Fast Axonal Transport Research Articles

The role of kinesin-1 in neuronal dense core vesicle transport, locomotion and lifespan regulation in C. elegans.

Fast axonal transport is crucial for neuronal function and is driven by kinesins and cytoplasmic dynein. Here, we investigated the role of kinesin-1 in dense core vesicle (DCV) transport in C. elegans, using mutants in the kinesin light chains (klc-1 and klc-2) and the motor subunit (unc-116) expressing an ida-1::gfp transgene that labels DCVs. DCV transport in both directions was greatly impaired in an unc-116 mutant and had reduced velocity in a klc-2 mutant. In contrast, the speed of retrograde DCV transport was increased in a klc-1 mutant whereas anterograde transport was unaffected. We identified striking differences between the klc mutants in their effects on worm locomotion and responses to drugs affecting neuromuscular junction activity. We also determined lifespan, finding that unc-116 mutant was short-lived whereas the klc single mutant lifespan was wild type. The ida-1::gfp transgenic strain was also short-lived, but surprisingly, klc-1 and klc-2 extended the ida-1::gfp lifespan beyond that of wild type. Our findings suggest that kinesin-1 not only influences anterograde and retrograde DCV transport but is also involved in regulating lifespan and locomotion, with the two kinesin light chains playing distinct roles.

Divergent Molecular Pathways for Toxicity of Selected Mutant C9ORF72-derived Dipeptide Repeats.

Expansion of a hexanucleotide repeat in a noncoding region of the C9ORF72 gene is responsible for a significant fraction of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) cases, but mechanisms linking mutant gene products to neuronal toxicity remain debatable. Pathogenesis was proposed to involve the production of toxic RNA species and/or accumulation of toxic dipeptide repeats (DPRs) but distinguishing between these mechanisms has been challenging. In this study, we first use complementary model systems for analyzing pathogenesis in adult-onset neurodegenerative diseases to characterize the pathogenicity of DPRs produced by Repeat Associated Non-ATG translation of C9ORF72 in specific cellular compartments: isolated axoplasm and giant synapse from the squid. Results showed selective axonal and presynaptic toxicity of GP-DPRs, independent of associated RNA. These effects involved a MAPK signaling pathway that affects fast axonal transport and synaptic function, a pathogenic mechanism shared with other mutant proteins associated with familial ALS, like SOD1 and FUS. In primary cultured neurons, GP but not other DPRs promote the "dying-back" axonopathy seen in ALS. Interestingly, GR- and PR-DPRs, which had no effect on axonal transport or synaptic transmission, were found to disrupt the nuclear membrane, promoting "dying-forward" neuropathy. All C9-DPR-mediated toxic effects observed in these studies are independent of whether the corresponding mRNAs contained hexanucleotide repeats or alternative codons. Finally, C9ORF72 human tissues confirmed a close association between GP and active P38 in degenerating motor neurons as well as GR-associated nuclear damage in the cortex. Collectively, our studies establish compartment-specific toxic effects of C9-DPRs associated with degeneration, suggesting that two independent pathogenic mechanisms may contribute to disease heterogeneity and/or synergize on disease progression in C9ORF72 patients with ALS and/or FTD symptoms.

Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport

BackgroundBioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson’s, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function.MethodsWe generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos.ResultsWe provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.ConclusionNMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons.

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-β, α-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions. Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellulardomain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport. Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

A vesicular Warburg effect: Aerobic glycolysis occurs on axonal vesicles for local NAD+ recycling and transport.

In neurons, fast axonal transport (FAT) of vesicles occurs over long distances and requires constant and local energy supply for molecular motors in the form of adenosine triphosphate (ATP). FAT is independent of mitochondrial metabolism. Indeed, the glycolytic machinery is present on vesicles and locally produces ATP, as well as nicotinamide adenine dinucleotide bonded with hydrogen (NADH) and pyruvate, using glucose as a substrate. It remains unclear whether pyruvate is transferred to mitochondria from the vesicles as well as how NADH is recycled into NAD+ on vesicles for continuous glycolysis activity. The optimization of a glycolytic activity test for subcellular compartments allowed the evaluation of the kinetics of vesicular glycolysis in the brain. This revealed that glycolysis is more efficient on vesicles than in the cytosol. We also found that lactate dehydrogenase (LDH) enzymatic activity is required for effective vesicular ATP production. Indeed, inhibition of LDH or the forced degradation of pyruvate inhibited ATP production from axonal vesicles. We found LDHA rather than the B isoform to be enriched on axonal vesicles suggesting a preferential transformation of pyruvate to lactate and a concomitant recycling of NADH into NAD+ on vesicles. Finally, we found that LDHA inhibition dramatically reduces the FAT of both dense-core vesicles and synaptic vesicle precursors in a reconstituted cortico-striatal circuit on-a-chip. Together, this shows that aerobic glycolysis is required to supply energy for vesicular transport in neurons, similar to the Warburg effect.

