Search

Mutations in the microtubule-binding motor protein kinesin 5 A (KIF5A) are implicated in several adult-onset motor neuron diseases, including Amyotrophic Lateral Sclerosis, Spastic Paraplegia Type 10 and Charcot-Marie-Tooth Disease Type 2. While KIF5 family members transport a variety of cargos along axons, the specific cargos affected by KIF5A mutations remain poorly understood. Here, we generated KIF5Anull mutant human motor neurons and analyzed the impact on axonal transport and motor neuron outgrowth and regeneration in vitro. KIF5A deficiency caused reduced neurite complexity in young neurons (DIV14) and defects in axonal regeneration. KIF5A deficiency did not affect neurofilament transport but impaired mitochondrial motility and anterograde speed at DIV42. Notably, KIF5A deficiency strongly reduced anterograde transport of splicing factor proline/glutamine-rich (SFPQ)-associated RNA granules in DIV42 axons. Hence, KIF5A plays a critical role in promoting axonal regrowth after injury and in driving the anterograde transport of mitochondria and especially SFPQ-associated RNA granules in mature neurons., Competing Interests: Declaration of competing interest K.E. is a cofounder of Q-State Biosciences, Quralis, Enclear Therapies and is group vice-president at BioMarin Pharmaceutical. The remaining authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuronal mitochondria are essential organelles to maintain synaptic activity due to the high calcium buffering capacity and ATP production. In neurons, mitochondria transport occurs along the microtubules mediated by motor proteins, kinesins and dynein, to drive mitochondria toward the synapses. Disruption of axonal transport is an early pathogenic event in neurodegenerative disorders and growing evidence supports that it may precede neurodegeneration. Here, we describe a method to label mitochondria with fluorescent proteins to monitor their movement along the axons in hiPSC-derived medium spiny neuron-like cells. We also included a detailed protocol for differentiation of hiPSC that produces electrophysiologically mature GABAergic striatal neurons with low amount of glial population., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The interactions between mechanical forces and neuronal dynamics have long intrigued researchers. Several studies revealed that force plays a pivotal role in shaping axonal outgrowth. However, the molecular mechanisms underpinning force-driven axonal plasticity remain not completely elucidated. This review explores the relationship between force and axonal plasticity, with a focus on local mechanisms, including local translation and axonal transport, and the emerging concept of force-driven cross-talk, a dialogue in which local dynamics are tightly regulated. Recent experimental evidence suggests that microtubules may serve as key mediators of this cross-talk, orchestrating the coordination between local mechanisms and facilitating mass addition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons (MNs), and despite progress, there is no effective treatment. A large body of evidence shows that astrocytes expressing ALS-linked mutant proteins cause non-cell autonomous toxicity of MNs. Although MNs innervate muscle fibers and ALS is characterized by the early disruption of the neuromuscular junction (NMJ) and axon degeneration, there are controversies about whether muscle contributes to non-cell-autonomous toxicity to MNs. In this study, we generated primary skeletal myotubes from myoblasts derived from ALS mice expressing human mutant SOD1 G93A (termed hereafter mutSOD1). Characterization revealed that mutSOD1 skeletal myotubes display intrinsic phenotypic and functional differences compared to control myotubes generated from non-transgenic (NTg) littermates. Next, we analyzed whether ALS myotubes exert non-cell-autonomous toxicity to MNs. We report that conditioned media from mutSOD1 myotubes (mutSOD1-MCM), but not from control myotubes (NTg-MCM), induced robust death of primary MNs in mixed spinal cord cultures and compartmentalized microfluidic chambers. Our study further revealed that applying mutSOD1-MCM to the MN axonal side in microfluidic devices rapidly reduces mitochondrial axonal transport while increasing Ca2 + transients and reactive oxygen species (i.e., H 2 O 2 ). These results indicate that soluble factor(s) released by mutSOD1 myotubes cause MN axonopathy that leads to lethal pathogenic changes., (© 2024. The Author(s).)

The axonal transport of synaptic vesicle precursors relies on KIF1A and UNC-104 ortholog motors. In mammals, KIF1Bβ is also responsible for the axonal transport of synaptic vesicle precursors. Mutations in KIF1A and KIF1Bβ lead to a wide range of neuropathies. Although previous studies have revealed the biochemical, biophysical and cell biological properties of KIF1A, and its defects in neurological disorders, the fundamental properties of KIF1Bβ remain elusive. In this study, we determined the motile parameters of KIF1Bβ through single-molecule motility assays. We found that the C-terminal region of KIF1Bβ has an inhibitory role in motor activity. AlphaFold2 prediction suggests that the C-terminal region blocks the motor domain. Additionally, we established simple methods for testing the axonal transport activity of human KIF1Bβ using Caenorhabditis elegans genetics. Taking advantage of these methods, we demonstrated that these assays enable the detection of reduced KIF1Bβ activities, both in vitro and in vivo, caused by a Charcot-Marie-Tooth disease-associated Q98L mutation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Mitochondrial dysfunction is a prominent hallmark of Alzheimer's disease (AD). The transcriptional coactivator PPARγ coactivator 1 (PGC-1a) has been identified as a key regulator of mitochondrial biogenesis and function. However, the precise structure/function relationship between PGC-1a and mitochondrial quality control remains incompletely understood. In this study, we investigated the impact of PGC-1a on AD pathology and its underlying mechanisms with a specific focus on mitochondrial axonal transport. Additionally, we generated two PGC-1α mutants by substituting leucine residues at positions 148 and 149 within the LKKLL motif or at positions 209 and 210 within the LLKYL motif with alanine. Subsequently, we examined the effects of these mutants on mutAPP-induced abnormalities in anterograde and retrograde axonal transport, disrupted mitochondrial distribution, and impaired mitophagy. Mutagenesis studies revealed that the LLKYL motif at amino acid position 209-210 within PGC-1α plays an essential role in its interaction with estrogen-related receptors (ERRα), which is necessary for restoring normal mitochondrial anterograde axonal transport, maintaining proper mitochondrial distribution, and ultimately preventing neuronal apoptosis. Furthermore, it was found that the Leu-rich motif at amino acids 209-210 within PGC-1α is crucial for rescuing mutAPP-induced impairment in mitophagy and loss of membrane potential by restoring normal mitochondrial retrograde axonal transport. Conversely, mutation of residues 148 and 149 in the LKKLL motif does not compromise the effectiveness of PGC-1α. These findings provide valuable insights into the molecular determinants governing specificity of action for PGC-1α involved in regulating mutAPP-induced deficits in mitochondrial axonal trafficking. Moreover, they suggest a potential therapeutic target for addressing Alzheimer's disease., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

