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Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho- RAB7A--positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation. [ABSTRACT FROM AUTHOR]

Axonal transport is required for neuronal development and survival. Transport from the axon to the soma is driven by the molecular motor cytoplasmic dynein, yet it remains unclear how dynein is spatially and temporally regulated. We find that the dynein effector Hook1 mediates transport of TrkB-BDNF-signaling endosomes in primary hippocampal neurons. Hook1 comigrates with a subpopulation of Rab5 endosomes positive for TrkB and BDNF, which exhibit processive retrograde motility with faster velocities than the overall Rab5 population. Knockdown of Hook1 significantly reduced the motility of BDNF-signaling endosomes without affecting the motility of other organelles. In microfluidic chambers, Hook1 depletion resulted in a significant decrease in the flux and processivity of BDNF-Qdots along the mid-axon, an effect specific for Hook1 but not Hook3. Hook1 depletion inhibited BDNF trafficking to the soma and blocked downstream BDNF- and TrkB-dependent signaling to the nucleus. Together, these studies support a model in which differential association with cargo-specific effectors efficiently regulates dynein in neurons. [ABSTRACT FROM AUTHOR]

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A Cterminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dyneindynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1's EFhands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1's EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 μM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface. [ABSTRACT FROM AUTHOR]

Myosin-19 (Myo19) controls the size, morphology, and distribution of mitochondria, but the underlying role of Myo19 motor activity is unknown. Complicating mechanistic in vitro studies, the identity of the light chains (LCs) of Myo19 remains unsettled. Here, we show by coimmunoprecipitation, reconstitution, and proteomics that the three IQ motifs of human Myo19 expressed in Expi293 human cells bind regulatory light chain (RLC12B) and calmodulin (CaM). We demonstrate that overexpression of Myo19 in HeLa cells enhances the recruitment of both Myo19 and RLC12B to mitochondria, suggesting cellular association of RLC12B with the motor. Further experiments revealed that RLC12B binds IQ2 and is flanked by two CaM molecules. In vitro, we observed that the maximal speed (~350 nm/s) occurs when Myo19 is supplemented with CaM, but not RLC12B, suggesting maximal motility requires binding of CaM to IQ-1 and IQ-3. The addition of calcium slowed actin gliding (~200 nm/s) without an apparent effect on CaM affinity. Furthermore, we show that small ensembles of Myo19 motors attached to quantum dots can undergo processive runs over several microns, and that calcium reduces the attachment frequency and run length of Myo19. Together, our data are consistent with a model where a few single-headed Myo19 molecules attached to a mitochondrion can sustain prolonged motile associations with actin in a CaM- and calcium-dependent manner. Based on these properties, we propose that Myo19 can function in mitochondria transport along actin filaments, tension generation on multiple randomly oriented filaments, and/or pushing against branched actin networks assembled near the membrane surface. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein drives the majority of minus end-directed vesicular and organelle motility in the cell. However, it remains unclear how dynein is spatially and temporally regulated given the variety of cargo that must be properly localized to maintain cellular function. Recent work has suggested that adaptor proteins provide a mechanism for cargo-specific regulation of motors. Of particular interest, studies in fungal systems have implicated Hook proteins in the regulation of microtubule motors. Here we investigate the role of mammalian Hook proteins, Hook1 and Hook3, as potential motor adaptors. We used optogenetic approaches to specifically recruit Hook proteins to organelles and observed rapid transport of peroxisomes to the perinuclear region of the cell. This rapid and efficient translocation of peroxisomes to microtubule minus ends indicates that mammalian Hook proteins activate dynein rather than kinesin motors. Biochemical studies indicate that Hook proteins interact with both dynein and dynactin, stabilizing the formation of a supramolecular complex. Complex formation requires the N-terminal domain of Hook proteins, which resembles the calponin-homology domain of end-binding (EB) proteins but cannot bind directly to microtubules. Single-molecule motility assays using total internal reflection fluorescence microscopy indicate that both Hook1 and Hook3 effectively activate cytoplasmic dynein, inducing longer run lengths and higher velocities than the previously characterized dynein activator bicaudal D2 (BICD2). Together, these results suggest that dynein adaptors can differentially regulate dynein to allow for organellespecific tuning of the motor for precise intracellular trafficking. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein is well characterized as an organelle motor, but dynein also acts to tether and stabilize dynamic microtubule plus-ends in vitro. Here we identify a novel and direct interaction between dynein and the 180-kDa isoform of the neural cell adhesion molecule (NCAM). Optical trapping experiments indicate that dynein bound to beads via the NCAM180 interaction domain can tether projecting microtubule plus-ends. Live cell assays indicate that the NCAM180-dependent recruitment of dynein to the cortex leads to the selective stabilization of microtubules projecting to NCAM180 patches at the cell periphery. The dynein-NCAM180 interaction also enhances cell-cell adhesion in heterologous cell assays. Dynein and NCAM180 co-precipitate from mouse brain extract and from synaptosomal fractions, consistent with an endogenous interaction in neurons. Thus, we examined microtubule dynamics and synaptic density in primary cortical neurons. We find that depletion of NCAM, inhibition of the dynein-NCAM180 interaction, or dampening of microtubule dynamics with low dose nocodazole all result in significantly decreased in synaptic density. Based on these observations, we propose a working model for the role of dynein at the synapse, in which the anchoring of the motor to the cortex via binding to an adhesion molecule mediates the tethering of dynamic microtubule plus-ends to potentiate synaptic stabilization. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein and dynactin interact to drive microtubule- based transport in the cell. The p150Glued subunit of dynactin binds to dynein, and directly to microtubules. We have identified alternatively spliced isoforms of p150Glued that are expressed in a tissue-specific manner and which differ significantly in their affinity for microtubules. Live cell assays indicate that these alternatively spliced isoforms also differ significantly in their microtubule plus end-tracking activity, suggesting a mechanism by which the cell may regulate the dynamic localization of dynactin. To test the function of the microtubule-binding domain of p150Glued, we used RNAi to deplete the endogenous polypeptide from HeLa cells, followed by rescue with constructs encoding either the full-length polypeptide or an isoform lacking the microtubule-binding domain. Both constructs fully rescued defects in Golgi morphology induced by depletion of p150Glued, indicating that an independent microtubule-binding site in dynactin may not be required for dynactin-mediated trafficking in some mammalian cell types. In neurons, however, a mutation within the microtubule-binding domain of p150Glued results in motor neuron disease; here we investigate the effects of four other mutations in highly conserved domains of the polypeptide (M571T, R785W, R1101K, and T1249I) associated in genetic studies with Amyotrophic Lateral Sclerosis. Both biochemical and cellular assays reveal that these amino acid substitutions do not result in functional differences, suggesting that these sequence changes are either allelic variants or contributory risk factors rather than causative for motor neuron disease. Together, these studies provide further insight into the regulation of dynein-dynactin function in the cell. [ABSTRACT FROM AUTHOR]