The AI‐based AlphaFold 2 algorithm predicts an open structure for tauC3

AbstractBackgroundTauC3 is an especially noxious C‐terminal tau fragment produced by caspase‐3 cleavage at Asp421. Two decades of accumulating evidence support its importance in the pathogenesis of AD and other tauopathies, notably FTD and PSP. TauC3 promotes aggregation and filament formation, recruits full length tau (FLT) to incipient fibrils, and despite low concentration in AD brain tissue relative to total tau, has been reported to account for substantially all the seeding activity in AD brain extracts. TauC3 has also been shown to slow fast axonal transport (FAT), with deleterious consequences. Although tau’s largely disordered structure prevents analysis by X‐ray crystallography, FRET analysis suggested the existence of a hairpin conformation in which the N‐terminus folds over the C‐terminus close to the MTBR. In this model, Ser432 is the point of closest contact with Pro301. Opening of the hairpin has been hypothesized to 1) make FLT prone to aggregation by exposing the MTBR, and 2) impair FAT by overexposing the phosphatase activating domain near the N‐terminus. We postulated that tauC3 cannot form the hairpin because it lacks Ser432 and thus has an “open” conformation. This would explain tauC3’s substantially increased pathogenicity compared to FLT. The AI‐based tool AlphaFold 2 (AF2) is being used to predict the conformation of many proteins with great accuracy. Applying it to FLT and tauC3 could provide valuable data about the hairpin and whether caspase cleavage alters it.MethodWe tested AF2’s ability to predict the conformation of 4‐repeat FLT and tauC3 from FASTA sequences, aligning the models produced with the MTBR.ResultAF2 modeling revealed a dramatically different conformation for tauC3 compared to FLT, specifically, an open conformation with the MTBR and N‐terminus far apart (Figure). Interestingly, it also revealed that the entire C‐terminus region of FLT starting at Gln423 is composed of a single α‐helix.ConclusionAF2 predictions support the existence of the hairpin conformation for tau and underscore the critical importance of residue 432 in maintaining it. The extended C‐terminus α‐helix may have functional implications. TauC3’s open conformation is consistent with its slowing of FAT and its ability to seed aggregation even at low concentrations.

Axon-Autonomous Effects of the Amyloid Precursor Protein Intracellular Domain (AICD) on Kinase Signaling and Fast Axonal Transport.

The amyloid precursor protein (APP) is a key molecular component of Alzheimer's disease (AD) pathogenesis. Proteolytic APP processing generates various cleavage products, including extracellular amyloid beta (Aβ) and the cytoplasmic APP intracellular domain (AICD). Although the role of AICD in the activation of kinase signaling pathways is well established in the context of full-length APP, little is known about intracellular effects of the AICD fragment, particularly within discrete neuronal compartments. Deficits in fast axonal transport (FAT) and axonopathy documented in AD-affected neurons prompted us to evaluate potential axon-autonomous effects of the AICD fragment for the first time. Vesicle motility assays using the isolated squid axoplasm preparation revealed inhibition of FAT by AICD. Biochemical experiments linked this effect to aberrant activation of selected axonal kinases and heightened phosphorylation of the anterograde motor protein conventional kinesin, consistent with precedents showing phosphorylation-dependent regulation of motors proteins powering FAT. Pharmacological inhibitors of these kinases alleviated the AICD inhibitory effect on FAT. Deletion experiments indicated this effect requires a sequence encompassing the NPTY motif in AICD and interacting axonal proteins containing a phosphotyrosine-binding domain. Collectively, these results provide a proof of principle for axon-specific effects of AICD, further suggesting a potential mechanistic framework linking alterations in APP processing, FAT deficits, and axonal pathology in AD.

Toxic effects of mutant huntingtin in axons are mediated by its proline-rich domain.

