Axonal Trafficking Research Articles

Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome

Long-distance axonal trafficking plays a critical role in neuronal function and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.

Chain length of saturated fatty acids regulates mitochondrial trafficking and function in sensory neurons

Dyslipidemia associated with T2D leads to diabetic neuropathy, a complication characterized by sensory neuronal dysfunction and peripheral nerve damage. Sensory dorsal root ganglion (DRG) neurons are dependent on axonal mitochondrial energy production facilitated by mitochondrial transport mechanisms that distribute mitochondria throughout the axon. Because long-chain saturated FAs (SFAs) damage DRG neurons and medium-chain SFAs are reported to improve neuronal function, we evaluated the impact of SFA chain length on mitochondrial trafficking, mitochondrial function, and apoptosis. DRG neurons were exposed to SFAs with C12:0-C18:0 chain lengths and evaluated for changes in mitochondrial trafficking, mitochondrial polarization, and apoptosis. DRG neurons treated with C16:0 and C18:0 SFAs showed a significant decrease in the percentage of motile mitochondria and velocity of mitochondrial trafficking, whereas C12:0 and C14:0 SFAs had no impact on motility. Treatment with C16:0 and C18:0 SFAs exhibited mitochondrial depolarization correlating with impaired mitochondrial motility; the C12:0- and C14:0-treated neurons retained mitochondrial polarization. The reduction in mitochondrial trafficking and function in C16:0- and C18:0-treated DRG neurons correlated with apoptosis that was blocked in C12:0 and C14:0 SFA treatments. These results suggest that SFA chain length plays an important role in regulating axonal mitochondrial trafficking and function in DRG neurons.

Mitochondrial distribution violation and nuclear indentations in neurons differentiated from iPSCs of Huntington's disease patients

AIM: Huntington's disease (HD) is an inherited disease caused by an expansion of cytosine-adenine-guanine (CAG) repeats in the huntingtin gene (HTT) that ultimately leads to neurodegeneration. To study the molecular basis of this disease, induced pluripotent stem cells (iPSCs) generated from patients' fibroblasts were used to investigate axonal mitochondrial trafficking and the nature of nuclear indentations. METHODS: Pathological and control iPSCs generated from patients with a low number of repeats were differentiated in striatal neurons of the brain. Mitochondrial density was measured along the axon using tubulin beta 3 co-staining in pathological and control neurons. To investigate the connection of nuclear roundness with calcium dysregulation, several calcium inhibitors were used. Proteasome system inhibition was applied to mimic premature neuronal ageing. RESULTS: We found that the mitochondrial density was approximately 7.6 ± 0.2 in neurites in control neurons but was only 5.3 ± 0.2 in mutant neurons with 40-44 CAG repeats (p-value <0.005). Neuronal ageing induced by proteasome inhibitor MG132 significantly decreased the mitochondrial density by 15% and 25% in control and mutant neurons to 6.5 ± 0.1 (p-value < 0.005) and 4.0 ± 0.3 (p-value < 0.005), respectively. Thus, for the first time, an impairment of mitochondrial trafficking in pathological neurons with endogenous mutant huntingtin was demonstrated. We found that inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), the ryanodine-receptor (RyR) or the inositol 1,4,5-trisphosphate receptor (IP3R) by specific inhibitors did not specifically affect the nuclear roundness or survival of pathological neurons differentiated from patient iPSCs. Therefore, nuclear calcium homeostasis is not directly associated with HD pathology. CONCLUSION: Identifying HD iPSCs and differentiating from them neurons provide a unique system for modelling the disease in vitro. Impairments of mitochondrial trafficking and nuclear roundness manifest long before the disease onset, while premature neuronal ageing enhances differences in mitochondrial distribution.

