Mitochondria are critical organelles that continually remodel to carry out their many cellular functions. This remodeling is particularly important in neurons due to the specialized shape and high energy demands inherent to these cells. Mitochondrial network remodeling is determined in part by kinesin-1 and dynein-dynactin, which drive mitochondrial transport in opposite directions along microtubules. Interestingly, mitochondrial dynamics and microtubule motors both have close causal links to neurodegenerative and neurodevelopmental diseases, such as Amyotrophic Lateral Sclerosis (ALS) and Fragile X Syndrome. In this work, we describe molecular mechanisms connecting these diseases to mitochondrial dynamics and microtubule motors, and dissect the regulation of microtubule-based mitochondrial transport by TRAK-MIRO motor-adaptor complexes. First, we utilize in vitro single-molecule imaging and biochemistry to demonstrate that TRAK2 activates kinesin-1 and dynein-dynactin, and show that TRAK2 scaffolds an interaction between these motors to link their function within a single complex. In Chapter 3, we collaborate with the Dominguez lab to characterize a direct interaction between TRAK1 and MIRO1 that facilitates recruitment of TRAK1 to mitochondria. In Chapter 4, we collaborate with the Landers lab to investigate the effects of ALS-associated mutations on the kinesin-1 isoform KIF5A. We find that multiple ALS-linked mutations result in skipping of exon 27, which produce a novel C-terminal sequence within KIF5A that impairs its autoinhibition, creating a hyperactive motor protein that is toxic to neurons. In Chapter 5, we characterize mitochondrial morphological and metabolic defects in a Drosophila model of Fragile X Syndrome and show phenotypic links between this model, insulin signaling, and circadian behavior. Last, we show that FMRP, the protein whose dysfunction causes Fragile X Syndrome, is central to a mechanism by which protein synthesis is locally orchestrated to efficiently guide mitochondrial fission in neurons. We characterize the precise spatiotemporal dynamics underlying this process with spinning-disk confocal microscopy and collaborate with the Lakadamyali and Chang labs to describe the nanoscale architecture of FMRP-mitochondria interactions with DNA-PAINT super-resolution microscopy and cryo-electron tomography. Together, these studies provide detailed insight into mechanisms underlying mitochondrial network organization in neurons and connect this organization to the regulation of microtubule motors and human neurological disease.