Huntington's disease results from expansion of a polyglutamine tract (polyQ) in mutant huntingtin (mHTT) protein, but mechanisms underlying polyQ expansion-mediated toxic gain-of-mHTT function remain elusive. Here, deletion and antibody-based experiments revealed that a proline-rich domain (PRD) adjacent to the polyQ tract is necessary for mHTT to inhibit fast axonal transport and promote axonal pathology in cultured mammalian neurons. Further, polypeptides corresponding to subregions of the PRD sufficed to elicit the toxic effect on fast axonal transport, which was mediated by c-Jun N-terminal kinases (JNKs) and involved PRD binding to one or more SH3-domain containing proteins. Collectively, these data suggested a mechanism whereby polyQ tract expansion in mHTT promotes aberrant PRD exposure and interactions of this domain with SH3 domain-containing proteins including some involved in activation of JNKs. In support, biochemical and immunohistochemical experiments linked aberrant PRD exposure to increased JNK activation in striatal tissues of the zQ175 mouse model and from post-mortem Huntington's disease patients. Together, these findings support a critical role of PRD on mHTT toxicity, suggesting a novel framework for the potential development of therapies aimed to halt or reduce axonal pathology in Huntington's disease.

Axonal Transport Defect in Gigaxonin Deficiency Rescued by Tubastatin A

SummaryGiant axonal neuropathy (GAN) is a disease caused by a deficiency of gigaxonin, a mediator of the degradation of intermediate filament (IF) proteins. A lack of gigaxonin alters the turnover of IF proteins, provoking accumulation and disorganization of neurofilaments (NFs) in neurons, a hallmark of the disease. However, the effects of IF disorganization on neuronal function remain unknown. Here, we report that cultured embryonic dorsal root ganglia (DRG) neurons derived from Gan−/− mice exhibit accumulations of IF proteins and defects in fast axonal transport of organelles. Kymographs generated by time-lapse microscopy revealed substantial reduction of anterograde movements of mitochondria and lysosomes in axons of Gan−/− DRG neurons. Treatment of Gan−/− DRG neurons with Tubastatin A (TubA) increased the levels of acetylated tubulin and it restored the normal axonal transport of these organelles. Furthermore, we tested the effects of TubA in a new mouse model of GAN consisting of Gan−/− mice with overexpression of peripherin (Prph) transgene. Treatment of 12-month-old Gan−/−;TgPer mice with TubA led to a slight amelioration of motor function, especially a significant improvement of gait performance as measured by footprint analyses. Moreover, TubA treatment reduced the abnormal accumulations of Prph and NF proteins in spinal neurons and it boosted the levels of Prph transported into peripheral nerve axons. These results suggest that drug inhibitors of histone deacetylase aiming to enhance axonal transport should be considered as a potential treatment for GAN disease.

Phosphomimetics at Ser199/Ser202/Thr205 in Tau Impairs Axonal Transport in Rat Hippocampal Neurons.

Our understanding of the biological functions of the tau protein now includes its role as a scaffolding protein involved in signaling regulation, which also has implications for tau-mediated dysfunction and degeneration in Alzheimer's disease and other tauopathies. Recently, we found that pseudophosphorylation at sites linked to the pathology-associated AT8 phosphoepitope of tau disrupts normal fast axonal transport through a protein phosphatase 1 (PP1)-dependent pathway in squid axoplasm. Activation of the pathway and the resulting transport deficits required tau's N-terminal phosphatase-activating domain (PAD) and PP1 but the connection between tau and PP1 was not well defined. Here, we studied functional interactions between tau and PP1 isoforms and their effects on axonal transport in mammalian neurons. First, we found that wild-type tau interacted with PP1α and PP1γ primarily through its microtubule-binding repeat domain. Pseudophosphorylation of tau at S199/S202/T205 (psTau) increased PAD exposure, enhanced interactions with PP1γ, and increased active PP1γ levels in mammalian cells. Expression of psTau also significantly impaired axonal transport in primary rat hippocampal neurons. Deletion of PAD in psTau significantly reduced the interaction with PP1γ, eliminated increases of active PP1γ levels, and rescued axonal transport impairment in neurons. These data suggest that a functional consequence of phosphorylation within S199-T205 in tau, which occurs in AD and several other tauopathies, may be aberrant interaction with and activation of PP1γ and subsequent axonal transport disruption in a PAD-dependent fashion.

Tau phosphorylation and PAD exposure in regulation of axonal growth.