Lysosomes are central to the maintenance of protein and organelle homeostasis in cells. Optimal lysosome function is particularly critical for neurons which are long-lived, non-dividing and highly polarized with specialized compartments such as axons and dendrites with distinct architecture, cargo, and turnover requirements. In recent years, there has been a growing appreciation for the role played by axonal lysosome transport in regulating neuronal development, its maintenance and functioning. Perturbations to optimal axonal lysosome abundance leading to either strong accumulations or dearth of lysosomes are both linked to altered neuronal health and functioning. In this review we highlight how two critical regulators of axonal lysosome transport and abundance, the small GTPase Arl8 and the adaptor protein JIP3, aid in maintaining axonal lysosome homeostasis and how alterations to their levels and activity could contribute to neurodevelopmental and neurodegenerative diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) from patients with amyotrophic lateral sclerosis (ALS) and the C9ORF72 hexanucleotide repeat expansion (HRE) have multiple cellular phenotypes, but which of these accurately reflect the biology underlying the cell-specific vulnerability of ALS is uncertain. We therefore compared phenotypes due to the C9ORF72 HRE in MNs with sensory neurons (SNs), which are relatively spared in ALS. The iPSC models were able to partially reproduce the differential gene expression seen between adult SNs and MNs. We demonstrated that the typical hallmarks of C9ORF72-ALS, including RNA foci and dipeptide formation, as well as specific axonal transport defects, occurred equally in MNs and SNs, suggesting that these in vitro phenotypes are not sufficient to explain the cell-type selectivity of ALS in isolation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Disruption of synapse assembly and maturation leads to a broad spectrum of neurodevelopmental disorders. Presynaptic proteins are largely synthesized in the soma, where they are packaged into precursor vesicles and transported into distal axons to ensure precise assembly and maintenance of presynapses. Due to their morphological features, neurons face challenges in the delivery of presynaptic cargos to nascent boutons. Thus, targeted axonal transport is vital to build functional synapses. A growing number of mutations in genes encoding the transport machinery have been linked to neurodevelopmental disorders. Emerging lines of evidence have started to uncover presynaptic mechanisms underlying axonal transport defects, thus broadening the view of neurodevelopmental disorders beyond postsynaptic mechanisms. In this review, we discuss presynaptic perspectives of neurodevelopmental disorders by focusing on impaired axonal transport and disturbed assembly and maintenance of presynapses. We also discuss potential strategies for restoring axonal transport as an early therapeutic intervention., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRS E196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of Yars E196K mice modelling DI-CMT. We determined that Yars E196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRS E196K , but not TyrRS WT , into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy., Competing Interests: Declaration of competing interest The technology described in this work has been protected in the patent GB2303495.2 (patent applicant, UCL Business Ltd., status pending), in which GS and JNS are named as inventors. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurons pose a particular challenge to degradative processes like autophagy due to their long and thin processes. Autophagic vesicles (AVs) are formed at the tip of the axon and transported back to the soma. This transport is essential since the final degradation of the vesicular content occurs only close to or in the soma. Here, we established an in vivo live-imaging model in the rat optic nerve using viral vector mediated LC3-labeling and two-photon-microscopy to analyze axonal transport of AVs. Under basal conditions in vivo, 50% of the AVs are moving with a majority of 85% being transported in the retrograde direction. Transport velocity is higher in the retrograde than in the anterograde direction. A crush lesion of the optic nerve results in a rapid breakdown of retrograde axonal transport while the anterograde transport stays intact over several hours. Close to the lesion site, the formation of AVs is upregulated within the first 6 h after crush, but the clearance of AVs and the levels of lysosomal markers in the adjacent axon are reduced. Expression of p150Glued, an adaptor protein of dynein, is significantly reduced after crush lesion. In vitro, fusion and colocalization of the lysosomal marker cathepsin D with AVs are reduced after axotomy. Taken together, we present here the first in vivo analysis of axonal AV transport in the mammalian CNS using live-imaging. We find that axotomy leads to severe defects of retrograde motility and a decreased clearance of AVs via the lysosomal system., (© 2024. The Author(s).)

Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required., (© 2024 MRC Laboratory of Molecular Biology.)

Mitochondria transport is crucial for axonal mitochondria distribution and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 binding to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1, and metaxin2. We conclude that transport complexes containing kinesin-1 and RIC-7 polarize at the leading edge of mitochondria and are required for anterograde axonal transport in C. elegans., (© 2024 Wu et al.)

