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The axon initial segment (AIS) is fundamental for neuronal communication and action potential initiation, a characteristic which has been shown to be disrupted in inflammatory diseases such as Multiple Sclerosis (MS). Previous work from our lab has shown AIS breakdown in layer 5 of the cortex in a mouse model of MS known as experimental autoimmune encephalomyelitis (EAE). Moreover, it was shown that AIS breakdown was independent of demyelination but temporally correlated with microglial inflammatory reactivity. In order to determine if this pathology is specific to the cortex or affects other regions of the brain, we exploited these EAE induced mice and investigated AIS integrity in the hippocampus, a region associated with cognitive dysfunction in inflammatory diseases. Additionally, we used a second model of microglial activation that was shown to have AIS breakdown in the cortex. This model relies on the injection of lipopolysaccharide (LPS). Although LPS activates microglia, there is little to no direct effect on other immune cells, which is not the case for EAE. To test AIS stability in the hippocampus, EAE was induced in 12 week old c57bl/6 mice; LPS was injected into 11-12 week old c57bl/6 mice. AnkyrinG, which is an essential cytoskeletal scaffolding protein necessary for proper AIS structure and function, was used to examine AIS integrity through immunohistochemistry (IHC) combined with laser scanning confocal microscopy. Qualitative analysis of AIS produced by confocal imaging, displayed no prevalent signs of AIS shortening in early or late stages of inflammation in the EAE mouse model. The breakdown of βIV spectrin, a cytoskeletal protein that is also clustered in the AIS and is known to link ankyrinG-NaV to the actin cytoskeleton, was correlated with structural disruption of AIS. βIV spectrin breakdown products can be assessed by western blot analysis; however, no breakdown products were observed from the hippocampus of either LPS injected or EAE induced mice. These findings are preliminary but they indicate that the AIS has differential stabilities throughout the CNS, which may provide a tentative explanation for regional differences within the brain. Future studies will include quantification of microglial activation; we believe the depletion of microglial cells play a vital role in both maintaining AIS stability and AIS disruption. These cells are enigmatic and are known to play different roles in different regions of the brain. We are also interested in isolating these cells from the hippocampus and from the cortex in order to compare their expression profiles. Findings from these studies should shed light on the role microglia play in different brain regions during disease.
EAE, myelin, Multiple Sclerosis, hippocampus, axon initial segment, demyelination, beta IV spectrin
Molecular and Cellular Neuroscience | Other Neuroscience and Neurobiology
Current Academic Year
Dr. Jeffrey Dupree
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