Tips Of Neurites Research Articles

Neurons dispose of hyperactive kinesin into glial cells for clearance

Microtubule-based kinesin motor proteins are crucial for intracellular transport, but their hyperactivation can be detrimental for cellular functions. This study investigated the impact of a constitutively active ciliary kinesin mutant, OSM-3CA, on sensory cilia in C. elegans. Surprisingly, we found that OSM-3CA was absent from cilia but underwent disposal through membrane abscission at the tips of aberrant neurites. Neighboring glial cells engulf and eliminate the released OSM-3CA, a process that depends on the engulfment receptor CED-1. Through genetic suppressor screens, we identified intragenic mutations in the OSM-3CA motor domain and mutations inhibiting the ciliary kinase DYF-5, both of which restored normal cilia in OSM-3CA-expressing animals. We showed that conformational changes in OSM-3CA prevent its entry into cilia, and OSM-3CA disposal requires its hyperactivity. Finally, we provide evidence that neurons also dispose of hyperactive kinesin-1 resulting from a clinic variant associated with amyotrophic lateral sclerosis, suggesting a widespread mechanism for regulating hyperactive kinesins.

ALS Causative Mutations in KIF5A Disrupt Autoinhibition Leading to Toxic Gain of Function (P8-8.002)

<h3>Objective:</h3> The objective of this study was to elucidate the pathogenic mechanisms of ALS causative mutations in motor protein KIF5A. <h3>Background:</h3> KIF5A is a neuronal specific subunit of Kinesin-1, which is a microtubule motor protein that plays roles in axonal transport and cytoskeletal regulation. Mutations in distinct regions of Kinesin-1 lead to a broad range of neurologic diseases. KIF5A enables Kinesin-1 to walk along microtubules and transport various cargos such as mitochondria and RNA granules into the distal axon. ALS-causative mutations in KIF5A affect the structure of the domain of the protein that functions in cargo binding and autoregulation. However, the consequences of ALS-causative mutations on disease pathogenesis are not fully understood. Our central hypothesis was that KIF5A ALS mutations disrupt cargo binding and/or autoregulation leading to neurodegeneration. <h3>Design/Methods:</h3> We generated <i>in vitro</i> models of KIF5A ALS using transient transfection of disease causative variants into neuroblastoma cell lines. We performed immunofluorescence staining and confocal imaging to visualize the distribution of motor proteins KIF5A, KIF5B, and Dynein. We utilized Drosophila S2 cell lines to study mitochondrial motility using live-imaging. <h3>Results:</h3> Whereas wild type (WT) KIF5A displayed relatively homogeneous distribution, ALS mutant KIF5A developed dramatic accumulation within distal neurites. Co-expression of the ALS mutant protein with the WT caused it to accumulate as well. Mitochondria displayed similar mislocalization to distal neurites. No change was seen in the distribution of KIF5B or Dynein. <h3>Conclusions:</h3> We found that ALS mutant KIF5A caused dramatic accumulation of mutant and WT protein as well as mitochondrial within distal neurite tips. These findings suggest that ALS mutation disrupts its autoinhibition causing mislocalization of its cargos through gain of function. These changes in distribution are specific to KIF5A containing motors. Our findings establish dysregulation of KIF5A activity as the underlying pathogenic mechanism for KIF5A ALS. <b>Disclosure:</b> Dr. Brent has received research support from The American Academy of Neurology. Mr. Sterling-Angus has nothing to disclose. Dr. Deng has nothing to disclose.

Expression of ATP-binding cassette transporter A1 is induced by nerve injury and its deficiency affects neurite tip morphology and elongation in cultured neurons

Axonal regeneration requires changes in the lipid dynamics of the axon membrane for growth and extension. Here, we examined the expression of genes associated with lipid transport after nerve injury. The expression of ATP-binding cassette transporter-A1 (ABCA1), which participates in the transport of cholesterol from the plasma membrane, was markedly upregulated in motor and sensory neurons after nerve injury. Stimulation of PC12 cells with the nerve growth factor induced neurite extension and ABCA1 expression predominantly in regions proximal to the neurite tip. To clarify the functional role of ABCA1 in neurite elongation, we examined the morphology of neurons cultured from conditionally-injured dorsal root ganglia from ABCA1-deficient mice. We found a significant increase in neurite branch formation in these neurons. In addition, the neurite tips of ABCA1-deficient neurons appeared excessively ruffled, and the direction of neurite elongation was unsteady. In contrast, the neurite tips of wild-type neurons were not excessively ruffled, and the neurites elongated rapidly in a stable directionally-oriented manner. Together, these findings suggest that ABCA1 plays an important role in regulating the membrane lipid composition of injured neurons and in axonal regeneration following nerve injury.