Super-resolving botulinum neurotoxin type A molecules: from surface landing to internalization in synaptic vesicles

Botulinum neurotoxin type-A (BoNT/A) is internalized into motor nerve terminals as part of its intoxication strategy to incapacitate nerve-muscle communication. The “dual receptor” model explained how BoNT/A initially interacts with GT1b gangliosides, thereby concentrating the toxin on the presynaptic membrane to foster the subsequent interaction with a proteinaceous co-receptor SV2 which triggers receptor-mediated endocytosis. I will revisit the “dual receptor” concept using two single-molecule imaging strategies,1-3 allowing tracking of single Atto647N-labeled BoNT/A molecules upon (i) landing on the plasma membrane (by uPAINT) and (ii) internalization in synaptic vesicles (by sdTIM) of living mature hippocampal neurons. With 30 to 40 nm localization precision, we revealed that once internalized in synaptic vesicles, Atto647N-tagged BoNT/A exhibits a markedly lower mobility than on the plasma membrane. I will discuss how individual genetic inactivation of the GT1b and SV2 binding sites of the neurotoxin affects the diffusion states of BoNT/A mutants on the plasma membrane and axonal trafficking. Single neurotoxin super-resolution imaging uncovers an updated dual receptor model taking into consideration the diffusive patterns generated by each of the co-receptors and leading to defined nanoscale dynamic organizations at key steps of the intoxication journey.

Intrinsic Determinants of Axon Regeneration.

The failure of axons to regenerate in the damaged mammalian CNS is the main impediment to functional recovery. There are many molecules and structures in the environment of the injured nervous system that can inhibit regeneration, but even when these are removed or replaced with a permissive environment, most CNS neurons exhibit little regeneration of their axons. This contrasts with the extensive and vigorous axon growth that may occur when embryonic neurons are transplanted into the adult CNS. In the peripheral nervous system, the axons usually respond to axotomy with a vigorous regenerative response accompanied by a regenerative program of gene expression, usually referred to as the regeneration-associated gene (RAG) program. These different responses to axotomy in the mature and immature CNS and the PNS lead to the concept of the intrinsic regenerative response of axons. Analysis of the many mechanisms and issues that affect the intrinsic regenerative response is the topic of this special issue of Developmental Neurobiology. The review articles highlight the control of expression of growth and regeneration-associated genes, emphasizing the role of epigenetic mechanisms. The reviews also discuss changes within axons that lead to the developmental loss of regenerative ability. This is caused by changes in axonal transport and trafficking, in the cytoskeleton and in signaling pathways.

Single-molecule analysis of endogenous β-actin mRNA trafficking reveals a mechanism for compartmentalized mRNA localization in axons.

During embryonic nervous system assembly, mRNA localization is precisely regulated in growing axons, affording subcellular autonomy by allowing controlled protein expression in space and time. Different sets of mRNAs exhibit different localization patterns across the axon. However, little is known about how mRNAs move in axons or how these patterns are generated. Here, we couple molecular beacon technology with highly inclined and laminated optical sheet microscopy to image single molecules of identified endogenous mRNA in growing axons. By combining quantitative single-molecule imaging with biophysical motion models, we show that β-actin mRNA travels mainly as single copies and exhibits different motion-type frequencies in different axonal subcompartments. We find that β-actin mRNA density is fourfold enriched in the growth cone central domain compared with the axon shaft and that a modicum of directed transport is vital for delivery of mRNA to the axon tip. Through mathematical modeling we further demonstrate that directional differences in motor-driven mRNA transport speeds are sufficient to generate β-actin mRNA enrichment at the growth cone. Our results provide insight into how mRNAs are trafficked in axons and a mechanism for generating different mRNA densities across axonal subcompartments.

Cue-Polarized Transport of β-actin mRNA Depends on 3'UTR and Microtubules in Live Growth Cones.

Guidance cues trigger fast responses in axonal growth cones such as directional turning and collapse that require local protein synthesis. An attractive cue-gradient, such as Netrin-1, triggers de novo synthesis of β-actin localized to the near-side compartment of the growth cone that promotes F-actin assembly and attractive steering. How this precise spatial asymmetry in mRNA translation arises across the small expanse of the growth cone is poorly understood. Pre-localized mRNAs in the vicinity of activated receptors could be selectively translated and/or new mRNAs could be trafficked into the area. Here we have performed live imaging of fluorescent-tagged β-actin mRNA to investigate mRNA trafficking dynamics in Xenopus retinal ganglion cell (RGC) axons and growth cones in response to Netrin-1. A Netrin-1 gradient was found to elicit the transport of β-actin mRNA granules to the near-side of growth cones within a 4–7 min window. This polarized mRNA trafficking depended on the 3′ untranslated region (UTR) since mRNA-Δ3′UTR mutant failed to exhibit cue-induced localization. Global application of Netrin-1 significantly increased the anterograde movement of β-actin mRNA along axons and also promoted microtubule-dependent mRNA excursions from the central domain of the growth cone into the periphery (filopodia and lamellipodia). Dual channel imaging revealed β-actin mRNA riding behind the microtubule plus-end tracking protein, EB1, in movements along dynamic microtubules into filopodia. The mRNA-EB1 movements were unchanged by a Netrin-1 gradient indicating the dynamic microtubules themselves do not underlie the cue-induced polarity of RNA movement. Finally, fast-moving elongated “worm-like” trains of Cy3-RNA, distinct from mitochondria, were seen transporting RNA along axons in vitro and in vivo suggesting the existence of a novel transport organelle. Overall, the results provide evidence that the axonal trafficking of β-actin mRNA can be regulated by the guidance cue Netrin-1 to transduce the polarity of an extracellular stimulus and that the 3′UTR is essential for this cue-induced regulation.

