Mitochondria are highly dynamic organelles with strict quality control processes that maintain cellular homeostasis. Within axons, coordinated cycles of fission-fusion mediated by dynamin related GTPase protein (DRP1) and mitofusins (MFN), together with regulated motility of healthy mitochondria anterogradely and damaged/oxidized mitochondria retrogradely, control mitochondrial shape, distribution and size. Disruption of this tight regulation has been linked to aberrant oxidative stress and mitochondrial dysfunction causing mitochondrial disease and neurodegeneration. Although pharmacological induction of Parkinson’s disease (PD) in humans/animals with toxins or in mice overexpressing α-synuclein (α-syn) exhibited mitochondrial dysfunction and oxidative stress, mice lacking α-syn showed resistance to mitochondrial toxins; yet, how α-syn influences mitochondrial dynamics and turnover is unclear. Here, we isolate the mechanistic role of α-syn in mitochondrial homeostasis in vivo in a humanized Drosophila model of Parkinson’s disease (PD). We show that excess α-syn causes fragmented mitochondria, which persists with either truncation of the C-terminus (α-syn1–120) or deletion of the NAC region (α-synΔNAC). Using in vivo oxidation reporters Mito-roGFP2-ORP1/GRX1 and MitoTimer, we found that α-syn-mediated fragments were oxidized/damaged, but α-syn1–120-induced fragments were healthy, suggesting that the C-terminus is required for oxidation. α-syn-mediated oxidized fragments showed biased retrograde motility, but α-syn1–120-mediated healthy fragments did not, demonstrating that the C-terminus likely mediates the retrograde motility of oxidized mitochondria. Depletion/inhibition or excess DRP1-rescued α-syn-mediated fragmentation, oxidation, and the biased retrograde motility, indicating that DRP1-mediated fragmentation is likely upstream of oxidation and motility changes. Further, excess PINK/Parkin, two PD-associated proteins that function to coordinate mitochondrial turnover via induction of selective mitophagy, rescued α-syn-mediated membrane depolarization, oxidation and cell death in a C-terminus-dependent manner, suggesting a functional interaction between α-syn and PINK/Parkin. Taken together, our findings identify distinct roles for α-syn in mitochondrial homeostasis, highlighting a previously unknown pathogenic pathway for the initiation of PD.

Proper transport of the Parkinson's disease (PD) protein, α-synuclein (α-syn), is thought to be crucial for its localization and function at the synapse. Previous work has shown that defects in long distance transport within narrow caliber axons occur early in PD, but how such defects contribute to PD is unknown. Here we test the hypothesis that the NAC region is involved in facilitating proper transport of α-syn within axons via its association with membranes. Excess α-syn or fPD mutant α-syn A53T accumulates within larval axons perturbing the transport of synaptic proteins. These α-syn expressing larvae also show synaptic morphological and larval locomotion defects, which correlate with the extent of α-syn-mediated axonal accumulations. Strikingly, deletion of the NAC region (α-syn 71−82) prevented α-syn accumulations and axonal blockages, and reduced its synaptic localization due to decreased axonal entry and axonal transport of α-syn, due to less α-syn bound to membranes. Intriguingly, co-expression α-syn 71−82 with full-length α-syn rescued α-syn accumulations and synaptic morphological defects, and decreased the ratio of the insoluble higher molecular weight (HMW)/soluble low molecular weight (LMW) α-syn, indicating that this region is perhaps important for the dimerization of α-syn on membranes. Together, our observations suggest that under physiological conditions, α-syn associates with membranes via the NAC region, and that too much α-syn perturbs axonal transport via aggregate formation, instigating synaptic and behavioral defects seen in PD.

Neurons require intracellular transport of essential components for function and viability and defects in transport has been implicated in many neurodegenerative diseases including Alzheimer's disease (AD). One possible mechanism by which transport defects could occur is by improper regulation of molecular motors. Previous work showed that reduction of presenilin (PS) or glycogen synthase kinase 3 beta (GSK3β) stimulated amyloid precursor protein vesicle motility. Excess GSK3β caused transport defects and increased motor binding to membranes, while reduction of PS decreased active GSK3β and motor binding to membranes. Here, we report that functional PS and the catalytic loop region of PS is essential for the rescue of GSK3β-mediated axonal transport defects. Disruption of PS loop (PSΔE9) or expression of the non-functional PS variant, PSD447A, failed to rescue axonal blockages in vivo. Further, active GSK3β associated with and phosphorylated kinesin-1 in vitro. Our observations together with previous work that showed that the loop region of PS interacts with GSK3β propose a scaffolding mechanism for PS in which the loop region sequesters GSK3β away from motors for the proper regulation of motor function. These findings are important to uncouple the complex regulatory mechanisms that likely exist for motor activity during axonal transport in vivo.

Unlike virtually any other cells in the human body, neurons are tasked with the unique problem of transporting important factors from sites of synthesis at the cell bodies, across enormous distances, along narrow-caliber projections, to distally located nerve terminals in order to maintain cell viability. As a result, axonal transport is a highly regulated process whereby necessary cargoes of all types are packaged and shipped from one end of the neuron to the other. Interruptions in this finely tuned transport have been linked to many neurodegenerative disorders including Alzheimer’s (AD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) suggesting that this pathway is likely perturbed early in disease progression. Therefore, developing therapeutics targeted at modifying transport defects could potentially avert disease progression. In this review, we examine a variety of potential compounds identified from marine aquatic species that affect the axonal transport pathway. These compounds have been shown to function in microtubule (MT) assembly and maintenance, motor protein control, and in the regulation of protein degradation pathways, such as the autophagy-lysosome processes, which are defective in many degenerative diseases. Therefore, marine compounds have great potential in developing effective treatment strategies aimed at early defects which, over time, will restore transport and prevent cell death.

It is more than 25 years since the discovery that kinesin 1 is phosphorylated by several protein kinases. However, fundamental questions still remain as to how specific protein kinase(s) contribute to particular motor functions under physiological conditions. Because, within an whole organism, kinase cascades display considerable crosstalk and play multiple roles in cell homeostasis, deciphering which kinase(s) is/are involved in a particular process has been challenging. Previously, we found that GSK3β plays a role in motor function. Here, we report that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3β in vivo. The GSK3β-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3β phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3β non-phosphorylatable-KHCS314A Drosophila larval axons, owing to decreased kinesin 1 attachment to microtubules and/or membranes, and reduced ATPase activity. Together, we propose that GSK3β phosphorylation fine-tunes kinesin 1 movement in vivo via differential phosphorylation, unraveling the complex in vivo regulatory mechanisms that exist during axonal motility of cargos attached to multiple kinesin 1 and dynein motors.