Splice variants of microtubule-associated protein 4 regulate the efficiency of organelle transport in dendrites

Project Summary/Abstract In vertebrate neurons, axons have a nearly uniform microtubule polarity pattern with mostly plus-end-out microtubules, while dendrites have a mixed microtubule polarity pattern with about half of the microtubules having each orientation. These distinct microtubule polarity patterns are fundamental to the morphological and compositional differences that define these two types of neuronal processes. However, a question that often arises is how organelle transport can function in dendrites if microtubules of both orientations are interspersed. Would organelles not move back and forth futilely as they come off one microtubule and then associate with another of the opposite orientation? Recent studies have shown that in hippocampal neurons, the microtubules of each orientation are spatially segregated, with the minus-end-out microtubules coalesced into centralized bundles and the plus-end-out microtubules dispersed around the bundles. This appears not to be the case for the dendrites of all types of neurons, but only the dendrites of certain types of neurons, presumably those with the need for higher efficiency in organelle transport. What accounts for the segregation of microtubules of opposite orientation in these dendrites? Posited here is that the answer lies with a newly discovered slice variant of MAP4, called oMAP4, which has the same microtubule-binding domain as the classic MAP4, called uMAP4, but an entirely different projection domain. uMAP4 has been shown to regulate which molecular motor proteins can interact with microtubules. Unlike uMAP4, oMAP4 is endowed with strong microtubule crosslinking properties. Preliminary data obtained by the applicant, Julie Schaub, indicate that oMAP4 is strongly expressed in hippocampal neurons, where it is highly enriched on the centralized bundles of microtubules. The overarching hypothesis of this proposal is that uMAP4 regulates transport via the motor proteins that move organelles along plus-end-out microtubules, while oMAP4 bundles the minus-end-out microtubules and thereby segregates them from microtubules of the opposite orientation. To investigate this hypothesis, rat brain tissue as well as primary cultures of rat neurons will be probed with antibodies to uMAP4 and oMAP4, the latter of which were generated and validated by the applicant. In addition, the impact of experimental manipulation of uMAP4 and oMAP4 levels will be assessed, and cutting-edge microscopy techniques will be used to evaluate the hypothesis. The proposed work has medical relevance because little is known about how corruption of the dendrite’s microtubule array contributes to dendritic atrophy in neurodegenerative diseases like Alzheimer’s, where it is already known that MAP4 expression is altered. In addition to conducting the proposed research, the applicant will undergo a rigorous training program in the neurosciences that includes scientific meetings, journal club, lab meetings, one- on-one meetings with her sponsor, twice annual meetings with her dissertation committee, career education, a panoply of seminars/discussions on skills needed for success, and a rich environment of collaborators and colleagues from across the biomedical sciences.