Introduction: Tau is a microtubule associated phosphoprotein found principally in neurons. Prevailing dogma continues to define microtubule stabilization as the major function of tau in vivo, despite several lines of evidence suggesting this is not the case. Most importantly, tau null mice have deficits in axonal outgrowth and neuronal migration while still possessing an extensive microtubule network. Instead, mounting evidence suggests that tau may have a major function in the regulation of fast axonal transport (FAT) through activation of neuronal signaling pathways. Previous studies identified a phosphatase activating domain (PAD) at the tau N-terminal that is normally sequestered, but is constitutively exposed in tauopathies. When exposed, the PAD activates a signaling cascade involving PP1 and GSK3β which affects cellular functions including release of cargo from kinesin. Furthermore, we discovered that PAD exposure can be regulated by a single phosphorylation at T205. Exposure of the PAD is an early event in multiple tauopathies and a major contributing factor to neurodegeneration associated with tau hyperphosphorylation. However, effects of tau PAD exposure on anterograde FAT raised the interesting possibility that this pathway may be a mechanism for physiological regulation of cargo delivery through site-specific phosphorylation of tau and transient activation of PP1 and GSK3β. Significantly, there is already evidence of local control of PP1 and GSK3β at sites which require cargo delivery. Methods: To investigate this hypothesis, first we evaluated cellular localization of tau PAD exposure, pT205 tau phosphorylation, and active GSK3β in primary hippocampal neurons during development. Second, we analyzed the axonal outgrowth of tau knockout neurons following transfection with full length hTau40-WT, hTau40-ΔPAD, or hTau40-T205A. Results and Discussion: The results presented here suggest that transient activation of a PP1-GSK3β signaling pathway through locally regulated PAD exposure is a mechanism for cargo delivery, and thereby important for neurite outgrowth of developing neurons.

In Vivo Fast Nonlinear Microscopy Reveals Impairment of Fast Axonal Transport Induced by Molecular Motor Imbalances in the Brain of Zebrafish Larvae.

Cargo transport by molecular motors along microtubules is essential for the function of eukaryotic cells, in particular neurons in which axonal transport defects constitute the early pathological features of neurodegenerative diseases. Mainly studied in motor and sensory neurons, axonal transport is still difficult to characterize in neurons of the brain in absence of appropriate in vivo tools. Here, we measured fast axonal transport by tracing the second harmonic generation (SHG) signal of potassium titanyl phosphate (KTP) nanocrystals (nanoKTP) endocytosed by brain neurons of zebrafish (Zf) larvae. Thanks to the optical translucency of Zf larvae and to the perfect photostability of nanoKTP SHG, we achieved a high scanning speed of 20 frames (of ≈90 μm × 60 μm size) per second in Zf brain. We focused our study on endolysosomal vesicle transport in axons of known polarization, separately analyzing kinesin and dynein motor-driven displacements. To validate our assay, we used either loss-of-function mutations of dynein or kinesin 1 or the dynein inhibitor dynapyrazole and quantified several transport parameters. We successfully demonstrated that dynapyrazole reduces the nanoKTP mobile fraction and retrograde run length consistently, while the retrograde run length increased in kinesin 1 mutants. Taking advantage of nanoKTP SHG directional emission, we also quantified fluctuations of vesicle orientation. Thus, by combining endocytosis of nanocrystals having a nonlinear response, fast two-photon microscopy, and high-throughput analysis, we are able to finely monitor fast axonal transport in vivo in the brain of a vertebrate and reveal subtle axonal transport alterations. The high spatiotemporal resolution achieved in our model may be relevant to precisely investigate axonal transport impairment associated with disease models.

Quantum-dot-labeled synuclein seed assay identifies drugs modulating the experimental prion-like transmission

Synucleinopathies are neurodegenerative disorders including Parkinson disease (PD), dementia with Lewy body (DLB), and multiple system atrophy (MSA) that involve deposits of the protein alpha-synuclein (α-syn) in the brain. The inoculation of α-syn aggregates derived from synucleinopathy or preformed fibrils (PFF) formed in vitro induces misfolding and deposition of endogenous α-syn. This is referred to as prion-like transmission, and the mechanism is still unknown. In this study, we label α-syn PFF with quantum dots and visualize their movement directly in acute slices of brain tissue inoculated with α-syn PFF seeds. Using this system, we find that the trafficking of α-syn seeds is dependent on fast axonal transport and the seed spreading is dependent on endocytosis and neuronal activity. We also observe pharmacological effects on α-syn seed spreading; clinically available drugs including riluzole are effective in reducing the spread of α-syn seeds and this effect is also observed in vivo. Our quantum-dot-labeled α-syn seed assay system combined with in vivo transmission experiment reveals an early phase of transmission, in which uptake and spreading of seeds occur depending on neuronal activity, and a later phase, in which seeds induce the propagation of endogenous misfolded α-syn.