In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination, and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated and detyrosinated MTs for proper AIS length and axonal transport processes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Experimental studies in flies, mice, and humans suggest a significant role of impaired axonal transport in the pathogenesis of Alzheimer's disease (AD). The mechanisms underlying these impairments in axonal transport, however, remain poorly understood. Here we report that the Swedish familial AD mutation causes a standstill of the amyloid precursor protein (APP) in the axons at the expense of its reduced anterograde transport. The standstill reflects the perturbed directionality of the axonal transport of APP, which spends significantly more time traveling in the retrograde direction. This ineffective movement is accompanied by an enhanced association of dynactin-1 with APP, which suggests that reduced anterograde transport of APP is the result of enhanced activation of the retrograde molecular motor dynein by dynactin-1. The impact of the Swedish mutation on axonal transport is not limited to the APP vesicles since it also reverses the directionality of a subset of early endosomes, which become enlarged and aberrantly accumulate in distal locations. In addition, it also reduces the trafficking of lysosomes due to their less effective retrograde movement. Altogether, our experiments suggest a pivotal involvement of retrograde molecular motors and transport in the mechanisms underlying impaired axonal transport in AD and reveal significantly more widespread derangement of axonal transport pathways in the pathogenesis of AD., Competing Interests: Conflict of interest The authors declare no that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Axonal transport in neurons is essential for cargo movement between the cell body and synapses. Caenorhabditis elegans UNC-104 and its homolog KIF1A are kinesin-3 motors that anterogradely transport precursors of synaptic vesicles (pre-SVs) and are degraded at synapses. However, in C. elegans, touch neuron-specific knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to UNC-104 accumulation at neuronal ends and synapses. Here, we performed an RNAi screen and identified that depletion of fbxb-65, which encodes an F-box protein, leads to UNC-104 accumulation at neuronal distal ends, and alters UNC-104 net anterograde movement and levels of UNC-104 on cargo without changing synaptic UNC-104 levels. Split fluorescence reconstitution showed that UNC-104 and FBXB-65 interact throughout the neuron. Our theoretical model suggests that UNC-104 might exhibit cooperative cargo binding that is regulated by FBXB-65. FBXB-65 regulates an unidentified post-translational modification (PTM) of UNC-104 in a region beside the cargo-binding PH domain. Both fbxb-65 and UNC-104, independently of FBXB-65, regulate axonal pre-SV distribution, transport of pre-SVs at branch points and organismal lifespan. FBXB-65 regulates a PTM of UNC-104 and the number of motors on the cargo surface, which can fine-tune cargo transport to the synapse., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Slow axonal transport (SAT) moves multiple proteins from the soma, where they are synthesized, to the axon terminal. Due to the great lengths of axons, SAT almost exclusively relies on active transport, which is driven by molecular motors. The puzzling feature of slow axonal transport is its bidirectionality. Although the net direction of SAT is anterograde, from the soma to the terminal, experiments show that it also contains a retrograde component. One of the proteins transported by SAT is the microtubule-associated protein tau. To better understand why the retrograde component in tau transport is needed, we used the perturbation technique to analyze how the full tau SAT model can be simplified for the specific case when retrograde motor-driven transport and diffusion-driven transport of tau are negligible and tau is driven only by anterograde (kinesin) motors. The solution of the simplified equations shows that without retrograde transport the tau concentration along the axon length stays almost uniform (decreases very slightly), which is inconsistent with the experimenal tau concentration at the outlet boundary (at the axon tip). Thus kinesin-driven transport alone is not enough to explain the empirically observed distribution of tau, and the retrograde motor-driven component in SAT is needed.

Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. Consequently, loss of axonal transport impairs neuronal growth and function. Studying axonal transport therefore improves our understanding of neuronal cell biology. With recent improvements in CRISPR Cas9 genome editing, endogenous labeling of axonal cargos has become accessible, enabling to move beyond ectopic expression-based visualization of transport. However, endogenous labeling often comes at the cost of low signal intensity and necessitates optimization strategies to obtain robust data. Here, we describe a protocol to optimize the visualization of axonal transport by discussing acquisition parameters and a bleaching approach to improve the signal of endogenous labeled cargo over diffuse cytoplasmic background. We apply our protocol to optimize the visualization of synaptic vesicle precursors (SVPs) labeled by green fluorescent protein (GFP)-tagged RAB-3 to highlight how fine-tuning acquisition parameters can improve the analysis of endogenously labeled axonal cargo in Caenorhabditis elegans (C. elegans).

Background: Bioenergetic 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., Methods: We 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., Results: We 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., Conclusion: NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport., (© 2024. The Author(s).)

The specialized function and extreme geometry of neurons necessitates a unique reliance upon long-distance microtubule-based transport. Appropriate trafficking of axonal cargos by motor proteins is essential for establishing circuitry during development and continuing function throughout a lifespan. Visualizing and quantifying cargo movement provides valuable insight into how axonal organelles are replenished, recycled, and degraded during the dynamic dance of outgoing and incoming axonal traffic. Long-distance axonal trafficking is of particular importance as it encompasses a pathway commonly disrupted in developmental and degenerative disease states. Here, we describe neuronal organelles and outline methods for live imaging and quantifying their movement throughout the axon via transient expression of fluorescently labeled organelle markers. This resource provides recommendations for target proteins/domains and appropriate acquisition time scales for visualizing distinct neuronal cargos in cultured neurons derived from human induced pluripotent stem cells (iPSCs) and primary rat neurons., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 extracellular domain 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.

