Assessment of Neuronal Cell Death in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a powerful experimental platform for cell biology studies. The molecular mechanisms that mediate cell death and neurodegeneration have been characterized extensively in the nematode. In addition, the availability of a wide arsenal of genetic and molecular tools and methodologies renders C. elegans an organism of choice for modeling human neurodegenerative diseases. Indeed, neuronal necrosis can readily be observed and examined in vivo, in the worm. In this chapter, we describe the two main approaches that are routinely used for monitoring and quantifying neuronal cell death in C. elegans. The first is based on direct visualization of dying cells via Nomarski differential interference contrast (DiC) microscopy, and the second on the assessment of neuronal survival by fluorescence microscopy.

Assessment of dopaminergic neuron degeneration in a C. elegans model of Parkinson's disease.

Transgenic Caenorhabditis elegans that expresses the full-length wild-type human α-synuclein in dopaminergic neurons provides a well-established Parkinson's disease (PD) nematode model. Here, we present a detailed protocol to monitor and dissect the molecular underpinnings of age-associated neurodegeneration using this PD nematode model. This protocol includes preparation of nematode growth media and bacterial food sources, as well as procedures for nematode growth, synchronization, and treatment. We then describe procedures to assess dopaminergic neuronal death in vivo using fluorescence imaging. For complete details on the use and execution of this protocol, please refer to SenGupta et al. (2021).

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP).

Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.

Intracellular Assessment of ATP Levels in Caenorhabditis elegans.

Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans.