NPY-Induced Kinase Activation in C. elegans Effects on Signaling Nodes

Jessica Díaz Rivera

Francisco Fuster

Ricardo Chiesa, PhD

RISE Program

Background information

• Neuropeptides are small proteins that act on the cells surface receptor to communicate between neurons.

• NPY is short for Neuropeptide Y.– Is a neurotransmitter found in the brain.– 36 amino acids

• It regulates:– Food intake– Memory and learning process– Anxiolytic effects

NPY receptor 1Terminal N Extracellular

Intracellular C Terminus

Caenorhabditis elegans

• C. elegans is a free living non-parasitic nematode.• Nervous system with cerebral ganglia.• Neurotransmitters:– Acetylcholine (Ach)– Serotonin (5HT)– Dopamine (DA)– Tyramine (TA)– Octopamine (OA)– Glutamate (Glu)– Gamma-aminobutyric acid (GABA)

Caenorhabditis elegans

• Maturity: 3-4 days.Adult hermaphrodite

Egg

Objectives

• Monitor changes in signaling nodes with the presence of NPY.

• Monitor the effect that NPY causes on GABA synthesis.

• Validate the proposed pathway.

NPY Proposed Pathway

Nucleus Por: María Elena Castelló Toro

γβ

GTP

PLC

Ca+ Ca+Ca+

Ca+

Ca+

Ca+Ca+

Ca+Ca+

Ca+

Ca+

Ca+

Ca+PKC

cRaf

MEK 1/2

HRas

ERK 1/2

RSK 90

CREBP

Nucleus

β

P

P

P

GABRA gene

GABAA

Receptor

HRas

Hypothesis

• In the presence of NPY, there will be an up regulation of the GABRA gene and increase in the levels of proteins of the proposed pathway.

Experimental Design

Cultivation of C. elegans Protein Extraction with Heat Schock

Protein Quantification BCAELISA Assay

Spectrophotometry

Enzyme-linked Immunosorbent Assay (ELISA) mechanism

• Por: María Elena Castelló Toro

Spectrophotometry and Results

Conclusion

• The levels of total Akt-1 are higher (statistical significance) in the +NPY protein extracts. However, phospho-Akt-1 levels are the same both in the control and experimental group. We have to corroborate if a CRE element is present in the Akt-1 gene. If future experiments show an increase in the levels of phospho-CREB, this could account for the increase in the levels of total Akt-1.

• Phospho-Stat-3 and phospho-p38 levels are the same both in control group (no NPY) and the experimental group (with NPY).

Future Experiments

• Repeat ELISA Assays with C. elegans proteins.

• Work with cells to compare in vitro results with in vivo.

References• Domschke K, Dannlowski U, Hohoff C, et all. (2010). Neuropeptide Y (NPY) gene: Impact on emotional

processing and treatment response in anxious depression. Eur Neuropsychopharmacol, 5, 301-9.• Bacchi F, Mathe AA, Jimenez P, et. all. (2006). Anxiolytic-like effect of the selective Neuropeptide Y Y2

receptor antagonist BIIE0246 in the elevated plus-maze. Peptides,12, 3202-7.• Geary, T. G., Kubiak, T. M. (2005). Neuropeptide G-protein couple receptors, their cognate ligands and

behavior in Caernorhabditis elegans. TRENDS in Pharmacological Sciencies, 26, 56-58.

Questions