Neuronal autophagosomes form and engulf cargos at presynaptic sites in the axon and are then transported to the soma to recycle their cargo. Autophagic vacuoles (AVs) mature en route via fusion with lysosomes to become degradatively competent organelles; transport is driven by the microtubule motor protein cytoplasmic dynein, with motor activity regulated by a sequential series of adaptors. Using lysate-based single-molecule motility assays and live-cell imaging in primary neurons, we show that JNK-interacting proteins 3 (JIP3) and 4 (JIP4) are activating adaptors for dynein that are regulated on autophagosomes and lysosomes by the small GTPases ARF6 and RAB10. GTP-bound ARF6 promotes formation of the JIP3/4-dynein-dynactin complex. Either knockdown or overexpression of RAB10 stalls transport, suggesting that this GTPase is also required to coordinate the opposing activities of bound dynein and kinesin motors. These findings highlight the complex coordination of motor regulation during organelle transport in neurons., (© 2023 Cason and Holzbaur.)

In this study, we consider axonal transport of large cargo vesicles characterised by transient expansion of the axon shaft. Our goal is to formulate a mathematical model which captures the dynamic mechanical interaction of such cargo vesicles with the membrane associated periodic cytoskeletal structure (MPS). It consists of regularly spaced actin rings that are transversal to the longitudinal direction of the axon and involved in the radial contraction of the axon. A system of force balance equations is formulated by which we describe the transversal rings as visco-elastic Kelvin-Voigt elements. In a homogenisation limit, we reformulate the model as a free boundary problem for the interaction of the submembranous MPS with the large vesicle. We derive a non-linear force-velocity relation as a quasi-steady state solution. Computationally we analyse the vesicle size dependence of the transport speed and use an asymptotic approximation to formulate it as a power law that can be tested experimentally., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We describe a compartmental model of mitochondrial transport in axons, which we apply to compute mitochondrial age at different distances from the soma. The model predicts that at the tip of an axon that has a length of 1 cm, the average mitochondrial age is approximately 22 h. The mitochondria are youngest closest to the soma and their age scales approximately linearly with distance from the soma. To the best of the authors' knowledge, this is the first attempt to predict the spatial distribution of mitochondrial age within an axon. A sensitivity study of the mean age of mitochondria to various model parameters is also presented.

Crayfish axons contain a system of parallel membranous cisternae spaced by ~2 μm and oriented perpendicularly to the axon's long axis. Each cisterna is composed of two roughly parallel membranes, separated by a 150-400 Å wide space. The cisternae are interrupted by 500-600 Å pores, each occupied by a microtubule. Significantly, filaments, likely made of kinesin, often bridge the gap between the microtubule and the edge of the pore. Neighboring cisternae are linked by longitudinal membranous tubules. In small axons, the cisternae seem to be continuous across the axon, while in large axons they are intact only at the axon's periphery. Due to the presence of pores, we have named these structures "Fenestrated Septa" (FS). Similar structures are also present in vertebrates, including mammals, proving that they are widely expressed in the animal kingdom. We propose that FS are components of the "anterograde transport" mechanism that moves cisternae of the Golgi apparatus (GA) toward the nerve ending by means of motor proteins, likely to be kinesins. In crayfish lateral giant axons, we believe that vesicles that bud off FS at the nerve ending contain gap junction hemichannels (innexons) for gap junction channel and hemichannel formation and function.

Neuronal function depends on axonal transport by kinesin superfamily proteins (KIFs). KIF1A is the molecular motor that transports synaptic vesicle precursors, synaptic vesicles, dense core vesicles and active zone precursors. KIF1A is regulated by an autoinhibitory mechanism; many studies, as well as the crystal structure of KIF1A paralogs, support a model whereby autoinhibited KIF1A is monomeric in solution, whereas activated KIF1A is dimeric on microtubules. KIF1A-associated neurological disorder (KAND) is a broad-spectrum neuropathy that is caused by mutations in KIF1A. More than 100 point mutations have been identified in KAND. In vitro assays show that most mutations are loss-of-function mutations that disrupt the motor activity of KIF1A, whereas some mutations disrupt its autoinhibition and abnormally hyperactivate KIF1A. Studies on disease model worms suggests that both loss-of-function and gain-of-function mutations cause KAND by affecting the axonal transport and localization of synaptic vesicles. In this Review, we discuss how the analysis of these mutations by molecular genetics, single-molecule assays and force measurements have helped to reveal the physiological significance of KIF1A function and regulation, and what physical parameters of KIF1A are fundamental to axonal transport., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Axonal transport is a major cellular process that mediates bidirectional signaling between the soma and synapse, enabling both intracellular and intercellular communications. Cellular materials, such as proteins, RNAs, and organelles, are transported by molecular motor proteins along cytoskeletal highways in a highly regulated manner. Several studies have demonstrated that axonal transport is central to normal neuronal function, plasticity, and memory storage. Importantly, disruptions in axonal transport result in neuronal dysfunction and are associated with several neurodegenerative disorders. However, we do not know much about axonal transport deficits in neuropsychiatric disorders. Here, we briefly discuss our current understanding of the role of axonal transport in schizophrenia, bipolar and autism., Competing Interests: Declaration of competing interest The authors of this review article do not have any competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules that fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin in neurons both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers like guanosine triphosphate (GTP)-tubulin. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin velocity and density on microtubules consisting of elongated dimers add to the increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin contributes to the regulation of mitochondrial motility and as such to the local distribution of mitochondria along axons.

