Glycogen synthase kinase 3ß (GSK3ß) and presenilin (PS) are key regulators of kinesin-1-mediated cargo motility within axons.

It has been a quarter century since the discovery that molecular motors are phosphorylated, but fundamental questions still remain as to how specific kinases contribute to particular motor functions, particularly in vivo, and to what extent these processes have been evolutionarily conserved. Such questions remain largely unanswered because there is no cohesive strategy to unravel the likely complex spatial and temporal mechanisms that control motility in vivo. Since diverse cargoes are transported simultaneously within cells and along narrow long neurons to maintain intracellular processes and cell viability, and disruptions in these processes can lead to cancer and neurodegeneration, there is a critical need to better understand how kinases regulate molecular motors. Here, we review our current understanding of how phosphorylation can control kinesin-1 motility and provide evidence for a novel regulatory mechanism that is governed by a specific kinase, glycogen synthase kinase 3ß (GSK3ß), and a scaffolding protein presenilin (PS).

Early Mitochondrial Defects in the 5xFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: Mitochondrial (MT) dysfunction is a hallmark of Alzheimer's disease (AD). Amyloid-ß protein precursor and amyloid-ß peptides localize to MT and lead to MT dysfunction in familial forms of AD. This dysfunction may trigger subsequent types of pathology. OBJECTIVE: To identify the MT phenotypes that occur early in order to help understand the cascade of AD pathophysiology. METHODS: The 5xFAD mouse model was used to explore the time course of MT pathologies in both sexes. Protein biomarkers for MT dynamics were measured biochemically and MT function was measured using oxygen consumption and ATP assays. RESULTS: We discovered progressive alterations in mitochondrial dynamics (biogenesis, fission, fusion, and mitophagy) and function (O2 consumption, ATP generation, and Ca2+ import) in the hippocampus of 5xFAD mice in both sexes as early as 2 months of age. Thus, mitochondrial dynamics and function become altered at young ages, consistent with an early role for mitochondria in the AD pathological cascade. CONCLUSION: Our study offers the baseline information required to understand the hierarchical relationship between the multiple pathologies that develop in this mouse model and provides early biomarkers for MT dysfunction. This will aid in dissecting the temporal cascade of pathologies, understanding sex-specific differences, and in testing the efficacy of putative mitochondrial therapeutics.

A stop or go switch: glycogen synthase kinase 3ß phosphorylation of the kinesin 1 motor domain at Ser314 halts motility without detaching from microtubules.

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 GSK3ß plays a role in motor function. Here, we report that a particular site on kinesin 1 motor domain (KHC), S314, is phosphorylated by GSK3ß in vivo. The GSK3ß-phosphomimetic-KHCS314D stalled kinesin 1 motility without dissociating from microtubules, indicating that constitutive GSK3ß phosphorylation of the motor domain acts as a STOP. In contrast, uncoordinated mitochondrial motility was observed in CRISPR/Cas9-GSK3ß 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 GSK3ß 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.

Differential mitochondrial roles for α-synuclein in DRP1-dependent fission and PINK1/Parkin-mediated oxidation.

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.

In Vivo Visualization of Moving Synaptic Cargo Complexes within Drosophila Larval Segmental Axons.

Identifying moving synaptic vesicle complexes and isolating specific proteins present within such complexes in vivo is challenging. Here we detail a protocol that we have developed that is designed to simultaneously visualize the axonal transport of two fluorescently tagged synaptic vesicle proteins in living Drosophila larval segmental nerves in real time. Using a beam-splitter and split view software, larvae expressing GFP-tagged Synaptobrevin (Syb) and mRFP-tagged Rab4-GTPase or YFP-tagged Amyloid Precursor protein (APP) and mRFP-tagged Rab4-GTPase are imaged simultaneously using separate wavelengths. Merged kymographs from the two wavelengths are evaluated for colocalization analysis. Vesicle velocity analysis can also be done. Such analysis enables us to visualize the motility behaviors of two synaptic proteins present on a single vesicle complex and identify candidate proteins moving on synaptic vesicles in vivo, under physiological conditions.

Excess active P13K rescues huntingtin-mediated neuronal cell death but has no effect on axonal transport defects.

High levels of oxidative stress is detected in neurons affected by many neurodegenerative diseases, including huntington's disease. Many of these diseases also show neuronal cell death and axonal transport defects. While nuclear inclusions/accumulations likely cause cell death, we previously showed that cytoplasmic axonal accumulations can also contribute to neuronal death. However, the cellular mechanisms responsible for activating cell death is unclear. One possibility is that perturbations in normal axonal transport alter the function of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-pathway, a signal transduction pathway that promotes survival/growth in response to extracellular signals. To test this proposal in vivo, we expressed active PI3K in the context of pathogenic huntingtin (HTT-138Q) in Drosophila larval nerves, which show axonal transport defects and neuronal cell death. We found that excess expression of active P13K significantly suppressed HTT-138Q-mediated neuronal cell death, but had no effect on HTT-138Q-mediated axonal transport defects. Expression of active PI3K also rescued Paraquat-mediated cell death. Further, increased levels of pSer9 (inactive) glycogen synthase kinase 3ß was seen in HTT-138Q-mediated larval brains, and in dynein loss of function mutants, indicating the modulation of the pro-survival pathway. Intriguingly, proteins in the PI3K/AKT-pathway showed functional interactions with motor proteins. Taken together our observations suggest that proper axonal transport is likely essential for the normal function of the pro-survival PI3K/AKT-signaling pathway and for neuronal survival in vivo. These results have important implications for targeting therapeutics to early insults during neurodegeneration and death.

The Non-amyloidal Component Region of α-Synuclein Is Important for α-Synuclein Transport Within Axons.

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-synA53T 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-synΔ71-82) 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-synΔ71-82 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.

The presenilin loop region is essential for glycogen synthase kinase 3 ß (GSK3ß) mediated functions on motor proteins during axonal transport.

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 (GSK3ß) stimulated amyloid precursor protein vesicle motility. Excess GSK3ß caused transport defects and increased motor binding to membranes, while reduction of PS decreased active GSK3ß and motor binding to membranes. Here, we report that functional PS and the catalytic loop region of PS is essential for the rescue of GSK3ß-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 GSK3ß associated with and phosphorylated kinesin-1 in vitro. Our observations together with previous work that showed that the loop region of PS interacts with GSK3ß propose a scaffolding mechanism for PS in which the loop region sequesters GSK3ß 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.

Axonal Transport and Neurodegeneration: How Marine Drugs Can Be Used for the Development of Therapeutics.

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.