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.

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.

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.

Ethanol stimulates the in vivo axonal movement of neuropeptide dense-core vesicles in Drosophila motor neurons.

Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. In this study we test how the axonal movement of a neuropeptide-containing dense-core vesicle (DCV) responds to alcohol stressors. We found that ethanol induces a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide-DCV while high doses inhibit bi-directional movement. This process required the presence of functional kinesin-1 motors as reduction in kinesin prevented the ethanol-induced stimulation of the anterograde movement of neuropeptide-DCV. Furthermore, expression of inactive glycogen synthase kinase 3 (GSK-3ß) also prevented ethanol-induced stimulation of neuropeptide-DCV movement, similar to pharmacological inhibition of GSK-3ß with lithium. Conversely, inhibition of PI3K/AKT signaling with wortmannin led to a partial prevention of ethanol-stimulated transport of neuropeptide-DCV. Taken together, we conclude that GSK-3ß signaling mediates the stimulatory effects of ethanol. Therefore, our study provides new insight into the physiological response of the axonal movement of neuropeptide-DCV to exogenous stressors. Cover Image for this Issue: doi: 10.1111/jnc.14165.

Huntingtin differentially regulates the axonal transport of a sub-set of Rab-containing vesicles in vivo.

Loss of huntingtin (HTT), the Huntington's disease (HD) protein, was previously shown to cause axonal transport defects. Within axons, HTT can associate with kinesin-1 and dynein motors either directly or via accessory proteins for bi-directional movement. However, the composition of the vesicle-motor complex that contains HTT during axonal transport is unknown. Here we analyze the in vivo movement of 16 Rab GTPases within Drosophila larval axons and show that HTT differentially influences the movement of a particular sub-set of these Rab-containing vesicles. While reduction of HTT perturbed the bi-directional motility of Rab3 and Rab19-containing vesicles, only the retrograde motility of Rab7-containing vesicles was disrupted with reduction of HTT. Interestingly, reduction of HTT stimulated the anterograde motility of Rab2-containing vesicles. Simultaneous dual-view imaging revealed that HTT and Rab2, 7 or 19 move together during axonal transport. Collectively, our findings indicate that HTT likely influences the motility of different Rab-containing vesicles and Rab-mediated functions. These findings have important implications for our understanding of the complex role HTT plays within neurons normally, which when disrupted may lead to neuronal death and disease.

prickle modulates microtubule polarity and axonal transport to ameliorate seizures in flies.

Recent analyses in flies, mice, zebrafish, and humans showed that mutations in prickle orthologs result in epileptic phenotypes, although the mechanism responsible for generating the seizures was unknown. Here, we show that Prickle organizes microtubule polarity and affects their growth dynamics in axons of Drosophila neurons, which in turn influences both anterograde and retrograde vesicle transport. We also show that enhancement of the anterograde transport mechanism is the cause of the seizure phenotype in flies, which can be suppressed by reducing the level of either of two Kinesin motor proteins responsible for anterograde vesicle transport. Additionally, we show that seizure-prone prickle mutant flies have electrophysiological defects similar to other fly mutants used to study seizures, and that merely altering the balance of the two adult prickle isoforms in neurons can predispose flies to seizures. These data reveal a previously unidentified pathway in the pathophysiology of seizure disorders and provide evidence for a more generalized cellular mechanism whereby Prickle mediates polarity by influencing microtubule-mediated transport.

Presenilin influences glycogen synthase kinase-3 ß (GSK-3ß) for kinesin-1 and dynein function during axonal transport.

Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of control and regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. We previously showed that presenilin (PS), a gene involved in Alzheimer's disease (AD), influences kinesin-1 and dynein function in vivo. Here, we show that these PS-mediated effects on motor protein function are via a pathway that involves glycogen synthase kinase-3ß (GSK-3ß). PS genetically interacts with GSK-3ß in an activity-dependent manner. Excess of active GSK-3ß perturbed axonal transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein. These GSK-3ß-mediated axonal defects do not appear to be caused by disruptions or alterations in microtubules (MTs). Excess of non-functional GSK-3ß did not affect axonal transport. Strikingly, GSK-3ß-activity-dependent axonal transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3ß are required for normal motor protein function. Our observations propose a model, in which PS likely plays a role in regulating GSK-3ß activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during the motility of vesicles within axons.

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.

Presenilin controls kinesin-1 and dynein function during APP-vesicle transport in vivo.

Neurons and other cells require intracellular transport of essential components for viability and function. Previous work has shown that while net amyloid precursor protein (APP) transport is generally anterograde, individual vesicles containing APP move bi-directionally. This discrepancy highlights our poor understanding of the in vivo regulation of APP-vesicle transport. Here, we show that reduction of presenilin (PS) or suppression of gamma-secretase activity substantially increases anterograde and retrograde velocities for APP vesicles. Strikingly, PS deficiency has no effect on an unrelated cargo vesicle class containing synaptotagmin, which is powered by a different kinesin motor. Increased velocities caused by PS or gamma-secretase reduction require functional kinesin-1 and dynein motors. Together, our findings suggest that a normal function of PS is to repress kinesin-1 and dynein motor activity during axonal transport of APP vesicles. Furthermore, our data suggest that axonal transport defects induced by loss of PS-mediated regulatory effects on APP-vesicle motility could be a major cause of neuronal and synaptic defects observed in Alzheimer's Disease (AD) pathogenesis. Thus, perturbations of APP/PS transport could contribute to early neuropathology observed in AD, and highlight a potential novel therapeutic pathway for early intervention, prior to neuronal loss and clinical manifestation of disease.