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150[sup Glued] subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150[sup Glued] for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150[sup Glued] protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150[sup Glued] causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death. [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein and kinesin I are both unidirectional intracellular motors. Dynein moves cargo toward the cell center, and kinesin moves cargo toward the cell periphery. There is growing evidence that bi-directional motility is regulated in the cell, potentially through direct interactions between oppositely oriented motors. We have identified a direct interaction between cytoplasmic dynein and kinesin I. Using the yeast two-hybrid assay and affinity chromatography, we demonstrate that the intermediate chain of dynein binds to kinesin light chains 1 and 2. The interaction is both direct and specific. Co-immunoprecipitation experiments demonstrate an interaction between endogenous proteins in rat brain cytosol. Double-label immunocytochemistry reveals a partial co-localization of vesicle-associated motor proteins. Together these observations suggest that soluble motors can interact, potentially allowing kinesin I to actively localize dynein to cellular sites of function. There is also a vesicle population with both dynein and kinesin I bound that may be capable of bi-directional motility along cellular microtubules. [ABSTRACT FROM AUTHOR]

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms. [ABSTRACT FROM AUTHOR]

Studies of human induced pluripotent stem cell (iPSC)-derived neurons promise important insights into neurodegenerative diseases. Here, we present a protocol for live imaging of axonal transport in glutamatergic iPSC-derived neurons (iNeurons). We describe steps for the differentiation of iPSCs into iNeurons via PiggyBac-mediated neurogenin 2 (NGN2) delivery, iNeuron culture and transfection, and the acquisition and analysis of time-lapse images. Our protocol is optimized for the widely available catalog of KOLF2.1J iPSCs with mutations relevant to neurodegenerative diseases but is also applicable to other iPSC lines. For complete details on the use and execution of this protocol, please refer to Dou et al. 1 , 2 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Maintenance of lysosomal integrity is essential for cell viability. Upon injury, lysosomes may be targeted for degradation via a selective form of autophagy known as lysophagy. The engulfment of a damaged lysosome by an autophagosome is mediated by the recruitment of adaptor proteins, including SQSTM1/p62. p62 promotes lysophagy via the formation of phase-separated condensates in a mechanism that is regulated by the heat shock protein HSP27. Here, we demonstrate a direct interaction between HSP27 and p62. We used structural modeling to predict the binding interface between HSP27 and p62 and identify several disease-associated mutations that map to this interface. We used proteomics to identify post-translational modifications of HSP27 that regulate HSP27 recruitment to stressed lysosomes, finding robust phosphorylation at several serine residues. Next, we characterized the upstream signaling mechanism leading to HSP27 phosphorylation and found that p38 mitogen-activated protein kinase (MAPK) and its effector kinase MAP kinase-activated protein kinase 2 (MK2) are activated upon lysosomal damage by the kinase mTOR and the production of intracellular reactive oxygen species (ROS). Increased ROS activates p38 MAPK, which in turn allows MK2-dependent phosphorylation of HSP27. Depletion of HSP27 or the inhibition of HSP27 phosphorylation alters the dynamics of p62 condensates on stressed lysosomes, significantly inhibiting p62-dependent lysophagy. Thus, we define a novel lysosomal quality control mechanism in which lysosomal injury triggers a p38 MAPK/MK2 signaling cascade promoting p62-dependent lysophagy. Further, this signaling cascade is activated by many cellular stressors, including oxidative and heat stress, suggesting that other forms of selective autophagy may be regulated by p38 MAPK/MK2/HSP27., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fragile X messenger ribonucleoprotein (FMRP) is a critical regulator