measuring paralysis in the presence of exogenous dopamine, which revealed clear differences between cat-2 dopamine deficient mutants, wildtype worms, and dat-1 dopamine abundant mutants. Next, we integrated an automated image processing algorithm and a microfluidic device to segregate worm populations by their cephalic dendrite morphologies. We show that nematodes with dopaminergic dendrite degeneration markers, such as blebbing or breakage, paralyze at higher rates in a dopamine solution, providing evidence that dopaminergic neurodegeneration morphologies are correlated with functional neuronal outputs.

Endogenous DAF-16 spatiotemporal activity quantitatively predicts lifespan extension induced by dietary restriction.

In many organisms, dietary restriction (DR) leads to lifespan extension through the activation of cell protection and pro-longevity gene expression programs. In the nematode C. elegans, the DAF-16 transcription factor is a key aging regulator that governs the Insulin/IGF-1 signaling pathway and undergoes translocation from the cytoplasm to the nucleus of cells when animals are exposed to food limitation. However, how large is the influence of DR on DAF-16 activity, and its subsequent impact on lifespan has not been quantitatively determined. In this work, we assess the endogenous activity of DAF-16 under various DR regimes by coupling CRISPR/Cas9-enabled fluorescent tagging of DAF-16 with quantitative image analysis and machine learning. Our results indicate that DR regimes induce strong endogenous DAF-16 activity, although DAF-16 is less responsive in aged individuals. DAF-16 activity is in turn a robust predictor of mean lifespan in C. elegans, accounting for 78% of its variability under DR. Analysis of tissue-specific expression aided by a machine learning tissue classifier reveals that, under DR, the largest contribution to DAF-16 nuclear intensity originates from the intestine and neurons. DR also drives DAF-16 activity in unexpected locations such as the germline and intestinal nucleoli.

Morphological hallmarks of dopaminergic neurodegeneration are associated with altered neuron function in Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) is an excellent model system to study neurodegenerative diseases, such as Parkinson's disease, as it enables analysis of both neuron morphology and function in live animals. Multiple structural changes in neurons, such as cephalic dendrite morphological abnormalities, have been considered hallmarks of neurodegeneration in this model, but their relevance to changes in neuron function are not entirely clear. We sought to test whether hallmark morphological changes associated with chemically induced dopaminergic neuron degeneration, such as dendrite blebbing, breakage, and loss, are indicative of neuronal malfunction and result in changes in behavior. We adapted an established dopaminergic neuronal function assay by measuring paralysis in the presence of exogenous dopamine, which revealed clear differences between cat-2 dopamine deficient mutants, wildtype worms, and dat-1 dopamine abundant mutants. Next, we integrated an automated image processing algorithm and a microfluidic device to segregate worm populations by their cephalic dendrite morphologies. We show that nematodes with dopaminergic dendrite degeneration markers, such as blebbing or breakage, paralyze at higher rates in a dopamine solution, providing evidence that dopaminergic neurodegeneration morphologies are correlated with functional neuronal outputs.

Increased cytotoxicity of Pb2+ with co-exposures to a mitochondrial uncoupler and mitochondrial calcium uniporter inhibitor.

Lead (Pb2+) is an important developmental toxicant. The mitochondrial calcium uniporter (MCU) imports calcium ions using the mitochondrial membrane potential (MMP), and also appears to mediate the influx of Pb2+ into the mitochondria. Since our environment contains mixtures of toxic agents, it is important to consider multi-chemical exposures. To begin to develop generalizable, predictive models of interactive toxicity, we developed mechanism-based hypotheses about interactive effects of Pb2+ with other chemicals. To test these hypotheses, we exposed HepG2 (human liver) cells to Pb2+ alone and in mixtures with other mitochondria-damaging chemicals: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler that reduces MMP, and Ruthenium Red (RuRed), a dye that inhibits the MCU. After 24 hours, Pb2+ alone, the mixture of Pb2+ and RuRed, and the mixture of Pb2+ and FCCP caused no decrease in cell viability. However, the combination of all three exposures led to a significant decrease in cell viability at higher Pb2+ concentrations. After 48 hours, the co-exposure to elevated Pb2+ concentrations and FCCP caused a significant decrease in cell viability, and the mixture of all three showed a clear dose-response curve with significant decreases in cell viability across a range of Pb2+ concentrations. We performed ICP-MS analyses on isolated mitochondrial and cytosolic fractions and found no differences in Pb2+ uptake across exposure groups, ruling out altered cellular uptake as the mechanism for interactive toxicity. We assessed MMP following exposure and observed a decrease in membrane potential that corresponds to loss of cell viability but is likely not sufficient to be the causative mechanistic driver of cell death. This research provides a mechanistically-based framework for understanding Pb2+ toxicity in mixtures with mitochondrial toxicants.

Identifying C. elegans lifespan mutants by screening for early-onset protein aggregation.

Genetic screens are widely used to identify genes that control specific biological functions. In Caenorhabditis elegans, forward genetic screens rely on the isolation of reproductively active mutants that can self-propagate clonal populations. Screens that target post-reproductive phenotypes, such as lifespan, are thus challenging. We combine microfluidic technologies and image processing to perform high-throughput automated screening for short-lived mutants using protein aggregation as a marker for aging. We take advantage of microfluidics for maintaining a reproductively active adult mutagenized population and for performing serial high-throughput analysis and sorting of animals with increased protein aggregation, using fluorescently-labeled PAB-1 as a readout. We demonstrate that lifespan mutants can be identified by screening for accelerated protein aggregation through quantitative analysis of fluorescently labeled aggregates while avoiding conditional sterilization or manual separation of parental and progeny populations. We also show that aged wildtypes and premature aggregation mutants differ in aggregate morphology, suggesting aggregate growth is time-dependent.

A microfluidic platform for lifelong high-resolution and high throughput imaging of subtle aging phenotypes in C. elegans.

Aging produces a number of changes in the neuronal structure and function throughout a variety of organisms. These aging-induced changes encompass a wide range of phenotypes, from loss of locomotion ability to defective production of synaptic vesicles. C. elegans is one of the primary systems used to elucidate phenotypes associated with aging processes. Conventional aging studies in C. elegans are typically labor-intensive, low-throughput, and incorporate fluorodeoxyuridine (FUdR) as a sterilizing agent to keep the population age-synchronized throughout the assay. However, FUdR exposure induces lifespan extension, and can potentially mask the phenotypes associated with the natural aging process. In addition, studying cellular or subcellular structures requires anesthetics or adhesives to immobilize nematodes while acquiring high-resolution images. In this platform, we are able to maintain a population (∼1000 worms) age-synchronized throughout its lifespan and perform a series of high-resolution microscopy studies in a drug-free environment. The device is composed of two main interconnected sections, one with the purpose of filtering progeny while keeping the parent population intact, and one for trapping nematodes in individual compartments for microscopy. Immobilization is carried out by decreasing the temperature of the device where nematodes are trapped by placing a heat sink on top of the chip. We were able to perform periodic high-resolution microscopy of fluorescently tagged synapses located at the dorsal side of the nematode's tail throughout the worms' lifespan. To characterize the subtle phenotypes that emerge as nematodes age, computer vision was implemented to perform automated unbiased detection of synapses and quantitative analysis of aging-induced synaptic changes.