The use of translational profiling for gaining a deeper molecular understanding of memory storage in the brain.
translation is essential to synaptic plasticity and memory storage. Specifically, local
protein translation within dendrites provides specific proteins needed at sites of
synaptic activation, thereby supporting highly localized synaptic plasticity. Despite
this important role for dendritic translation, there is no complete profile of
dendritically translated mRNAs. Identifying ... read morethe types of mRNAs that are translated in
dendrites following learning will offer insight into the molecular processes that
underlie synaptic plasticity, thereby providing a basis for better understanding how we
acquire and store new information. Here we introduce a novel transgenic mouse that
allows for the collection of ribosome-bound mRNAs from defined cell-types. We created a
mouse that expresses EGFP-L10a under the tetracycline operator (tetO-TRAP). By crossing
this mouse with driver specific tTA mouse lines, EGFP-L10a can be expressed in a
cell-type specific manner. Expression of EGFP-L10a then allows for the collection of
ribosome-bound mRNA, thereby providing a translational profile of the cell-type of
interest. We showed that this mouse can be crossed with the Camk2a-tTA and Fos-tTA
driver mice to induce expression of EGFP-L10a in both a cell-type and functionally
defined population of neurons, respectively. We show that the Camk2a-TRAP mouse enables
successful collection of high quality ribosome-bound mRNA from CA1 pyramidal neurons in
the hippocampus. Using the Camk2a-TRAP mouse line, we created a novel method for
collecting ribosome-bound mRNA from in vivo neuronal dendrites. By combining this novel
method with RNA-seq analysis, we generated a list of ribosome-bound mRNAs found within
neuronal dendrites. This list greatly adds to our understanding of what genes can be
locally translated within neuronal dendrites, as well as what genes are regulated during
a learning paradigm. We looked more closely at one of the dendritically translated mRNAs
found in our screen. We found that both Med8 mRNA and protein are localized to neuronal
dendrites, a surprising finding as the only known function of Med8 protein is within the
nucleus of the cell. Besides its well characterized function as part of the
transcriptional Mediator complex, we showed that Med8 protein can endogenously
incorporate into an E3 ubiquitin ligase. Med8's association with the ubiquitin ligase
was most strongly detected in the cytoplasm of brain tissue, and not within other
tissues of the body that were examined. This greatly adds to our understanding of where
Med8 can function as part of an E3 ubiquitin ligase. Together, our work contributes
important new tools to the field of translational profiling, as well as adds to our
understanding of local dendritic protein translation and the types of mRNAs that are
found within dendrites.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Neuroscience.
Advisor: Leon Reijmers.
Committee: Rob Jackson, Michele Jacob, and Grace Gill.
Keyword: Neurosciences.read less