of translation, whose dysfunction causes fragile X syndrome. FMRP dysfunction disrupts mitochondrial health in neurons, but it is unclear how FMRP supports mitochondrial homoeostasis. Here we demonstrate that FMRP granules are recruited to the mitochondrial midzone, where they mark mitochondrial fission sites in axons and dendrites. Endolysosomal vesicles contribute to FMRP granule positioning around mitochondria and facilitate FMRP-associated fission via Rab7 GTP hydrolysis. Cryo-electron tomography and real-time translation imaging reveal that mitochondria-associated FMRP granules are ribosome-rich structures that serve as sites of local protein synthesis. Specifically, FMRP promotes local translation of mitochondrial fission factor (MFF), selectively enabling replicative fission at the mitochondrial midzone. Disrupting FMRP function dysregulates mitochondria-associated MFF translation and perturbs fission dynamics, resulting in increased peripheral fission and an irregular distribution of mitochondrial nucleoids. Thus, FMRP regulates local translation of MFF in neurons, enabling precise control of mitochondrial fission., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Autophagic dysfunction is a hallmark of neurodegenerative disease, leaving neurons vulnerable to the accumulation of damaged organelles and proteins. However, the late onset of diseases suggests that compensatory quality control mechanisms may be engaged to delay the deleterious effects induced by compromised autophagy. Neurons expressing common familial Parkinson's disease (PD)-associated mutations in LRRK2 kinase exhibit defective autophagy. Here, we demonstrate that both primary murine neurons and human iPSC-derived neurons harboring pathogenic LRRK2 upregulate the secretion of extracellular vesicles. We used unbiased proteomics to characterize the secretome of LRRK2 G2019S neurons and found that autophagic cargos including mitochondrial proteins were enriched. Based on these observations, we hypothesized that autophagosomes are rerouted toward secretion when cell-autonomous degradation is compromised, likely to mediate clearance of undegraded cellular waste. Immunoblotting confirmed the release of autophagic cargos and immunocytochemistry demonstrated that secretory autophagy was upregulated in LRRK2 G2019S neurons. We also found that LRRK2 G2019S neurons upregulate the release of exosomes containing miRNAs. Live-cell imaging confirmed that this upregulation of exosomal release was dependent on hyperactive LRRK2 activity, while pharmacological experiments indicate that this release staves off apoptosis. Finally, we show that markers of both vesicle populations are upregulated in plasma from mice expressing pathogenic LRRK2. In sum, we find that neurons expressing pathogenic LRRK2 upregulate the compensatory release of secreted autophagosomes and exosomes, to mediate waste disposal and transcellular communication, respectively. We propose that this increased secretion contributes to the maintenance of cellular homeostasis, delaying neurodegenerative disease progression over the short term while potentially contributing to increased neuroinflammation over the longer term.

First identified in dividing cells as revolving clusters of actin filaments, these are now understood as mitochondrially-associated actin waves that are active throughout the cell cycle. These waves are formed from the polymerization of actin onto a subset of mitochondria. Within minutes, this F-actin depolymerizes while newly formed actin filaments assemble onto neighboring mitochondria. In interphase, actin waves locally fragment the mitochondrial network, enhancing mitochondrial content mixing to maintain organelle homeostasis. In dividing cells actin waves spatially mix mitochondria in the mother cell to ensure equitable partitioning of these organelles between daughter cells. Progress has been made in understanding the consequences of actin cycling as well as the underlying molecular mechanisms, but many questions remain, and here we review these elements. Also, we draw parallels between mitochondrially-associated actin cycling and cortical actin waves. These dynamic systems highlight the remarkable plasticity of the actin cytoskeleton., 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 Elsevier Ltd. All rights reserved.)

