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theses

The small Rab GTPases are an evolutionary conserved family of proteins that have been shown to play critical roles in all stages of vesicle trafficking including the formation of new vesicles, mobilization, and fusion of vesicles with the target membrane. As with other families of small GTPases, their function is dependent on the cycling of the GTPases from an inactive GDP-bound form to an active GTP-bound form. Transition from inactive to active states enable the Rab GTPases to interact with specific effector proteins to perform a variety of functions, such as binding to motor proteins for travel along specific microtubule tracks. Within the mammalian system, over 60 members of the Rab family have been identified. Interestingly, it has been shown that each member resides within a specific subcellular location. This restricts the region in which each Rab GTPase can function in addition to restricting the effector proteins they can interact with. Together, this creates specialized routes within the cell for specific sorting of proteins and other molecules. The formation and maintenance of these specialized routes are essential for the maintenance and function of the cell.

Although there is an abundance of Rab GTPases within the mammalian cell, my study will focus on one member of the Rab GTPase family called Rab11. Rab11 resides within the recycling endosome and is responsible for the trafficking of internalized proteins back to the surface of the cell. The role of Rab11 has been identified through numerous studies within non-neuronal cells relying on experiments that manipulated the state of the GTPase with materials such as constitutively active, dominant negative, or overexpression constructs. Manipulation of Rab11 in the non-neuronal cell types has shown its importance for critical cell functions such as proliferation, differentiation, and survival. These tools have also been used to begin to understand the role of Rab11 in neuronal development. In neuronal cells, perturbing the function of Rab11 has resulted in impairments of neurite outgrowth in dorsal root ganglion (DRG) neurons and migration of cortical neurons. However, the precise in vivo role of Rab11 is unclear. In vivo studies have been limited to the Drosophila model system since only one isoform is present. Loss of Rab11 in the Drosophila has been reported to disrupt spinal cord axon guidance, wing, and eye development. In the mammalian system, three isoforms of Rab11 (Rab11a, Rab11b and Rab11c) are present and it has been reported that they function in a redundant manner making studies in vivo more complicated. Recently, there was a study examining human patients harboring a mutation to the Rab11b gene. Mutation to Rab11b gene in this individuals resulted in distinct brain phenotypes that included a thin corpus callossum and a smaller cerebellum. As a consequence of the severity of anatomical changes due to the Rab11b mutation, these individuals also display autistic like behavior. Previous studies showed that while Rab11c has a restricted expression in only epithelial cells, both Rab11a and Rab11b are present in the nervous system. Based on these studies, the main question I asked was what is the role of Rab11a and Rab11b in vivo during brain development and how do we study it?

Although several regions of the brain are impacted in the study of human individuals harboring a mutation within the Rab11b gene, the focus of my study will be on the role of Rab11 during cerebellum development, since the cerebellum was one area affected in humans harboring mutations to the Rab11 genes. Using mouse genetics, I have generated a mouse line that harbors a conditional mutation to Rab11a either in the cerebellar granule cells, using a Math1-Cre line, or the Purkinje cells, using a Pcp2-Cre line, in addition to a global mutation to the Rab11b gene. This marks the first time that both isoforms, Rab11a and Rab11b, have been deleted within a mammalian system in vivo. In addition, this will represent the first in vivo study within the mammalian cerebellum investigating the role of Rab11. Using these mouse lines, I will analyze the impact that loss of Rab11 has on the anatomy of the cerebellum, the cellular processes required for cerebellum development, and the behavior of the mouse. Results collected in this study will aid in the understanding of Rab11 dependent events required for neural development.

ETD_11404

Ph.D.

Includes bibliographical references

External ETD doctoral

doi:10.7282/t3-nst7-1w58

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