Neuronal network maturation differently affects secretory vesicles and mitochondria transport in axons

Studying intracellular dynamics in neurons is crucial to better understand how brain circuits communicate and adapt to environmental changes. In neurons, axonal secretory vesicles underlie various functions from growth during development to plasticity in the mature brain. Similarly, transport of mitochondria, the power plant of the cell, regulates both axonal development and synaptic homeostasis. However, because of their submicrometric size and rapid velocities, studying the kinetics of these organelles in projecting axons in vivo is technically challenging. In parallel, primary neuronal cultures are adapted to study axonal transport but they lack the physiological organization of neuronal networks, which in turn may bias observations. We previously developed a microfluidic platform to reconstruct a physiologically-relevant and functional corticostriatal network in vitro that is compatible with high-resolution videorecording of axonal trafficking. Here, using this system we report progressive changes in axonal transport kinetics of both dense core vesicles and mitochondria that correlate with network development and maturation. Interestingly, axonal flow of both types of organelles change in opposite directions, with rates increasing for vesicles and decreasing for mitochondria. Overall, our observations highlight the need for a better spatiotemporal control for the study of intracellular dynamics in order to avoid misinterpretations and improve reproducibility.

Secretory vesicle trafficking in awake and anaesthetized mice: differential speeds in axons versus synapses.

Despite their immense physiological and pathophysiological importance, we know very little about the biology of dense core vesicle (DCV) trafficking in the intact mammalian brain. DCVs are transported at similar average speeds in the anaesthetized and awake mouse brain compared to neurons in culture, yet maximal speed and pausing fraction of transport were higher. Microtubule plus (+)-end extension imaging visualized microtubular growth at 0.12μm/s and revealed that DCVs were transported faster in the anterograde direction. DCV transport slowed down upon presynaptic bouton approach, possibly promoting synaptic localization and cargo release. Our work provides a basis to extrapolate DCV transport properties determined in cultured neurons to the intact mouse brain and reveals novel features such as slowing upon bouton approach and brain state-dependent trafficking directionality. Neuronal dense core vesicles (DCVs) transport many cargo molecules like neuropeptides and neurotrophins to their release sites in dendrites or axons. The transport properties of DCVs in axons of the intact mammalian brain are unknown. We used viral expression of a DCV cargo reporter (NPY-Venus/Cherry) in the thalamus and two-photon in vivo imaging to visualize axonal DCV trafficking in thalamocortical projections of anaesthetized and awake mice. We found an average speed of 1μm/s, maximal speeds of up to 5μm/s and a pausing fraction of ∼11%. Directionality of transport differed between anaesthetized and awake mice. In vivo microtubule +-end extension imaging using MACF18-GFP revealed microtubular growth at 0.12μm/s and provided positive identification of antero- and retrograde axonal transport. Consistent with previous reports, anterograde transport was faster (∼2.1μm/s) than retrograde transport (∼1.4μm/s). In summary, DCVs are transported with faster maximal speeds and lower pausing fraction in vivo compared to previous results obtained in vitro. Finally, we found that DCVs slowed down upon presynaptic bouton approach. We propose that this mechanism promotes synaptic localization and cargo release.

MFN2 agonists reverse mitochondrial defects in preclinical models of Charcot-Marie-Tooth disease type 2A.

