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Research Mentor and Department
Dr. Joseph Dougherty, Departments of Genetics & Psychiatry
While it is well understood that neurons utilize ribosomes in presynaptic neuron components to synthesize proteins required for neurotransmission, it has been unclear whether astrocytes also carry out localized translation in their processes. Since a hallmark of astrocyte function is responding to changes at the tripartite synapse, our initial hypothesis was that astrocytes utilize the same physiological mechanism of local translation that neurons do to respond to synaptic changes. Preliminary data supports this hypothesis by showing that ribosomes and ribosome-bound mRNAs exist in peripheral astrocyte processes (PAPs). I have previously focused on validating the presence of specific mRNAs at PAPs by utilizing fluorescent in situ hybridization (FISH). To do so, we created mRNA target probes and added them to coronal slices of AAV9 GFP virus-injected mouse brain tissue, followed by probe detection via fluorescent labelling. Through these experiments, we observed clear localization of multiple mRNAs in PAPs labelled sparsely by the virus GFP. We have also visualized protein translation in peripheral processes by quantifying puromycylation experiments. Puromycin (PMY) works by attaching to the terminal end of actively translating peptide chains since part of the PMY molecule resembles the 3' end of an aminoacylated tRNA. This results in the premature termination of translation. We have used antibodies that recognize puromycin to fluorescently label, image, and quantify translation in PAPs. Much of the aforementioned data is currently under review for publication.
Our proposed next steps are to test the activity dependence of astrocyte local translation to see if astrocytes do indeed respond to synaptic changes by translating proteins in their processes, as neurons do. To do this, we will treat acute mouse brain slices with KCl, an mGluR5 antagonist, an adenylate cyclase activator, and endocannabinoids to modulate activity. These physiological stimuli will cause synaptic changes due to shifts in neuronal activity, thereby leading to an astrocyte response of some sort. With these treated slices, we will repeat the aforementioned puromycylation experiment to examine post-treatment translation levels. My hypothesis is that astrocytes will have modulated local translation in response to changes at the tripartite synapse.