Neurological disorders are prevalent in horses, but their study is challenging due to anatomic constraints and the large body size; very few host-specific in vitro models have been established to study these types of diseases, particularly from adult donor tissue. Here we report the generation of primary neuronal dorsal root ganglia (DRG) cultures from adult horses: the mixed, dissociated cultures, containing neurons and glial cells, remained viable for at least 90 days. Similar to DRG neurons in vivo, cultured neurons varied in size, and they developed long neurites. The mitochondrial movement was detected in cultured cells and was significantly slower in glial cells compared to DRG-derived neurons. In addition, mitochondria were more elongated in glial cells than those in neurons. Our culture model will be a useful tool to study the contribution of axonal transport defects to specific neurodegenerative diseases in horses as well as comparative studies aimed at evaluating species-specific differences in axonal transport and survival., (© 2022 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Axonal transport plays a significant role in the establishment of neuronal polarity, axon growth, and synapse formation during neuronal development. The axon of a naturally growing neuron is a highly complex and multifurcated structure with a large number of bends and branches. Nowadays, the study of dynamic axonal transport in morphologically complex neurons is greatly limited by the technological barrier. Here, a sparse gene transfection strategy was developed to locate fluorescent mCherry in the lysosome of primary neurons, thus enabling us to track the lysosome-based axonal transport with a single-particle resolution. Thereby, several axonal transport models were observed, including the forward or backward transport model, stop-and-go model, repeated back-and-forth transport model, and cross-branch transport model. Then, the accurate single-particle velocity quantification by TrackMate revealed a highly heterogeneous and discontinuous transportation process of lysosome-based axonal transport in freely orientated axons. And, multiple physical factors, such as the axonal structure and the size of particles, were disclosed to affect the velocity of particle transporting in freely orientated axons. The combined single-particle fluorescence tracking and TrackMate assay can be served as a facile tool for evaluating axonal transport in neuronal development and axonal transport-related diseases., (© 2022 Wiley-VCH GmbH.)

Neurofilaments are abundant space-filling cytoskeletal polymers that are transported into and along axons. During postnatal development, these polymers accumulate in myelinated axons causing an expansion of axon caliber, which is necessary for rapid electrical transmission. Studies on cultured nerve cells have shown that axonal neurofilaments move rapidly and intermittently along microtubule tracks in both anterograde and retrograde directions. However, it is unclear whether neurofilament transport is also bidirectional in vivo Here, we describe a pulse-spread fluorescence photoactivation method to address this in peripheral nerves dissected from hThy1-paGFP-NFM transgenic mice, which express a photoactivatable fluorescent neurofilament protein. Neurofilaments were photoactivated in short segments of myelinated axons in tibial nerves at 2, 4, 8, and 16 weeks of age. The proximal and distal spread of the fluorescence due to the movement of the fluorescent neurofilaments was measured over time. We show that the directional bias and velocity of neurofilament transport can be calculated from these measurements. The directional bias was ∼60% anterograde and 40% retrograde and did not change significantly with age or distance along the nerve. The net velocity decreased with age and distance along the nerve, which is consistent with previous studies using radioisotopic pulse labeling. This decrease in velocity was caused by a decrease in both anterograde and retrograde movement. Thus, neurofilament transport is bidirectional in vivo , with a significant fraction of the filaments moving retrogradely in both juvenile and adult mice., (Copyright © 2022 Boyer et al.)

Axonal transport ensures long-range delivery of essential cargoes between proximal and distal compartments, and is needed for neuronal development, function, and survival. Deficits in axonal transport have been detected at pre-symptomatic stages in the SOD1 G93A and TDP-43 M337V mouse models of amyotrophic lateral sclerosis (ALS), suggesting that impairments in this critical process are fundamental for disease pathogenesis. Strikingly, in ALS, fast motor neurons (FMNs) degenerate first whereas slow motor neurons (SMNs) are more resistant, and this is a currently unexplained phenomenon. The main aim of this investigation was to determine the effects of brain-derived neurotrophic factor (BDNF) on in vivo axonal transport in different α-motor neuron (MN) subtypes in wild-type (WT) and SOD1 G93A mice. We report that despite displaying similar basal transport speeds, stimulation of wild-type MNs with BDNF enhances in vivo trafficking of signalling endosomes specifically in FMNs. This BDNF-mediated enhancement of transport was also observed in primary ventral horn neuronal cultures. However, FMNs display selective impairment of axonal transport in vivo in symptomatic SOD1 G93A mice, and are refractory to BDNF stimulation, a phenotype that was also observed in primary embryonic SOD1 G93A neurons. Furthermore, symptomatic SOD1 G93A mice display upregulation of the classical non-pro-survival truncated TrkB and p75 NTR receptors in muscles, sciatic nerves, and Schwann cells. Altogether, these data indicate that cell- and non-cell autonomous BDNF signalling is impaired in SOD1 G93A MNs, thus identifying a new key deficit in ALS., (© 2022. The Author(s).)

In this study a manganese-enhanced magnetic resonance imaging (MEMRI) method was developed for mice for measuring axonal transport (AXT) rates in real time in olfactory receptor neurons, which project from the olfactory epithelium to the olfactory neuronal layer of the olfactory bulb. Using this MEMRI method, two major experiments were conducted: 1) an evaluation of the effects of age on AXT rates and 2) an evaluation of the brain-penetrant, microtubule-stabilizing agent, Epothilone D for effect on AXT rates in aged mice. In these studies, we improved upon previous MEMRI approaches to develop a method where real-time measurements (32 time points) of AXT rates in mice can be determined over a single (approximately 100 min) scanning session. In the age comparisons, AXT rates were significantly higher in young (mean age ∼4.0 months old) versus aged (mean age ∼24.5 months old) mice. Moreover, in aged mice, eight weeks of treatment with Epothilone D, (0.3 and 1.0 mg/kg) was associated with statistically significant increases in AXT rates compared to vehicle-treated subjects. These experiments conducted in a living mammalian model (i.e., wild type, C57BL/6 mice), using a new modified MEMRI method, thus provide further evidence that the process of aging leads to decreases in AXT rates in the brain and they further support the argument that microtubule-based therapeutic strategies designed to improve AXT rates have potential for age-related neurological disorders., (Copyright © 2022 Elsevier B.V. All rights reserved.)