Mitofusins (MFNs) promote fusion-mediated mitochondrial content exchange and subcellular trafficking. Mutations in Mfn2 cause neurodegenerative Charcot-Marie-Tooth disease type 2A (CMT2A). We showed that MFN2 activity can be determined by Met376 and His380 interactions with Asp725 and Leu727 and controlled by PINK1 kinase-mediated phosphorylation of adjacent MFN2 Ser378 Small-molecule mimics of the peptide-peptide interface of MFN2 disrupted this interaction, allosterically activating MFN2 and promoting mitochondrial fusion. These first-in-class mitofusin agonists overcame dominant mitochondrial defects provoked in cultured neurons by CMT2A mutants MFN2 Arg94→Gln94 and MFN2 Thr105→Met105, as demonstrated by amelioration of mitochondrial dysmotility, fragmentation, depolarization, and clumping. A mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of MFN2 Thr105→Met105 mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism and/or trafficking.

Age-dependent neurodegeneration and organelle transport deficiencies in mutant TDP43 patient-derived neurons are independent of TDP43 aggregation

TAR DNA-binding protein 43 (TDP43) plays a significant role in familiar and sporadic amyotrophic lateral sclerosis (ALS). The diverse postulated mechanisms by which TDP43 mutations cause the disease are not fully understood. Human wildtype and TDP43 S393L and G294V mutant spinal motor neuron cultures were differentiated from patient-derived iPSCs. Mutant hTDP43 and wildtype motor neuron cultures did not differ in neuron differentiation capacity during early maturation stage. During aging we detected a dramatic neurodegeneration including neuron loss and pathological neurofilament abnormalities only in TDP43 mutant cultures. Additionally mitochondria and lysosomes of aging spinal motor neurons revealed robust TDP43 mutation dependent abnormal phenotypes in size, shape, speed and motility which all appeared without TDP43 mislocalization or aggregation formation. Furthermore, D-sorbitol – known to induce stress granules and cytoplasmic mislocalization of TDP43 – rescued axonal trafficking phenotypes without signs of TDP43 mislocalization or aggregation formation. Our data indicate TDP43 mutation-dependent but cytosolic aggregation-independent mechanisms of motor neuron degeneration in TDP43 ALS.

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype.

Soluble A\u03b21\u201342 increases the heterogeneity in synaptic vesicle pool size among synapses by suppressing intersynaptic vesicle sharing

Growing evidence has indicated that prefibrillar form of soluble amyloid beta (sAβ1–42) is the major causative factor in the synaptic dysfunction associated with AD. The molecular changes leading to presynaptic dysfunction caused by sAβ1–42, however, still remains elusive. Recently, we found that sAβ1–42 inhibits chemically induced long-term potentiation-induced synaptogenesis by suppressing the intersynaptic vesicle trafficking through calcium (Ca2+) dependent hyperphosphorylation of synapsin and CaMKIV. However, it is still unclear how sAβ1–42 increases intracellular Ca2+ that induces hyperphosphorylation of CaMKIV and synapsin, and what is the functional consequences of sAβ1–42-induced defects in intersynaptic vesicle trafficking in physiological conditions. In this study, we showed that sAβ1–42elevated intracellular Ca2+ through not only extracellular Ca2+ influx but also Ca2+ release from mitochondria. Surprisingly, without Ca2+ release from mitochondria, sAβ1–42 failed to increase intracellular Ca2+ even in the presence of normal extracellular Ca2+. We further found that sAβ1–42-induced mitochondria Ca2+ release alone sufficiently increased Serine 9 phosphorylation of synapsin. By blocking synaptic vesicle reallocation, sAβ1–42 significantly increased heterogeneity of total synaptic vesicle pool size among synapses. Together, our results suggested that by disrupting the axonal vesicle trafficking, sAβ1–42 disabled neurons to adjust synaptic pool sizes among synapses, which might prevent homeostatic rescaling in synaptic strength of individual neurons.

VEGF Axonal Transport Dependent on Kinesin-1B and Microtubules Dynamics.