Nanoparticles in the brain: a potential therapeutic system targeted to an early defect observed in many neurodegenerative diseases.

Currently, there are no effective treatments or cures for many neurodegenerative diseases affecting an aging baby-boomer generation. The ongoing problem with many of the current therapeutic treatments is that most are aimed at dissolving or dissociating aggregates and preventing cell death, common neuropathology often seen towards the end stage of disease. Often such treatments have secondary effects that are more devastating than the disease itself. Thus, effective therapeutics must be focused on directly targeting early events such that global deleterious effects of drugs are minimized while beneficial therapeutic effects are maximized. Recent work indicates that in many neurodegenerative diseases long distance axonal transport is perturbed, leading to axonal blockages. Axonal blockages are observed before pathological or behavioral phenotypes are seen indicating that this pathway is perturbed early in disease. Thus, developing novel therapeutic treatments to an early defect is critical in curing disease. Here I review neurodegenerative disease and current treatment strategies, and discuss a novel nanotechnology based approach that is aimed at targeting an early pathway, with the rationale that restoring an early problem will prevent deleterious downstream effects. To accomplish this, knowledge exchange between biologists, chemists, and engineers will be required to manufacture effective novel biomaterials for medical use.

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).

PolyQ-Expansion Causes Mitochondria Fragmentation Independent of Huntingtin and Is Distinct from Traumatic Brain Injury (TBI)/Mechanical Stress-Mediated Fragmentation Which Results from Cell Death.

Mitochondrial dysfunction has been reported in many Huntington's disease (HD) models; however, it is unclear how these defects occur. Here, we test the hypothesis that excess pathogenic huntingtin (HTT) impairs mitochondrial homeostasis, using Drosophila genetics and pharmacological inhibitors in HD and polyQ-expansion disease models and in a mechanical stress-induced traumatic brain injury (TBI) model. Expression of pathogenic HTT caused fragmented mitochondria compared to normal HTT, but HTT did not co-localize with mitochondria under normal or pathogenic conditions. Expression of pathogenic polyQ (127Q) alone or in the context of Machado Joseph Disease (MJD) caused fragmented mitochondria. While mitochondrial fragmentation was not dependent on the cellular location of polyQ accumulations, the expression of a chaperone protein, excess of mitofusin (MFN), or depletion of dynamin-related protein 1 (DRP1) rescued fragmentation. Intriguingly, a higher concentration of nitric oxide (NO) was observed in polyQ-expressing larval brains and inhibiting NO production rescued polyQ-mediated fragmented mitochondria, postulating that DRP1 nitrosylation could contribute to excess fission. Furthermore, while excess PI3K, which suppresses polyQ-induced cell death, did not rescue polyQ-mediated fragmentation, it did rescue fragmentation caused by mechanical stress/TBI. Together, our observations suggest that pathogenic polyQ alone is sufficient to cause DRP1-dependent mitochondrial fragmentation upstream of cell death, uncovering distinct physiological mechanisms for mitochondrial dysfunction in polyQ disease and mechanical stress.

HTT (huntingtin) and RAB7 co-migrate retrogradely on a signaling LAMP1-containing late endosome during axonal injury.

ABBREVIATIONS: Atg5: Autophagy-related 5; Atg8a: Autophagy-related 8a; AL: autolysosome; AP: autophagosome; BAF1: bafilomycin A1; BDNF: brain derived neurotrophic factor; BMP: bone morphogenetic protein; Cyt-c-p: Cytochrome c proximal; CQ: chloroquine; DCTN1: dynactin 1; Dhc: dynein heavy chain; EE: early endosome; DYNC1I1: dynein cytoplasmic 1 intermediate chain 1; HD: Huntington disease; HIP1/Hip1: huntingtin interacting protein 1; HTT/htt: huntingtin; iNeuron: iPSC-derived human neurons; IP: immunoprecipitation; Khc: kinesin heavy chain; KIF5C: kinesin family member 5C; LAMP1/Lamp1: lysosomal associated membrane protein 1; LE: late endosome; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K12/DLK: mitogen-activated protein kinase kinase kinase 12; MAPK8/JNK/bsk: mitogen-activated protein kinase 8/basket; MAPK8IP3/JIP3: mitogen-activated protein kinase 8 interacting protein 3; NGF: nerve growth factor; NMJ: neuromuscular junction; NTRK1/TRKA: neurotrophic receptor tyrosine kinase 1; NRTK2/TRKB: neurotrophic receptor tyrosine kinase 2; nuf: nuclear fallout; PG: phagophore; PtdIns3P: phosphatidylinositol-3-phosphate; puc: puckered; ref(2)P: refractory to sigma P; Rilpl: Rab interacting lysosomal protein like; Rip11: Rab11 interacting protein; RTN1: reticulon 1; syd: sunday driver; SYP: synaptophysin; SYT1/Syt1: synaptotagmin 1; STX17/Syx17: syntaxin 17; tkv: thickveins; VF: vesicle fraction; wit: wishful thinking; wnd: wallenda.