Across the cell cycle, mitochondrial dynamics are regulated by a cycling wave of actin polymerization/depolymerization. In metaphase, this wave induces actin comet tails on mitochondria that propel these organelles to drive spatial mixing, resulting in their equitable inheritance by daughter cells. In contrast, during interphase the cycling actin wave promotes localized mitochondrial fission. Here, we identify the F-actin nucleator/elongator FMNL1 as a positive regulator of the wave. FMNL1-depleted cells exhibit decreased mitochondrial polarization, decreased mitochondrial oxygen consumption, and increased production of reactive oxygen species. Accompanying these changes is a loss of hetero-fusion of wave-fragmented mitochondria. Thus, we propose that the interphase actin wave maintains mitochondrial homeostasis by promoting mitochondrial content mixing. Finally, we investigate the mechanistic basis for the observation that the wave drives mitochondrial motility in metaphase but mitochondrial fission in interphase. Our data indicate that when the force of actin polymerization is resisted by mitochondrial tethering to microtubules, as in interphase, fission results., (© 2024. The Author(s).)

Neurons rely on the long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, as well as how the efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule (MT)-severing enzyme spastin in locally enhancing MT polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new MT growth and synaptic vesicle precursor (SVP) pausing/retention. The disruption of neuronal spastin level or activity, by CRISPRi-mediated depletion, transient overexpression, or pharmacologic inhibition of enzymatic activity, interrupts the localized enrichment of dynamic MT plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of MT polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Abbreviations: SQSTM1/p62: Sequestosome-1; HSP27: Heat shock protein 27; LLPS: liquid-liquid phase separation; iPSC: induced pluripotent stem cell; PB1: Phox and Bem1p; FRAP: fluorescence recovery after photo-bleaching; ATG: autophagy-related; ALS: amyotrophic lateral sclerosis.

An actin-spectrin lattice, the membrane periodic skeleton (MPS), protects axons from breakage. MPS integrity relies on spectrin delivery via slow axonal transport, a process that remains poorly understood. We designed a probe to visualize endogenous spectrin dynamics at single-axon resolution in vivo. Surprisingly, spectrin transport is bimodal, comprising fast runs and movements that are 100-fold slower than previously reported. Modeling and genetic analysis suggest that the two rates are independent, yet both require kinesin-1 and the coiled-coil proteins UNC-76/FEZ1 and UNC-69/SCOC, which we identify as spectrin-kinesin adaptors. Knockdown of either protein led to disrupted spectrin motility and reduced distal MPS, and UNC-76 overexpression instructed excessive transport of spectrin. Artificially linking spectrin to kinesin-1 drove robust motility but inefficient MPS assembly, whereas impairing MPS assembly led to excessive spectrin transport, suggesting a balance between transport and assembly. These results provide insight into slow axonal transport and MPS integrity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Failure to clear damaged mitochondria via mitophagy disrupts physiological function and may initiate damage signaling via inflammatory cascades, although how these pathways intersect remains unclear. We discovered that nuclear factor kappa B (NF-κB) essential regulator NF-κB effector molecule (NEMO) is recruited to damaged mitochondria in a Parkin-dependent manner in a time course similar to recruitment of the structurally related mitophagy adaptor, optineurin (OPTN). Upon recruitment, NEMO partitions into phase-separated condensates distinct from OPTN but colocalizing with p62/SQSTM1. NEMO recruitment, in turn, recruits the active catalytic inhibitor of kappa B kinase (IKK) component phospho-IKKβ, initiating NF-κB signaling and the upregulation of inflammatory cytokines. Consistent with a potential neuroinflammatory role, NEMO is recruited to mitochondria in primary astrocytes upon oxidative stress. These findings suggest that damaged, ubiquitinated mitochondria serve as an intracellular platform to initiate innate immune signaling, promoting the formation of activated IKK complexes sufficient to activate NF-κB signaling. We propose that mitophagy and NF-κB signaling are initiated as parallel pathways in response to mitochondrial stress., Competing Interests: Declaration of interests S.M. is a member of the Scientific Advisory Board of Casma Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurons rely on long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, and how efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule severing enzyme spastin in locally enhancing microtubule polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components, termed 'protosynapses', which are distributed along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new microtubule growth and synaptic vesicle precursor (SVP) pausing/retention. Disruption of neuronal spastin, either by CRISPRi-mediated depletion or transient overexpression, interrupts the localized enrichment of dynamic microtubule plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of microtubule polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.

Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2 -p.R1441H. Knockout of the opposing protein phosphatase 1H ( PPM1H ) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adapter MADD, potentially preventing formation of the RAB3A-MADD-KIF1A/1Bβ complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), increasing phosphorylation of RAB GTPases through hyperactive kinase activity. We find that LRRK2-hyperphosphorylated RABs disrupt the axonal transport of autophagosomes by perturbing the coordinated regulation of cytoplasmic dynein and kinesin. In iPSC-derived human neurons, knockin of the strongly hyperactive LRRK2-p.R1441H mutation causes striking impairments in autophagosome transport, inducing frequent directional reversals and pauses. Knockout of the opposing protein phosphatase 1H (PPM1H) phenocopies the effect of hyperactive LRRK2. Overexpression of ADP-ribosylation factor 6 (ARF6), a GTPase that acts as a switch for selective activation of dynein or kinesin, attenuates transport defects in both p.R1441H knockin and PPM1H knockout neurons. Together, these findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive "tug-of-war" between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In response to lysosomal damage, cells engage several quality-control mechanisms, including the selective isolation and degradation of damaged lysosomes by lysophagy. Here, we report that the selective autophagy adaptor SQSTM1/p62 is recruited to damaged lysosomes in both HeLa cells and neurons and is required for lysophagic flux. The Phox and Bem1p (PB1) domain of p62 mediates oligomerization and is specifically required for lysophagy. Consistent with this observation, we find that p62 forms condensates on damaged lysosomes. These condensates are precisely tuned by the small heat shock protein HSP27, which is phosphorylated in response to lysosomal injury and maintains the liquidity of p62 condensates, facilitating autophagosome formation. Mutations in p62 have been identified in patients with amyotrophic lateral sclerosis (ALS); ALS-associated mutations in p62 impair lysophagy, suggesting that deficits in this pathway may contribute to neurodegeneration. Thus, p62 condensates regulated by HSP27 promote lysophagy by forming platforms for autophagosome biogenesis at damaged lysosomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuronal autophagosomes, "self-eating" degradative organelles, form at presynaptic sites in the distal axon and are transported to the soma to recycle their cargo. During transit, autophagic vacuoles (AVs) mature through fusion with lysosomes to acquire the enzymes necessary to breakdown their cargo. AV transport is driven primarily by the microtubule motor cytoplasmic dynein in concert with dynactin and a series of activating adaptors that change depending on organelle maturation state. The transport of mature AVs is regulated by the scaffolding proteins JIP3 and JIP4, both of which activate dynein motility in vitro. AV transport is also regulated by ARF6 in a GTP-dependent fashion. While GTP-bound ARF6 promotes the formation of the JIP3/4-dynein-dynactin complex, RAB10 competes with the activity of this complex by increasing kinesin recruitment to axonal AVs and lysosomes. These interactions highlight the complex coordination of motors regulating organelle transport in neurons.

Microtubule-based transport is a highly regulated process, requiring kinesin and/or dynein motors, a multitude of motor-associated regulatory proteins including activating adaptors and scaffolding proteins, and microtubule tracks that also provide regulatory cues. While in vitro studies are invaluable, fully replicating the physiological conditions under which motility occurs in cells is not yet possible. Here, we describe two methods that can be employed to study motor-based transport and motor regulation in a cellular context. Live-cell imaging of organelle transport in neurons leverages the uniform polarity of microtubules in axons to better understand the factors regulating microtubule-based motility. Peroxisome recruitment assays allow users to examine the net effect of motors and motor-regulatory proteins on organelle distribution. Together, these methods open the door to motility experiments that more fully interrogate the complex cellular environment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Long-range transport of organelles and other cellular cargoes along microtubules is driven by kinesin and dynein motor proteins in complex with cargo-specific adaptors. While some adaptors interact exclusively with a single motor, other adaptors interact with both kinesin and dynein motors. However, the mechanisms by which bidirectional motor adaptors coordinate opposing microtubule motors are not fully understood. While single-molecule studies of adaptors using purified proteins can provide key insight into motor adaptor function, these studies may be limited by the absence of cellular factors that regulate or coordinate motor function. As a result, motility assays using cell lysates have been developed to gain insight into motor adaptor function in a more physiological context. These assays are a powerful means to dissect the regulation of motor adaptors as cell lysates contain endogenous microtubule motors and additional factors that regulate motor function. Further, this system is highly tractable as individual proteins can readily be added or removed via overexpression or knockdown in cells. Here, we describe a protocol for in vitro reconstitution of motor-driven transport along dynamic microtubules at single-molecule resolution using total internal reflection fluorescence microscopy of cell lysates., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field., Competing Interests: Declaration of interests S.W.S. is on the scientific advisory council of the Lewy Body Dementia Association and the MSA Coalition. S.W.S. is an editorial board member for the Journal of Parkinson Disease and JAMA Neurology. S.W.S. received research support from Cerevel Therapeutics. M.K. serves on the scientific advisory boards of Engine Biosciences, Casma Therapeutics, Cajal Neuroscience, and Alector and is a consultant to Modulo Bio and Recursion Therapeutics. Participation by researchers from Data Tecnica International, LLC in this project was part of a competitive contract awarded to Data Tecnica International, LLC by the National Institutes of Health to support open science research. M.A.N. also currently serves on the scientific advisory board for Clover Therapeutics and is an advisor to Neuron23 Inc. E.A. is founder, shareholder, and scientific advisor of Cholestenix, Ltd., (Published by Elsevier Inc.)

