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The mitogen activated protein kinase (MAPK) pathway plays a vital role in the cell cycle, growth, and differentiation. Germline monogenic (single gene) mutations in genes that code for proteins in the pathway can lead to a family of disorders called RASopathies. The family of RASopathies includes Noonan Syndrome, Neurofibromatosis Type 1, Cardio-facio-cutaneous Syndrome, and LEOPARD Syndrome among others, and they share many similar phenotypic characteristics such as dysmorphic facial features, cardiac defects, and a higher occurrence of cancer. Most of the mutations in genes that encode for proteins in the pathway lead to gain-of-function (hyperactivation) in the MAPK pathway [1]. A previous study has recently found that expression of a gain- of-function constitutively active MEK1 (a protein downstream in the MAPK pathway) mutation in mice leads to the loss of ~60% of the cortical parvalbumin (PV) subset of inhibitory interneurons by P14. We hypothesize that hyperactivation of the MAPK pathway disrupts the morphology of PV interneurons, a response that could contribute to neural circuit defects and possibly GABAergic neuron loss. A Search for the Dying Neurons: An AnalysisBy: Shiv Shah

So why exactly did I do this project? Well for a few reasons. RASopathies, syndromes induced by mutations in the MAPK pathway, have a combined incidence of about 1:2000-1:3000 births [1]. In order to understand and possibly reverse the effects of these syndromes, identification of the morphological and biochemical pathology is necessary.Dendritic morphology as well as somal morphology is a key factor that regulates the amount of input into neurons. Therefore determining whether changes in input occur in response to these mutations may assist in understanding deficits in neural circuit function.Defects in parvalbumin GABAergic neurons have been implicated in of ASDs, schizophrenia, and traumatic brain injury, so this research can extend far beyond the scope of determined RASopathies. In a subset of these conditions (ASDs, schizophrenia, traumatic brain injury) aberrant MAPK signaling has been observed. Thus, deciphering how MAPK regulates this critical neuronal cell type may further our understanding of a wide range of pathological states [2,3] After completing a fairly extensive procedure, I was able to do an analysis on the resulting images and scans. Below is an image of a vibrotome, the machine that I used in order to section my mouse brain tissue. I then used a 6-well tray to do my staining procedure. After conducting my experiment, I was able to draw some conclusions. They are outlined next. It was previously determined that CA-MEK1 caused a 50% decrease in parvalbumin (PV) neurons in P14 mice, and this study furthered the previous one by determining that not only did this 50% decrease in PV neurons occur, but there appeared to be an alteration in the dendritic morphology of these surviving PV interneurons. Although the somal sizes of the mutants did not seem altered compared to the controls, there was a definite increase in the dendritic morphology, both in the dendrite length and the number of dendritic nodes. These measures combined offer a measure of dendritic complexity.

PV interneurons, which are shaped like basket-cells, are known to be fast-spiking neurons in the brain. Previous research has revealed that size of a neuron often can clue into the function of these neurons. Therefore, we see that hyperactive MAPK function may cause increased dendritic branching in parvalbumin-expressing inhibitory interneurons. In terms of future research, further characterization of these parvalbumin interneurons extremely necessary. In addition, electrophysiological recordings should be utilized in the future, as they would give a more direct and accurate value that assesses the amount of synaptic input that cells are receiving. Also, on a larger scope, after identification of the effects of hyperactivation of MAPK on inhibitory interneurons is complete, possible therapeutic approaches towards these effects should be developed to reverse the effects of some of these syndromes.References 1. Tidyman, William E., and Katherine A. Rauen. "The RASopathies: Developmental Syndromes of Ras/MAPK Pathway Dysregulation."Current Opinion in Genetics & Development 19.3 (2009): 230-36. Web. 2. Snow, Wanda M., Kelly Hartle, and Tammy L. Ivanco. "Altered Morphology of Motor Cortex Neurons in the VPA Rat Model of Autism."Developmental Psychobiology 50.7 (2008): 633-39. Web 3. Mndez, Pablo, and Alberto Bacci. "Assortment of GABAergic Plasticity in the Cortical Interneuron Melting Pot." Neural Plasticity 2011 (2011): 1-14. Web. 4. Lawrence, Y. A., T. L. Kemper, M. L. Bauman, and G. J. Blatt. "Parvalbumin-, Calbindin-, and Calretinin-immunoreactive Hippocampal Interneuron Density in Autism." Acta Neurologica Scandinavica 121.2 (2010): 99-108. Web.