Axon-transport plays an important role in neuronal activity and survival. Reduced endogenous VEGF can cause neuronal damage and axon degeneration. It is unknown at this time if VEGF can be transported within the axon or whether it can be released by axonal depolarization. We transfected VEGF-eGFP plasmids in cultured hippocampal neurons and tracked their movement in the axons by live-cell confocal imaging. Then, we co-transfected phVEGF-eGFP and kinesin-1B-DsRed vectors into neurons and combined with immunoprecipitation and two-color imaging to study the mechanism of VEGF axon-trafficking. We found that VEGF vesicles morphologically co-localized and biochemically bounded with kinesin-1B, as well as co-trafficked with it in the axons. Moreover, the capacity for axonal trafficking of VEGF was reduced by administration of nocodazole, an inhibitor of microtubules, or kinesin-1B shRNA. In addition, we found that VEGF could release from the cultured neurons under acute depolarizing stimulation with potassium chloride. Therefore, present findings suggest that neuronal VEGF is stored in the vesicles, actively released, and transported in the axons, which depends on the presence of kinesin-1B and functional microtubules. These results further help us to understand the importance of neuronal VEGF in the maintenance of neuronal activity and survival throughout life.

Direct central nervous system effects of botulinum neurotoxin

Local intramuscular injections of botulinum neurotoxin type A (BoNT/A) are effective in the treatment of focal dystonias, muscle spasms, and spasticity. However, not all clinical effects of BoNT/A may be explained by its action at peripheral nerve terminals. For example, the therapeutic benefit may exceed the duration of the peripheral neuroparalysis induced by the neurotoxin. In cellular and animal models, evidence demonstrates retrograde transport of catalytically active BoNT/A in projection neurons. This process of long-range trafficking is followed by transcytosis and action at second-order synapses. In humans, several physiological changes have been described following intramuscular delivery of BoNT/A. In particular, clinical studies have documented a decrease in Renshaw cell-mediated inhibition (i.e., recurrent inhibition), which may be important therapeutically for normalizing uncoordinated movements and overflow of muscle activity. In this review, we present data obtained in animal and experimental models that support direct central actions of BoNT/A mediated via retrograde axonal trafficking. We also discuss the reorganization of central circuitry induced by BoNT/A in patients, and the potential contribution of these effects to the therapeutic efficacy of the neurotoxin.

Accessibility of axonal G protein coupled mu-opioid receptors requires conceptual changes of axonal membrane targeting for pain modulation

The mechanisms of axonal trafficking and membrane targeting are well established for sodium channels, which are the principle targets for perineurally applied local anaesthetics. However, they have not been thoroughly investigated for G protein coupled receptors such as mu-opioid receptors (MOR). Focusing on these axonal mechanisms, we found that axonal MOR functionality is quite distinct in two different pain states, i.e. hindpaw inflammation and nerve injury. We observed axonal membrane MOR binding and functional G protein coupling exclusively at sites of CCI nerve injury. Moreover at these axonal membrane sites, MOR exhibited extensive co-localization with the membrane proteins SNAP and Na/K-ATPase as well as NGF-dependent enhanced lipid rafts and L1CAM anchoring proteins. Silencing endogenous L1CAM with intrathecal L1CAM specific siRNA, disrupting lipid rafts with the perineurial cholesterol-sequestering agent MβCD, as well as suppressing NGF receptor activation with the perineurial NGF receptor inhibitor K252a abrogated MOR axonal membrane integration, functional coupling, and agonist-elicited antinociception at sites of nerve injury. These findings suggest that local conceptual changes resulting from nerve injury are required for the establishment of functional axonal membrane MOR. Axonal integration and subsequent accessibility of functionally coupled MOR are of great relevance particularly for patients suffering from severe pain due to nerve injury or tumour infiltration.

Paclitaxel Reduces Axonal Bclw to Initiate IP3R1-Dependent Axon Degeneration

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect of many cancer treatments. The hallmark of CIPN is degeneration of long axons required for transmission of sensory information; axonal degeneration causes impaired tactile sensation and persistent pain. Currently the molecular mechanisms of CIPN are not understood, and there are no available treatments. Here we show that the chemotherapeutic agent paclitaxel triggers CIPN by altering IP3 receptor phosphorylation and intracellular calcium flux, and activating calcium-dependent calpain proteases. Concomitantly paclitaxel impairs axonal trafficking of RNA-granules and reduces synthesis of Bclw (bcl2l2), a Bcl2 family member that binds IP3R1 and restrains axon degeneration. Surprisingly, Bclw or a stapled peptide corresponding to theBclw BH4 domain interact with axonal IP3R1 and prevent paclitaxel-induced degeneration, while Bcl2 andBclxL cannot do so. Together these data identify a Bclw-IP3R1-dependent cascade that causes axon degeneration and suggest that Bclw-mimetics could provide effective therapy to prevent CIPN.