Kinesin-1 transport reductions enhance human tau hyperphosphorylation, aggregation and neurodegeneration in animal models of tauopathies.

Neurodegeneration induced by abnormal hyperphosphorylation and aggregation of the microtubule-associated protein tau defines neurodegenerative tauopathies. Destabilization of microtubules by loss of tau function and filament formation by toxic gain of function are two mechanisms suggested for how abnormal tau triggers neuronal loss. Recent experiments in kinesin-1 deficient mice suggested that axonal transport defects can initiate biochemical changes that induce activation of axonal stress kinase pathways leading to abnormal tau hyperphosphorylation. Here we show using Drosophila and mouse models of tauopathies that reductions in axonal transport can exacerbate human tau protein hyperphosphorylation, formation of insoluble aggregates and tau-dependent neurodegeneration. Together with previous work, our results suggest that non-lethal reductions in axonal transport, and perhaps other types of minor axonal stress, are sufficient to induce and/or accelerate abnormal tau behavior characteristic of Alzheimer's disease and other neurodegenerative tauopathies.

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.

Amyloid precursor protein-induced axonopathies are independent of amyloid-beta peptides.

Overexpression of amyloid precursor protein (APP), as well as mutations in the APP and presenilin genes, causes rare forms of Alzheimer's disease (AD). These genetic changes have been proposed to cause AD by elevating levels of amyloid-beta peptides (Abeta), which are thought to be neurotoxic. Since overexpression of APP also causes defects in axonal transport, we tested whether defects in axonal transport were the result of Abeta poisoning of the axonal transport machinery. Because directly varying APP levels also alters APP domains in addition to Abeta, we perturbed Abeta generation selectively by combining APP transgenes in Drosophila and mice with presenilin-1 (PS1) transgenes harboring mutations that cause familial AD (FAD). We found that combining FAD mutant PS1 with FAD mutant APP increased Abeta42/Abeta40 ratios and enhanced amyloid deposition as previously reported. Surprisingly, however, this combination suppressed rather than increased APP-induced axonal transport defects in both Drosophila and mice. In addition, neuronal apoptosis induced by expression of FAD mutant human APP in Drosophila was suppressed by co-expressing FAD mutant PS1. We also observed that directly elevating Abeta with fusions to the Familial British and Danish Dementia-related BRI protein did not enhance axonal transport phenotypes in APP transgenic mice. Finally, we observed that perturbing Abeta ratios in the mouse by combining FAD mutant PS1 with FAD mutant APP did not enhance APP-induced behavioral defects. A potential mechanism to explain these findings was suggested by direct analysis of axonal transport in the mouse, which revealed that axonal transport or entry of APP into axons is reduced by FAD mutant PS1. Thus, we suggest that APP-induced axonal defects are not caused by Abeta.

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 Rab4 rescues synaptic and behavioral dysfunction caused by defective HTT-Rab4 axonal transport in Huntington's disease.

Huntington's disease (HD) is characterized by protein inclusions and loss of striatal neurons which result from expanded CAG repeats in the poly-glutamine (polyQ) region of the huntingtin (HTT) gene. Both polyQ expansion and loss of HTT have been shown to cause axonal transport defects. While studies show that HTT is important for vesicular transport within axons, the cargo that HTT transports to/from synapses remain elusive. Here, we show that HTT is present with a class of Rab4-containing vesicles within axons in vivo. Reduction of HTT perturbs the bi-directional motility of Rab4, causing axonal and synaptic accumulations. In-vivo dual-color imaging reveal that HTT and Rab4 move together on a unique putative vesicle that may also contain synaptotagmin, synaptobrevin, and Rab11. The moving HTT-Rab4 vesicle uses kinesin-1 and dynein motors for its bi-directional movement within axons, as well as the accessory protein HIP1 (HTT-interacting protein 1). Pathogenic HTT disrupts the motility of HTT-Rab4 and results in larval locomotion defects, aberrant synaptic morphology, and decreased lifespan, which are rescued by excess Rab4. Consistent with these observations, Rab4 motility is perturbed in iNeurons derived from human Huntington's Disease (HD) patients, likely due to disrupted associations between the polyQ-HTT-Rab4 vesicle complex, accessory proteins, and molecular motors. Together, our observations suggest the existence of a putative moving HTT-Rab4 vesicle, and that the axonal motility of this vesicle is disrupted in HD causing synaptic and behavioral dysfunction. These data highlight Rab4 as a potential novel therapeutic target that could be explored for early intervention prior to neuronal loss and behavioral defects observed in HD.

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.

Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila.

We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.