KIF1A is a kinesin superfamily motor protein that transports synaptic vesicle precursors in axons. Cargo binding stimulates the dimerization of KIF1A molecules to induce processive movement along microtubules. Mutations in human Kif1a lead to a group of neurodegenerative diseases called KIF1A-associated neuronal disorder (KAND). KAND mutations are mostly de novo and autosomal dominant; however, it is unknown if the function of wild-type KIF1A motors is inhibited by heterodimerization with mutated KIF1A. Here, we have established Caenorhabditis elegans models for KAND using CRISPR-Cas9 technology and analyzed the effects of human KIF1A mutation on axonal transport. In our C. elegans models, both heterozygotes and homozygotes exhibited reduced axonal transport. Suppressor screening using the disease model identified a mutation that recovers the motor activity of mutated human KIF1A. In addition, we developed in vitro assays to analyze the motility of heterodimeric motors composed of wild-type and mutant KIF1A. We find that mutant KIF1A significantly impaired the motility of heterodimeric motors. Our data provide insight into the molecular mechanism underlying the dominant nature of de novo KAND mutations.

Synapse formation is locally determined by transmembrane proteins, yet synaptic material is synthesized remotely and undergoes processive transport in axons. How local synaptogenic signals intercept synaptic cargo in transport to promote its delivery and synapse formation is unknown. We found that the control of synaptic cargo delivery at microtubule (MT) minus ends mediates pro- and anti-synaptogenic activities of presynaptic neurexin and frizzled in C. elegans and identified the atypical kinesin VAB-8/KIF26 as a key molecule in this process. VAB-8/KIF26 levels at synaptic MT minus ends are controlled by frizzled and neurexin; loss of VAB-8 mimics neurexin mutants or frizzled hyperactivation, and its overexpression can rescue synapse loss in these backgrounds. VAB-8/KIF26 is required for the synaptic localization of other minus-end proteins and promotes the pausing of retrograde transport to allow delivery to synapses. Consistently, reducing retrograde transport rescues synapse loss in vab-8 and neurexin mutants. These results uncover a mechanistic link between synaptogenic signaling and axonal transport., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Heroin is a highly addictive drug that causes axonal damage. Here, manganese-enhanced magnetic resonance imaging (MEMRI) was used to dynamically monitor axonal transport at different stages of heroin addiction. Rat models of heroin addiction (HA) and prolonged heroin addiction (PHA) were established by injecting rats with heroin at different stages. Heroin-induced learning and memory deficits were evaluated in the Morris water maze (MWM), and MEMRI was used to dynamically evaluate axonal transport in the olfactory pathway. The expression of proteins related to axonal structure and function was also assessed by Western blotting. Transmission electron microscopy (TEM) was used to observe ultrastructural changes, and protein levels of neurofilament heavy chain (NF-H) were analyzed by immunofluorescence staining. HA rats, especially PHA rats, exhibited worse spatial learning and memory than control rats. Compared with HA rats and control rats, PHA rats exhibited significantly longer escape latencies, significantly fewer platform-location crossings, and significantly more time in the target quadrant during the MWM test. Mn 2+ transport was accelerated in HA rats. PHA rats exhibited severely reduced Mn 2+ transport, and the axonal transport rate (ATR) was significantly lower in these rats than in control rats (P < 0.001). The levels of cytoplasmic dynein and kinesin-1 were significantly decreased in the PHA group than in the control group (P < 0.001); additionally, the levels of energy-related proteins, including cytochrome c oxidase (COX) IV and ATP synthase subunit beta (ATPB), were lower in the PHA group (P < 0.001). The brains of heroin-exposed rats displayed an abnormal ultrastructure, with neuronal apoptosis and mitochondrial dysfunction. Heroin exposure decreased the expression of NF-H, as indicated by significantly reduced staining intensities in tissues from HA and PHA rats (P < 0.05). MEMRI detected axonal transport dysfunction caused by long-term repeated exposure to heroin. The main causes of axonal transport impairment may be decreases in the levels of motor proteins and mitochondrial dysfunction. This study shows that MEMRI is a potential tool for visualizing axonal transport in individuals with drug addictions, providing a new way to evaluate addictive encephalopathy., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The aim of this study was to evaluate the effect of radiation-induced brain injury (RIBI) on axonal transport (AT) and sexual function., Methods and Materials: Adult male rats received whole-brain radiation with a total dose of 30 Gy (15 Gy with 2 fractions) to build a RIBI model. Foraging behavior and sexual function were assessed, and MRI was performed 8 weeks after brain irradiation. MRI was performed in the early and delayed phases after perfusion of MnCl 2 into the rat nostril. The levels of motor proteins and proteins involved in energy metabolism and AT were determined by Western blotting. The levels of sex hormones in the blood were measured by ELISA. Ultrastructural analysis was performed with a transmission electron microscope., Results: The foraging ability of rats was reduced after brain irradiation, and the foraging time of the radiation group was longer than that of the control group (P < 0.05). The sexual function of rats in the radiation group was markedly decreased. Compared with control rats, radiation-treated rats showed significant decreases in serum testosterone, FSH, LH, and GnRH levels (P < 0.001). Mn 2+ uptake in the olfactory bulb (OB) in the early phase and delayed phase was lower in the radiation group than in the control group (P < 0.05). The AT rate in the lateral olfactory tracts (LOT) and the transsynaptic AT rate were significantly lower in the irradiated rats than in the control rats (P < 0.05). The levels of the motor proteins kinesin-1 and cytoplasmic dynein were significantly decreased in the irradiation group (P < 0.05). The expression of the energy metabolism-related proteins ATPB and COX IV was significantly lower in the irradiated rats than in the control rats (P < 0.05). Apoptosis and synaptic damage were observed after irradiation., Conclusion: MRI of the olfactory pathway can be used to assess AT impairment in RIBI models. AT deficits secondary to radiation damage are the result of multiple factors, including declines in motor protein levels, neuronal apoptosis, synaptic damage and energy metabolism dysfunction. Cranial irradiation-induced sexual dysfunction was associated with decreased sex hormone levels secondary to hypothalamic-pituitary-gonadal axis injury., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Despite recent advancements in therapeutic options for disorders of the central nervous system (CNS), the lack of an efficient drug-delivery system (DDS) hampers their clinical application. We hypothesized that liposomes could be optimized for retrograde transport in axons as a DDS from peripheral tissues to the spinal cord and dorsal root ganglia (DRGs). Three types of liposomes consisting of DSPC, DSPC/POPC, or POPC in combination with cholesterol (Chol) and polyethylene glycol (PEG) lipid were administered to sciatic nerves or the tibialis anterior muscle of mature rats. Liposomes in cell bodies were detected with infrared fluorescence of DiD conjugated to liposomes. Three days later, all nerve-administered liposomes were retrogradely transported to the spinal cord and DRGs, whereas only muscle-administered liposomes consisting of DSPC reached the spinal cord and DRGs. Modification with Cholera toxin B subunit improved the transport efficiency of liposomes to the spinal cord and DRGs from 4.5% to 17.3% and from 3.9% to 14.3% via nerve administration, and from 2.6% to 4.8% and from 2.3% to 4.1% via muscle administration, respectively. Modification with octa-arginine (R8) improved the transport efficiency via nerve administration but abolished the transport capability via muscle administration. These findings provide the initial data for the development of a novel DDS targeting the spinal cord and DRGs via peripheral administration.