The active transport of organelles and other cargos along the axon is required to maintain neuronal health and function, but we are just beginning to understand the complex regulatory mechanisms involved. The molecular motors, cytoplasmic dynein and kinesins, transport cargos along microtubules; this transport is tightly regulated by adaptors and effectors. Here we review our current understanding of motor regulation in axonal transport. We discuss the mechanisms by which regulatory proteins induce or repress the activity of dynein or kinesin motors, and explore how this regulation plays out during organelle trafficking in the axon, where motor activity is both cargo specific and dependent on subaxonal location. We survey several well-characterized examples of membranous organelles subject to axonal transport - including autophagosomes, endolysosomes, signalling endosomes, mitochondria and synaptic vesicle precursors - and highlight the specific mechanisms that regulate motor activity to provide localized trafficking within the neuron. Defects in axonal transport have been implicated in conditions ranging from developmental defects in the brain to neurodegenerative disease. Better understanding of the underlying mechanisms will be essential to develop more-effective treatment options., (© 2022. Springer Nature Limited.)

How synaptogenic signals trigger the targeted delivery of synaptic material is a fundamental question in neuroscience. In this issue of Developmental Cell, Balseiro-Gomez et al. identify a mechanism through which local synatogenic pathways control synaptic cargo delivery., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Understanding the pathogenic mechanisms of disease mutations is critical to advancing treatments. ALS-associated mutations in the gene encoding the microtubule motor KIF5A result in skipping of exon 27 (KIF5A ΔExon27 ) and the encoding of a protein with a novel 39 amino acid residue C-terminal sequence. Here, we report that expression of ALS-linked mutant KIF5A results in dysregulated motor activity, cellular mislocalization, altered axonal transport, and decreased neuronal survival. Single-molecule analysis revealed that the altered C terminus of mutant KIF5A results in a constitutively active state. Furthermore, mutant KIF5A possesses altered protein and RNA interactions and its expression results in altered gene expression/splicing. Taken together, our data support the hypothesis that causative ALS mutations result in a toxic gain of function in the intracellular motor KIF5A that disrupts intracellular trafficking and neuronal homeostasis., Competing Interests: Declaration of interests J.E.L. is a member of the scientific advisory board for Cerevel Therapeutics, a consultant for ACI Clinical LLC sponsored by Biogen, Inc. and Ionis Pharmaceuticals, Inc. J.E.L. is also a consultant for Perkins Coie LLP and may provide expert testimony., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neurons depend on autophagy to maintain cellular homeostasis, and defects in autophagy are pathological hallmarks of neurodegenerative disease. To probe the role of basal autophagy in the maintenance of neuronal health, we isolated autophagic vesicles from mouse brain tissue and used proteomics to identify the major cargos engulfed within autophagosomes, validating our findings in rodent primary and human iPSC-derived neurons. Mitochondrial proteins were identified as a major cargo in the absence of mitophagy adaptors such as OPTN. We found that nucleoid-associated proteins are enriched compared with other mitochondrial components. In the axon, autophagic engulfment of nucleoid-enriched mitochondrial fragments requires the mitochondrial fission machinery Drp1. We proposed that localized Drp1-dependent fission of nucleoid-enriched fragments in proximity to the sites of autophagosome biogenesis enhances their capture. The resulting efficient autophagic turnover of nucleoids may prevent accumulation of mitochondrial DNA in the neuron, thus mitigating activation of proinflammatory pathways that contribute to neurodegeneration., Competing Interests: Declaration of interests J.W.H. is a consultant and founder of Caraway Therapeutics and is a founding board member of Interline Therapeutics. J.W.H. also receives compensation for editorial service at eLife and Science Advances. E.H. receives compensation for editorial service from Science Advances., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Parkinson disease (PD)-causing mutations in the LRRK2 (leucine rich repeat kinase 2) gene hyperactivate LRRK2 kinase activity. Here, we discuss our recent work linking LRRK2 hyperactivation to defective axonal autophagosome transport in neurons. In three different models, we observed that expression of the most common causative mutation for PD, LRRK2 G2019S , disrupts processive autophagosome transport in a kinase-dependent manner. Mechanistically, we found that hyperactive LRRK2 recruits SPAG9/JIP4, a motor adaptor known to bind to LRRK2-phosphorylated RAB proteins, to the autophagosomal membrane. Increased SPAG9/JIP4 levels induce abnormal recruitment and activation of kinesin-1, which we propose results in an unproductive tug-of-war between anterograde and retrograde motors bound to autophagosomes. Disruption of autophagosome transport correlates with defective autophagosome maturation, suggesting that hyperactive LRRK2 may impair efficient degradation of autophagosomal cargo. Our work demonstrates that LRRK2 hyperactivation is sufficient to induce defects in autophagosome transport and maturation, further establishing a role of defective autophagy in the pathogenesis of PD.

