James Martin Harris '08
Proteomic Analysis of Compensatory Dendritic Regeneration in the Cricket, Gryllus bimaculatus
Honors Project 2007-2008
Support of Mentors and Their Students in the Neurosciences Fellow summer 2007
INBRE Pre-Doctoral Fellowship summer 2008
In the nervous system, information from one neuron is send along an axon, which acts like a wire. When the axon connects with the dendrites from another cell, information can be transmitted between the cells. However, when neural tissue is injured, axons typically degenerate resulting in a disruption of the connection with dendrites leaving these dendrites “deafferented”, as is the case in paralysis. In response to deafferentation, dendrites typically degenerate as well. In this degenerated state, neurons cannot pass information to one another and thus cannot form functional circuits. In the central nervous system, the degenerated state persists, with a noticeable lack of substantive regrowth. Research, particularly on axon regrowth has suggested that this difference in regenerative capacity lies not in the neurons themselves, but rather their environment following injury.
However, dendrites in the cricket auditory system are unique and do not degenerate in response to deafferentation, but rather form new connections, which restore the original functionality of the neuron. Originally, we hypothesized that this compensatory growth is due to changes in gene regulation. To screen for such genes, we performed a genetic screening technique called suppressive subtractive hybridization (SSH), do identify genes differentially regulated in crickets undergoing regeneration compared with controls. We identified about 50 candidate genes. We were surprised to find that several of these differentially regulated genes coded for enzymes that were part of the ubiqutin proteasome system, which can selectively degrade proteins in a cell. This discovery turned us on to the idea that post-translational protein regulation, rather than gene expression may potentially mediate the dendritic regeneration phenomenon.
In order to identify which proteins might be modified, we are in the process of completing 2-D Differential Gel Electrophoresis (DIGE) experiments. This technique, developed by the Minden lab at Carnegie Mellon in 2006, allows for the comparison of protein expression levels in two protein samples. One sample is labeled with red fluorescent dye, the other with green, and then the two samples are combined. Proteins in the combined sample are then separated by gel electrophoresis, first by charge then by size. The result is a gel with different colored spots, in which red and green spots represent differentially regulated proteins more abundant in their respective sample and yellow spots represent proteins that are in equal abundance in both samples. This past winter, I visited the Minden lab to perform the first round of our DIGE experiments that compared protein samples from control crickets to samples from crickets in the process of dendritic regeneration. Thus far we have identified ATP-synthase and beta actin as unchanging proteins (yellow spots), to which we normalized our other data. Currently, we are in the process of identifying about 20 differentially expressed proteins using several forms of MALDI mass spectrometry (TOF, tandem and FTMS). We hope that our results will help elucidate the mechanisms by which the auditory system of crickets is uniquely adapted to compensate for neuronal injury.