Bidirectional, unlike unidirectional transport, allows transporting axonal cargos against their concentration gradient

Even though most axonal cargos are synthesized in the soma, the concentration of many of these cargos is larger at the presynaptic terminal than in the soma. This requires transport of these cargos from the soma to the presynaptic terminal or other active sites in the axon. Axons utilize both bidirectional (for example, slow axonal transport) and unidirectional (for example, fast anterograde axonal transport) modes of cargo transport. Bidirectional transport seems to be less efficient because it requires more time and takes more energy to deliver cargos. In this paper, we studied a family of models which differ by the modes of axonal cargo transport (such as anterograde and retrograde motor-driven transport and passive diffusion) as well as by the presence or absence of pausing states. The models are studied to investigate their ability to describe axonal transport against the cargo concentration gradient. We argue that bidirectional axonal transport is described by a higher-order mathematical model, which allows imposing cargo concentration not only at the axon hillock but also at the axon terminal. The unidirectional transport model allows only for the imposition of cargo concentration at the axon hillock. Due to the great lengths of the axons, anterograde transport mostly relies on molecular motors, such as kinesins, to deliver cargos synthesized in the soma to the terminal and other active sites in the axon. Retrograde transport can be also motor-driven, in which case cargos are transported by dynein motors. If cargo concentration at the axon tip is higher than at the axon hillock, retrograde transport can also occur by cargo diffusion. However, because many axonal cargos are large or they assemble in multiprotein complexes for axonal transport, the diffusivity of such cargos is very small. We investigated the case of a small cargo diffusivity using a perturbation technique and found that for this case the effect of diffusion is limited to a very thin diffusion boundary layer near the axon tip. If cargo diffusivity is decreased in the model, we show that without motor-driven retrograde transport the model is unable to describe a high cargo concentration at the axon tip. To the best of our knowledge, our paper presents the first explanation for the utilization of seemingly inefficient bidirectional transport in neurons.

Assessment of Mitochondrial Trafficking as a Surrogate for Fast Axonal Transport in Human Induced Pluripotent Stem Cell-Derived Spinal Motor Neurons.

Axonal transport is essential for the development, function, and survival of the nervous system. In an energy-demanding process, motor proteins act in concert with microtubules to deliver cargoes, such as organelles, from one end of the axon to the other. Perturbations in axonal transport are a prominent phenotype of many neurodegenerative diseases, including amyotrophic lateral sclerosis. Here, we describe a simple method tofluorescently label mitochondrial cargo, a surrogate for fast axonal transport, in human induced pluripotent stem cell-derived motor neurons. This method enables the sparse labeling of axons to track directionality of movement and can be adapted to assess not only the cell autonomous effects of a genetic mutation on axonal transport but also the cell non-autonomous effects, through the use of conditioned medium and/or co-culture systems.

Engagement of Neurotropic Viruses in Fast Axonal Transport: Mechanisms, Potential Role of Host Kinases and Implications for Neuronal Dysfunction.

Much remains unknown about mechanisms sustaining the various stages in the life cycle of neurotropic viruses. An understanding of those mechanisms operating before their replication and propagation could advance the development of effective anti-viral strategies. Here, we review our current knowledge of strategies used by neurotropic viruses to undergo bidirectional movement along axons. We discuss how the invasion strategies used by specific viruses might influence their mode of interaction with selected components of the host’s fast axonal transport (FAT) machinery, including specialized membrane-bounded organelles and microtubule-based motor proteins. As part of this discussion, we provide a critical evaluation of various reported interactions among viral and motor proteins and highlight limitations of some in vitro approaches that led to their identification. Based on a large body of evidence documenting activation of host kinases by neurotropic viruses, and on recent work revealing regulation of FAT through phosphorylation-based mechanisms, we posit a potential role of host kinases on the engagement of viruses in retrograde FAT. Finally, we briefly describe recent evidence linking aberrant activation of kinase pathways to deficits in FAT and neuronal degeneration in the context of human neurodegenerative diseases. Based on these findings, we speculate that neurotoxicity elicited by viral infection may involve deregulation of host kinases involved in the regulation of FAT and other cellular processes sustaining neuronal function and survival.