Autophagy is a degradative pathway required to maintain homeostasis. Neuronal autophagosomes form constitutively at the axon terminal and mature via lysosomal fusion during dynein-mediated transport to the soma. How the dynein-autophagosome interaction is regulated is unknown. Here, we identify multiple dynein effectors on autophagosomes as they transit along the axons of primary neurons. In the distal axon, JIP1 initiates autophagosomal transport. Autophagosomes in the mid-axon require HAP1 and Huntingtin. We find that HAP1 is a dynein activator, binding the dynein-dynactin complex via canonical and noncanonical interactions. JIP3 is on most axonal autophagosomes, but specifically regulates the transport of mature autolysosomes. Inhibiting autophagosomal transport disrupts maturation, and inhibiting autophagosomal maturation perturbs the association and function of dynein effectors; thus, maturation and transport are tightly linked. These results reveal a novel maturation-based dynein effector handoff on neuronal autophagosomes that is key to motility, cargo degradation, and the maintenance of axonal health., (© 2021 Cason et al.)

TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia, including missense mutations that disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type-specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Parkinson's disease-causing mutations in the leucine-rich repeat kinase 2 (LRRK2) gene hyperactivate LRRK2 kinase activity and cause increased phosphorylation of Rab GTPases, important regulators of intracellular trafficking. We found that the most common LRRK2 mutation, LRRK2-G2019S, dramatically reduces the processivity of autophagosome transport in neurons in a kinase-dependent manner. This effect was consistent across an overexpression model, neurons from a G2019S knockin mouse, and human induced pluripotent stem cell (iPSC)-derived neurons gene edited to express the G2019S mutation, and the effect was reversed by genetic or pharmacological inhibition of LRRK2. Furthermore, LRRK2 hyperactivation induced by overexpression of Rab29, a known activator of LRRK2 kinase, disrupted autophagosome transport to a similar extent. Mechanistically, we found that hyperactive LRRK2 recruits the motor adaptor JNK-interacting protein 4 (JIP4) to the autophagosomal membrane, inducing abnormal activation of kinesin that we propose leads to an unproductive tug of war between anterograde and retrograde motors. Disruption of autophagosome transport correlated with a significant defect in autophagosome acidification, suggesting that the observed transport deficit impairs effective degradation of autophagosomal cargo in neurons. Our results robustly link increased LRRK2 kinase activity to defects in autophagosome transport and maturation, further implicating defective autophagy in the pathogenesis of Parkinson's disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The development and proper function of the brain requires the formation of highly complex neuronal circuitry. These circuits are shaped from synaptic connections between neurons and must be maintained over a lifetime. The formation and continued maintenance of synapses requires accurate trafficking of presynaptic and postsynaptic components along the axon and dendrite, respectively, necessitating deliberate and specialized delivery strategies to replenish essential synaptic components. Maintenance of synaptic transmission also requires readily accessible energy stores, produced in part by localized mitochondria, that are tightly regulated with activity level. In this review, we focus on recent developments in our understanding of the cytoskeletal environment of axons and dendrites, examining how local regulation of cytoskeletal dynamics and organelle trafficking promotes synapse-specific delivery and plasticity. These new insights shed light on the complex and coordinated role that cytoskeletal elements play in establishing and maintaining neuronal circuitry., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Symmetric cell division requires the even partitioning of genetic information and cytoplasmic contents between daughter cells. Whereas the mechanisms coordinating the segregation of the genome are well known, the processes that ensure organelle segregation between daughter cells remain less well understood 1 . Here we identify multiple actin assemblies with distinct but complementary roles in mitochondrial organization and inheritance in mitosis. First, we find a dense meshwork of subcortical actin cables assembled throughout the mitotic cytoplasm. This network scaffolds the endoplasmic reticulum and organizes three-dimensional mitochondrial positioning to ensure the equal segregation of mitochondrial mass at cytokinesis. Second, we identify a dynamic wave of actin filaments reversibly assembling on the surface of mitochondria during mitosis. Mitochondria sampled by this wave are enveloped within actin clouds that can spontaneously break symmetry to form elongated comet tails. Mitochondrial comet tails promote randomly directed bursts of movement that shuffle mitochondrial position within the mother cell to randomize inheritance of healthy and damaged mitochondria between daughter cells. Thus, parallel mechanisms mediated by the actin cytoskeleton ensure both equal and random inheritance of mitochondria in symmetrically dividing cells.

