PROJECT SUMMARY With no effective treatments or cures, ageing-related neurodegenerative diseases (NDs) present a pressing threat to our society. Converging evidence links NDs to defects in endolysosomal trafficking, a vital cellular process used by neurons to maintain their tidiness during normal aging. As one of the most extensively studied NDs, autosomal dominant Huntington's disease (HD) is caused by an abnormal expansion of CAG repeats in the coding region of the Huntingtin (HTT) gene that leads to an abnormally long polyglutamine (polyQ) tract in the corresponding encoded HTT protein. This results in a dominant neurotoxicity and potential alterations of HTT’s normal physiological functions that together lead to the selective loss of medium spiny and cortical projection neurons in the brain. Interestingly, defective endolysosomal trafficking has been observed in HD, and HTT itself has been implicated in endosomal pathways from many in vitro studies. However, whether and how HTT functions in endolysosomal pathways in a physiologic condition remains unexplored. HTT forms a tight complex with its cognate binding partner HAP40, both of which are conserved in the model organism Drosophila. By characterizing these two genes in Drosophila, I found in vivo evidence that HAP40 controls HTT in regulating the functional convergence of endolysosomal recycling and degradation. Together with other evidence, they led to my central hypothesis that through HAP40, HTT regulates Rab4 and Rab4-mediated endolysosomal trafficking, a cellular process potentially disrupted by polyQ expansion in HD. For the F99 dissertation stage, I propose to elucidate the molecular mechanism and functional consequence of this novel HTT/HAP40-mediated endosomal trafficking by harnessing the power of multiple complementary model systems, including genetics and genomics in fruit flies, live/super-resolution imaging in mammalian cells, and brain structure and function analysis in mice (Aim1). Results from this study will define a novel and conserved role of HTT in endolysosomal processes, uncover its underlying mechanism, and its physiological importance. For the K00 post-doctoral stage, I plan to continue this direction of research but expand my training to new technologies such as human induced pluripotent stem cells (hiPSC)/brain organoids that are more related to humans and with more direct translational potential, complemented with more systematic training in mouse ND models, to study endolysosomal pathways such as the Retromer complex that are implicated in more prevalent NDs including Parkinson’s (PD) and Alzheimer’s (AD) diseases. My goals are to understand how endolysosomal pathways contribute to different NDs and whether membrane trafficking pathways can be targeted as novel effective therapeutic avenue to treat NDs (Aim2). For both the F99 and K00 stages, I plan to devote extensive effort towards research training and professional development. Overall, the proposed study wil...