Turnover of synaptic vesicle (SV) proteins is vital for the maintenance of healthy and functional synapses. SV protein turnover is driven by neuronal activity in an endosomal sorting complex required for transport (ESCRT)-dependent manner. Here, we characterize a critical step in this process: axonal transport of ESCRT-0 component Hrs, necessary for sorting proteins into the ESCRT pathway and recruiting downstream ESCRT machinery to catalyze multivesicular body (MVB) formation. We find that neuronal activity stimulates the formation of presynaptic endosomes and MVBs, as well as the motility of Hrs+ vesicles in axons and their delivery to SV pools. Hrs+ vesicles co-transport ESCRT-0 component STAM1 and comprise a subset of Rab5+ vesicles, likely representing pro-degradative early endosomes. Furthermore, we identify kinesin motor protein KIF13A as essential for the activity-dependent transport of Hrs to SV pools and the degradation of SV membrane proteins. Together, these data demonstrate a novel activity- and KIF13A-dependent mechanism for mobilizing axonal transport of ESCRT machinery to facilitate the degradation of SV membrane proteins., (© 2022 Birdsall et al.)

Following exposure and replication at mucosal surfaces, most alphaherpesviruses invade the peripheral nervous system by retrograde axonal transport and establish lifelong latent infections in the peripheral ganglia. Reactivation of ganglionic infections is followed by anterograde axonal transport of virions back to body surfaces where viral replication results in disease that can range from moderate to severe in presentation. In the case of bovine herpesvirus 1 (BoHV-1), replication in the epithelial mucosa presents as infectious bovine rhinotracheitis (IBR), a respiratory disease of significant economic impact. In this study, we provide a live-cell analysis of BoHV-1 retrograde axonal transport relative to the model alphaherpesvirus pathogen pseudorabies virus (PRV) and demonstrate that this critical neuroinvasive step is conserved between the two viruses. In addition, we report that the BoHV-1 pUL37 tegument protein supports processive retrograde motion in infected axons and invasion of the calf peripheral nervous system. IMPORTANCE A molecular and cellular understanding of the retrograde axonal transport process that underlies the neuroinvasive properties of the alphaherpesviruses is established from studies of herpes simplex virus and pseudorabies virus. The degree to which this phenotype is conserved in other related viruses has largely not been examined. We provide a time-lapse analysis of the retrograde axonal transport kinetics of bovine herpesvirus 1 and demonstrate that mutation of the pUL37 region 2 effector affords a strategy to produce live-attenuated vaccines for enhanced protection of cattle.