Dyslipidemia impairs mitochondrial trafficking and function in sensory neurons.

Mitochondrial trafficking plays a central role in dorsal root ganglion (DRG) neuronal cell survival and neurotransmission by transporting mitochondria from the neuronal cell body throughout the bundles of DRG axons. In type 2 diabetes (T2DM), dyslipidemia and hyperglycemia damage DRG neurons and induce mitochondrial dysfunction; however, the impact of free fatty acids and glucose on mitochondrial trafficking in DRG neurons remains unknown. To evaluate the impact of free fatty acids compared to hyperglycemia on mitochondrial transport, primary adult mouse DRG neuron cultures were treated with physiologic concentrations of palmitate and glucose and assessed for alterations in mitochondrial trafficking, mitochondrial membrane potential, and mitochondrial bioenergetics. Palmitate treatment significantly reduced the number of motile mitochondria in DRG axons, but physiologic concentrations of glucose did not impair mitochondrial trafficking dynamics. Palmitate-treated DRG neurons also exhibited a reduction in mitochondrial velocity, and impaired mitochondrial trafficking correlated with mitochondrial depolarization in palmitate-treated DRG neurons. Finally, we found differential bioenergetic effects of palmitate and glucose on resting and energetically challenged mitochondria in DRG neurons. Together, these results suggest that palmitate induces DRG neuron mitochondrial depolarization, inhibiting axonal mitochondrial trafficking and altering mitochondrial bioenergetic capacity.-Rumora, A. E., Lentz, S. I., Hinder, L. M., Jackson, S. W., Valesano, A., Levinson, G. E., Feldman, E. L. Dyslipidemia impairs mitochondrial trafficking and function in sensory neurons.

Lissencephaly-1 dependent axonal retrograde transport of L1-type CAM Neuroglian in the adult drosophila central nervous system.

Here, we established the Drosophila Giant Fiber neurons (GF) as a novel model to study axonal trafficking of L1-type Cell Adhesion Molecules (CAM) Neuroglian (Nrg) in the adult CNS using live imaging. L1-type CAMs are well known for their importance in nervous system development and we previously demonstrated a role for Nrg in GF synapse formation. However, in the adult they have also been implicated in synaptic plasticity and regeneration. In addition, to its canonical role in organizing cytoskeletal elements at the plasma membrane, vertebrate L1CAM has also been shown to regulate transcription indirectly as well as directly via its import to the nucleus. Here, we intend to determine if the sole L1CAM homolog Nrg is retrogradley transported and thus has the potential to relay signals from the synapse to the soma. Live imaging of c-terminally tagged Nrg in the GF revealed that there are at least two populations of retrograde vesicles that differ in speed, and either move with consistent or varying velocity. To determine if endogenous Nrg is retrogradely transported, we inhibited two key regulators, Lissencephaly-1 (Lis1) and Dynactin, of the retrograde motor protein Dynein. Similar to previously described phenotypes for expression of poisonous subunits of Dynactin, we found that developmental knock down of Lis1 disrupted GF synaptic terminal growth and that Nrg vesicles accumulated inside the stunted terminals in both mutant backgrounds. Moreover, post mitotic Lis1 knock down in mature GFs by either RNAi or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) induced mutations, resulted in normal length terminals with fully functional GF synapses which also exhibited severe accumulation of endogenous Nrg vesicles. Thus, our data suggests that accumulation of Nrg vesicles is due to failure of retrograde transport rather than a failure of terminal development. Together with the finding that post mitotic knock down of Lis1 also disrupted retrograde transport of tagged Nrg vesicles in GF axons, it demonstrates that endogenous Nrg protein is transported from the synapse to the soma in the adult central nervous system in a Lis1-dependent manner.

Microtubule-dependent ribosome localization in C. elegans neurons.

Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level. We found that axonal ribosomes change localization during neuronal development and after axonal injury. By examining mutants affecting axonal trafficking and performing a forward genetic screen, we showed that the microtubule cytoskeleton and the JIP3 protein UNC-16 exert distinct effects on localization of axonal and somatic ribosomes. Our data demonstrate the utility of tissue-specific visualization of ribosomes in vivo, and provide insight into the mechanisms of active regulation of ribosome localization in neurons.