Assessing Gastrointestinal Motility in Caenorhabditis elegans RAC1/CED-10 Mutants as a Tool to Study Early Parkinson's Disease

Our research investigates preclinical symptoms of early Parkinson's disease, and explores lipidomic fingerprints to detect early neurodegenerative events. Achieving reproducible lipidomic fingerprint and validating early biomarkers in models and human cohorts are major challenges. To begin, wash the nematode growth medium plates containing L4 worms with three milliliters of M9 buffer to recover them into two 1.5 milliliter centrifuge tubes.

Centrifuge the tubes for one minute at 2000 g. Discard the supernatant and add one milliliter of fresh M9 buffer. To remove any residual bacteria, plate the washed worms onto a nematode growth medium plate without Escherichia coli OP50.

Then transfer worms individually to a 12-well plate, each well seeded with five microliters of Escherichia coli OP50 culture. Allow the worms to adapt for one hour at 20 degrees Celsius. Place the 12-well plate with worms under the microscope at 20 degrees Celsius.

Locate a worm and start recording when the first defecation cycle is detected. Record the defecation cycles for 15 minutes, capturing approximately 15 cycles using the snipping tool, and click the Lapse button at the end of each defecation cycle. Open one recorded video file using a video player program.

Open the spreadsheet template provided for data entry and enter the video file name and worm size in the corresponding fields. Start video playback to begin analysis. Observe the anus region of the worm to identify the first excretion.

Define the first excretion event as time zero, marking the start of the recording. Record the time in seconds for this event in the cell labeled First Excretion in the spreadsheet. Continue playback and document each subsequent excretion sequentially, labeling them Excretion 2, Excretion 3 and so on, until 16 excretions have been recorded.

The spreadsheet automatically calculates intervals between consecutive excretions. Use these intervals to determine individual cycle lengths. Once all 16 excretion events are recorded, verify the automatically calculated total cycle time.

The spreadsheet displays this value in the cell labeled Total Time. Finally, determine the cycle length as the time interval between two consecutive excretions, and evaluate defecation regularity by calculating the coefficient of variation. Representative images of the RAC1/ced-10 mutant worm showed the sequential defecation motor steps at the L4 stage, including posterior body contraction, anterior body contraction, and enteric muscle contraction with expulsion.

Cycle duration data of individual worms revealed consistent timing in wild-type animals, but higher variability RAC1/ced-10 mutants across all experimental replicates. The mean defecation cycle length was significantly longer in RAC1/ced-10 mutant worms compared to wild-type worms. The cycle regularity expressed as mean coefficient of variation was higher in RAC1/ced-10 mutants than in wild-type worms, indicating less regular intestinal cycles.

We found RAC1 essential for the paminergic and GABAergic function, identify RAC1-dependent lipidomic fingerprint, and generated a RAC1/ced-10 C.Elegans model to investigate PD before neurogeneration occurs. The RAC1/ced-10 C.Elegans model enables temporal tracking, high-throughput genetic and pharmacologic manipulation, and alpha synuclein-free exploration of early PD mechanisms. Our future research will examine molecular mechanisms linking RAC1/ced-10 function with gut-brain mechanisms, identify early biomarkers, and test interventions to delay the progression of preclinical Parkinson's disease.