Rab2 drives axonal transport of dense core vesicles and lysosomal organelles.

Fast axonal transport of neuropeptide-containing dense core vesicles (DCVs), endolysosomal organelles, and presynaptic components is critical for maintaining neuronal functionality. How the transport of DCVs is orchestrated remains an important unresolved question. The small GTPase Rab2 mediates DCV biogenesis and endosome-lysosome fusion. Here, we use Drosophila to demonstrate that Rab2 also plays a critical role in bidirectional axonal transport of DCVs, endosomes, and lysosomal organelles, most likely by controlling molecular motors. We further show that the lysosomal motility factor Arl8 is required as well for axonal transport of DCVs, but unlike Rab2, it is also critical for DCV exit from cell bodies into axons. We also provide evidence that the upstream regulators of Rab2 and Arl8, Ema and BORC, activate these GTPases during DCV transport. Our results uncover the mechanisms underlying axonal transport of DCVs and reveal surprising parallels between the regulation of DCV and lysosomal motility.

Molecular Determinants of the Neurotoxicity of Acrylamide Through Covalent Docking

Acrylamide is formed during food processing by Maillard reaction between sugars and proteins at high temperatures. Besides, acrylamide is used in many industries, from water waste treatment to manufacture of paper, fabrics, dyes or cosmetics. Cumulative exposure to acrylamide, either from diet or in the workplace, may result in neurotoxicity, involving three possible mechanisms: inhibition of fast axonal transport, alteration of neurotransmitter levels, and direct inhibition of neurotransmission. From the molecular point of view, acrylamide may affect protein function by forming a covalent adduct with cysteine residues via a Michael addition reaction. However, the molecular determinants of acrylamide reactivity are not completely understood and further investigation is needed. As a follow-up of previous computational studies by Carloni and coworkers [1,2], here we have performed a systematic study of the acrylamide protein targets reported so far in the literature in connection with neurotoxicity using covalent docking. Comparison with drug protein targets of acrylamide warhead-containing inhibitors was also performed. Analysis of the acrylamide-protein complexes provides insights into the determinants of cysteine reactivity, as well as the residue composition of acrylamide binding sites. This information could be used to predict other potential protein targets mediating acrylamide neurotoxicity. This project was inspired by late Dr. Ernesto Illy and funded by the Ernesto Illy Foundation. [1] de Lima, E. F., & Carloni, P. (2011). Acrylamide Binding to Its Cellular Targets: Insights from Computational Studies. Computational Biology and Applied Bioinformatics, 431. [2] Papamokos, G., Dreyer, J., Navarini, L., & Carloni, P. (2014). Trapping acrylamide by a Michael addition: a computational study of the reaction between acrylamide and niacin. Int. J. Quantum Chem., 114(9), 553-559.

Defined Tau Phosphospecies Differentially Inhibit Fast Axonal Transport Through Activation of Two Independent Signaling Pathways.

Tau protein is subject to phosphorylation by multiple kinases at more than 80 different sites. Some of these sites are associated with tau pathology and neurodegeneration, but other sites are modified in normal tau as well as in pathological tau. Although phosphorylation of tau at residues in the microtubule-binding repeats is thought to reduce tau association with microtubules, the functional consequences of other sites are poorly understood. The AT8 antibody recognizes a complex phosphoepitope site on tau that is detectable in a healthy brain but significantly increased in Alzheimer’s disease (AD) and other tauopathies. Previous studies showed that phosphorylation of tau at the AT8 site leads to exposure of an N-terminal sequence that promotes activation of a protein phosphatase 1 (PP1)/glycogen synthase 3 (GSK3) signaling pathway, which inhibits kinesin-1-based anterograde fast axonal transport (FAT). This finding suggests that phosphorylation may control tau conformation and function. However, the AT8 includes three distinct phosphorylated amino acids that may be differentially phosphorylated in normal and disease conditions. To evaluate the effects of specific phosphorylation sites in the AT8 epitope, recombinant, pseudophosphorylated tau proteins were perfused into the isolated squid axoplasm preparation to determine their effects on axonal signaling pathways and FAT. Results from these studies suggest a mechanism where specific phosphorylation events differentially impact tau conformation, promoting activation of independent signaling pathways that differentially affect FAT. Implications of findings here to our understanding of tau function in health and disease conditions are discussed.