Defining the functional role of microRNA-128-3p in neurons as it pertains to prion disease
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Date
2017
Authors
Boese, Amrit
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Abstract
Central Nervous System neurodegenerative diseases include prion, Alzheimer’s, Huntington’s, and Parkinson’s, all characterized by accumulating aggregates of misfolded host protein. With disease progression, neuronal degeneration results in cognitive and physical symptoms leading to fatality. There are no cures for these diseases. In the brains of patients and animal models, changes in miR-128-3p levels have been described in the literature albeit at terminal disease in a mixed cell population. I hypothesized that miR-128-3p is altered in neurons at early stages of prion induced neurodegeneration, when synaptic dysfunction is commencing, and that miR-128-3p regulates gene targets vital to synaptic function. To determine this, miR-128-3p was detected in the CA1 of the hippocampus, a neuronal subpopulation affected by misfolded prions, during a timecourse of RML scrapie infection in mice. CA1 neuronal cell bodies were isolated using laser capture microdissection and miR-128-3p levels were determined using RT-qPCR. Subsequently, the 3` UTR targets for miR-128-3p were predicted using the TargetScan Human algorithm and a few with prion disease relevance were validated using Luciferase Reporter assays. Next, miR-128-3p levels were determined by RT-qPCR of RNA from synaptoneurosome fractions isolated from the hippocampi and forebrains of RML scrapie prion infected mice and mock infected controls at an early and late stage of disease. As a number of miR-128-3p targets were glutamate receptor subunits, which are found in synapses and integral to their function, the role of miR-128-3p in glutamate signaling pathways was explored. Hippocampal neurons were cultured from embryonic CD1 mice and used as a model to induce glutamate signaling and miR-128-3p response was quantified by RT-qPCR. Lastly, miR-128-3p gain of and loss of function experiments were performed in cultured hippocampal neurons to delineate global proteome changes using mass spectrometry. Results show that miR-128-3p had a trend towards deregulation in the CA1 throughout prion infection and was increased in hippocampal synapses during early disease. The validated gene targets included GRM5, GRIN2B, and GRIN2D. Prolonged synaptic glutamate signaling and toxic levels of glutamate reduced miR-128-3p levels in hippocampal neurons. No changes to glutamate receptor proteins were detected in miR-128-3p gain of function experiments. However, three proteins were downregulated significantly, SLIT3, KCC2A, and SF3A3. This is the first neuronal report of miR-128-3p alterations during early neurodegeneration in an animal model. It provides the basis for future work into therapeutic modulation of miR-128-3p in a region within which the reversal of synapse loss restored cognitive ability in prion infected mice.
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Keywords
microRNA, prion disease, prions, glutamate receptors, NMDAR, neurodegeneration, microRNA-128