The epidermis regenerates continually to maintain a protective barrier at the body's surface composed of differentiating keratinocytes. Maturation of this stratified tissue requires that keratinocytes undergo wholesale organelle degradation upon reaching the outermost tissue layers to form compacted, anucleate cells. Through live imaging of organotypic cultures of human epidermis, we find that regulated breakdown of mitochondria is critical for epidermal development. Keratinocytes in the upper layers initiate mitochondrial fragmentation, depolarization, and acidification upon upregulating the mitochondrion-tethered autophagy receptor NIX. Depleting NIX compromises epidermal maturation and impairs mitochondrial elimination, whereas ectopic NIX expression accelerates keratinocyte differentiation and induces premature mitochondrial fragmentation via the guanosine triphosphatase (GTPase) DRP1. We further demonstrate that inhibiting DRP1 blocks NIX-mediated mitochondrial breakdown and disrupts epidermal development. Our findings establish mitochondrial degradation as a key step in terminal keratinocyte differentiation and define a pathway operating via the mitophagy receptor NIX in concert with DRP1 to drive epidermal morphogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria form networks that continually remodel and adapt to carry out their cellular function. The mitochondrial network is remodeled through changes in mitochondrial morphology, number, and distribution within the cell. Mitochondrial dynamics depend directly on fission, fusion, shape transition, and transport or tethering along the cytoskeleton. Over the past several years, many of the mechanisms underlying these processes have been uncovered. It has become clear that each process is precisely and contextually regulated within the cell. Here, we discuss the mechanisms regulating each aspect of mitochondrial dynamics, which together shape the network as a whole., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this issue of Neuron, Orr et al. (2020) identify an evolutionarily conserved mechanism of presynaptic homeostatic plasticity induced by ALS-like motor neuron degeneration, which maintains excitatory potentials until a threshold of synaptic loss is reached. Past this tipping point, disease onset progresses rapidly., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Damaged mitochondria are selectively removed from the cell in a process termed mitophagy. This mitochondrial quality control mechanism is important for neuronal homeostasis, and mutations in pathway components are causative for Parkinson disease and amyotrophic lateral sclerosis (ALS). Here, we discuss our recent work using a novel mild induction paradigm to investigate the spatiotemporal dynamics of mitophagy in primary neurons. Using live-cell imaging, we find that mitophagy-associated proteins translocate to depolarized mitochondrial fragments. These mitophagic events were primarily localized to somatodendritic compartments, suggesting neuronal mitophagy is primarily a somal quality control mechanism. Damaged mitochondria were efficiently sequestered within autophagosomes, but lysosomal fusion or acidification was significantly delayed. Surprisingly, engulfed mitochondria persisted in non-acidified vesicular compartments for hours to days after initial damage. Expression of an ALS-associated mutation disrupted the membrane potential of the mitochondrial network, and oxidative stress exacerbated this effect. Importantly, our results highlight the slow kinetics of mitophagy and suggest that slow turnover of damaged mitochondria may increase neuronal susceptibility to neurodegeneration.

The cargo-specific removal of organelles via selective autophagy is important to maintain neuronal homeostasis. Genetic studies indicate that deficits in these pathways are implicated in neurodegenerative diseases, including Parkinson's and amyotrophic lateral sclerosis. Here, we review our current understanding of the pathways that regulate mitochondrial quality control, and compare these mechanisms to those regulating turnover of the endoplasmic reticulum and the clearance of protein aggregates. Research suggests that there are multiple mechanisms regulating the degradation of specific cargos, such as dysfunctional organelles and protein aggregates. These mechanisms are critical for neuronal health, as neurons are uniquely vulnerable to impairment in organelle quality control pathways due to their morphology, size, polarity, and postmitotic nature. We highlight the consequences of dysregulation of selective autophagy in neurons and discuss current challenges in correlating noncongruent findings from in vitro and in vivo systems., (Copyright © 2019. Published by Elsevier Ltd.)

The intracellular transport system in neurons is specialized to an extraordinary degree, enabling the delivery of critical cargo to sites in axons or dendrites that are far removed from the cell center. Vesicles formed in the cell body are actively transported by kinesin motors along axonal microtubules to presynaptic sites that can be located more than a meter away. Both growth factors and degradative vesicles carrying aged organelles or aggregated proteins take the opposite route, driven by dynein motors. Distance is not the only challenge; precise delivery of cargos to sites of need must also be accomplished. For example, localized delivery of presynaptic components to hundreds of thousands of "en passant" synapses distributed along the length of a single axon in some neuronal subtypes provides a layer of complexity that must be successfully navigated to maintain synaptic transmission. We review recent advances in the field of axonal transport, with a focus on conceptual developments, and highlight our growing quantitative understanding of neuronal trafficking and its role in maintaining the synaptic function that underlies higher cognitive processes such as learning and memory., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)