Development and function of nerve cells rely on the orchestration of microtubule-based transport from the cell body into distal axonal terminals. Neurons often have highly elaborate branches innervating multiple targets, but how protein or membrane cargos navigate through branch junctions to specific branch targets is unknown. Here, we demonstrate that anterograde transport of membrane vesicles through axonal branch junctions is highly selective, which is influenced by branch length and more strongly by growth cone motility. Using an optogenetic tool, we demonstrate that signaling from the growth cone can rapidly direct transport through branch junctions. We further demonstrate that such transport selectivity is differentially regulated for different vesicles and mediated by the KIF1/kinesin-3 family motors. We propose that this transport regulation through branch junctions could broadly impact neuronal development, function, and regeneration., Competing Interests: Declarations of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The application of graphene-family nanomaterials (GFNs) in neuromedicine has recently gained increased attention, but the associated exposure risk for synaptic function and the underlying mechanism remains obscure. The results of this study utilizing nanosized graphene oxide (nGO) suggest that they exert depressive effects on neurotransmission, mainly due to energy deficiency at synaptic contacts. Mitophagy is activated but fails to renew mitochondria and maintain mitochondrial-mediated energy metabolism because of blockage of autophagosome transport through the microtubule system from the axonal terminal to the soma. Further investigation of the underlying mechanism indicates that nGO increases the level of microtubule detyrosination, which restrains loading of the dynactin-dynein motor complex onto microtubules and subsequently inhibits the efficacy of the retrograde transport route. Thus, our study reveals the underlying mechanism by which nGO depresses neurotransmission, and contributes to our understanding of the neurobiological effects of GFNs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Lysosome axonal transport is important for the clearance of cargoes sequestered by the endocytic and autophagic pathways. Building on observations that mutations in the JIP3 (MAPK8IP3) gene result in lysosome-filled axonal swellings, we analyzed the impact of JIP3 depletion on the cytoskeleton of human neurons. Dynamic focal lysosome accumulations were accompanied by disruption of the axonal periodic scaffold (spectrin, F-actin and myosin II) throughout each affected axon. Additionally, axonal microtubule organization was locally disrupted at each lysosome-filled swelling. This local axonal microtubule disorganization was accompanied by accumulations of both F-actin and myosin II. These results indicate that transport of axonal lysosomes is functionally interconnected with mechanisms that control the organization and maintenance of the axonal cytoskeleton. They have potential relevance to human neurological disease arising from JIP3 mutations as well as for neurodegenerative diseases associated with the focal accumulations of lysosomes within axonal swellings such as Alzheimer's disease., (© 2022. The Author(s).)

Neuronal growth, differentiation, homeostasis, viability, and injury response heavily rely on functional axonal transport (AT). Erroneous and disturbed AT may lead to accumulation of "disease proteins" such as tau, α-synuclein, or amyloid precursor protein causing various neurological disorders. Changes in AT often lead to observable behavioral consequences in C. elegans such as impeded movements, defects in touch response, chemosensation, and even egg laying. Long C. elegans neurons with clear distinguishable axons and dendrites provide an excellent platform to analyze AT. The possibility to relate changes in AT to neuronal defects that in turn lead to quantifiable changes in worm behavior allows for the advancement of neuropathological disease models. Even more, subsequent suppressor screens may aid in identifying genes responsible for observed behavioral changes providing a target for drug development to eventually delay or cure neurological diseases. Thus, in this chapter, we summarize critical methods to identify and quantify defects in axonal transport as well as exemplified behavioral assays that may relate to these defects., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 to fluorescently 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., (© 2022. The Author(s).)

The development and functions of neurons are supported by axonal transport. Axonal transport is a complex process whose regulation involves multiple molecules, such as microtubules, microtubule-associated proteins, kinases, molecular motors, and motor binding proteins. Gain of function and loss of function mutations of genes that encode these proteins often lead to human axonal neuropathy. Caenorhabditis elegans provides a powerful genetic system to study the consequences of gene mutations for axonal transport. Here, we discuss advantages and limitations of using C. elegans, propose standard criteria, and describe methods to analyze the impact of gene mutations on axonal transport in C. elegans. To obtain solid conclusions, it is necessary to image single neurons in vivo labeled by a specific promoter and to confirm that a mutation changes the localization of a cargo. The motility parameters of the transported cargo should then be analyzed in the mutant. This method enables the axonal transport of proteins and organelles, such as synaptic vesicle precursors and mitochondria, to be analyzed., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Neurones are highly polarized cells with extensive axonal projections that rely on transport of proteins, RNAs, and organelles in a bidirectional manner to remain healthy. This process, known as axonal transport, can be imaged in real time through epifluorescent imaging of fluorescently labeled proteins, organelles, and other cargoes. While this is most conveniently done in primary neuronal cultures, it is more physiologically relevant when carried out in the context of a developed nerve containing both axons and glia. Here we outline how to image axonal transport ex vivo in sciatic and optic nerves, and the fimbria of the fornix. These methods could be altered to image other fluorescently labeled molecules, as well as different mechanisms of intracellular transport., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Motor protein-driven transport of mRNAs on microtubules and their local translation underlie important neuronal functions such as development, growth cone steering, and synaptic plasticity. While there is abundant data on how membrane-bound cargoes such as vesicles, endosomes, or mitochondria are coupled to motor proteins, surprisingly little is known on the direct interactions of RNA-protein complexes and kinesins or dynein. Provided the potential building blocks are identified, in vitro reconstitutions coupled to Total Internal Reflection Microscopy (TIRF-M) are a powerful and highly sensitive tool to understand how single molecules dynamically interact to assemble into functional complexes. Here we describe how we assemble TIRF-M imaging chambers suitable for the imaging of single protein-RNA complexes. We give advice on optimal sample preparation procedures and explain how a minimal axonal mRNA transport complex can be assembled in vitro. As these assays work at picomolar-range concentrations of proteins and RNAs, they allow the investigation of molecules that cannot be obtained at high concentrations, such as many large or disordered proteins. This now opens the possibility to study how RNA-binding proteins (RBPs), RNAs, and microtubule-associated proteins act together in real-time at single-molecule sensitivity to create cytoplasmic mRNA distributions., (© 2022. The Author(s).)