JIP3 interacts with dynein and kinesin-1 to regulate bidirectional organelle transport.

The MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. How JIP3's interaction with dynein and kinesin-1 contributes to organelle clearance is unclear. We show that human dynein light intermediate chain (DLIC) binds the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in RH1 abrogates DLIC binding without perturbing the interaction between JIP3's RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3's leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bidirectional organelle transport.

Unbalanced Regulation of Sec22b and Ykt6 Blocks Autophagosome Axonal Retrograde Flux in Neuronal Ischemia-Reperfusion Injury.

Cerebral ischemia–reperfusion (I/R) injury in ischemic penumbra is accountable for poor outcome of ischemic stroke patients receiving recanalization therapy. Compelling evidence previously demonstrated a dual role of autophagy in stroke. This study aimed to understand the traits of autophagy in the ischemic penumbra and the potential mechanism that switches the dual role of autophagy. We found that autophagy induction by rapamycin and lithium carbonate performed before ischemia reduced neurologic deficits and infarction, while autophagy induction after reperfusion had the opposite effect in the male murine middle cerebral artery occlusion/reperfusion (MCAO/R) model, both of which were eliminated in mice lacking autophagy (Atg7flox/flox; Nestin-Cre). Autophagic flux determination showed that reperfusion led to a blockage of axonal autophagosome retrograde transport in neurons, which then led to autophagic flux damage. Then, we found that I/R induced changes in the protein levels of Sec22b and Ykt6 in neurons, two autophagosome transport-related factors, in which Sec22b significantly increased and Ykt6 significantly decreased. In the absence of exogenous autophagy induction, Sec22b knock-down and Ykt6 overexpression significantly alleviated autophagic flux damage, infarction, and neurologic deficits in neurons or murine exposed to cerebral I/R in an autophagy-dependent manner. Furthermore, Sec22b knock-down and Ykt6 overexpression switched the outcome of rapamycin posttreatment from deterioration to neuroprotection. Thus, Sec22b and Ykt6 play key roles in neuronal autophagic flux, and modest regulation of Sec22b and Ykt6 may help to reverse the failure of targeting autophagy induction to improve the prognosis of ischemic stroke.SIGNIFICANCE STATEMENT The highly polarized architecture of neurons with neurites presents challenges for material transport, such as autophagosomes, which form at the neurite tip and need to be transported to the cell soma for degradation. Here, we demonstrate that Sec22b and Ykt6 act as autophagosome porters and play an important role in maintaining the integrity of neuronal autophagic flux. Ischemia–reperfusion (I/R)-induced excess Sec22b and loss of Ykt6 in neurons lead to axonal autophagosome retrograde trafficking failure, autophagic flux damage, and finally neuronal injury. Facilitated axonal autophagosome retrograde transport by Sec22b knock-down and Ykt6 overexpression may reduce I/R-induced neuron injury and extend the therapeutic window of pharmacological autophagy induction for neuroprotection.

Predicting neurite extension for varying extracellular matrix stiffness and topography

Neurite extension is a dynamic process and is dependent on the microenvironment. The mechanical properties of the extracellular matrix (ECM), such as stiffness and topography influence the microenvironment and affects neurite extension; however, the mechanistic basis for this dynamic response of neurite extension remains elusive. In this study, we develop a computational model that predicts neurite extension dynamics process as the stiffness and patterned topography of ECM changes. The model includes the contribution of receptors integrin and neural cellular adhesion molecule toward the growth of neurite tip. We use non-linear finite element analysis (FEA) to model the neuronal cell, neurite, and the ECM, which is then coupled to the force–deformation receptor properties obtained from molecular dynamics simulations. Using an empirical relation, we develop a neurite extension algorithm that simulates the dynamic process of growth cone induced by growth cone extension, receptor density, and rupture. We investigate the dependence of neurite extension on ECM stiffness using three distinct materials, the effect of width and spacing of continuous (cylindrical) and discontinuous (pillar) patterned topography, as well as the topography steepness and stiffness gradient. We find that an increasing stiffness and width of patterned topography results in increased neurite extension, but the magnitude of the increase differs depending on the growth cone extension and receptor density between them. These findings will aid in vitro studies in determining an ECM with appropriate mechanical properties, such as stiffness and topography that will improve neurite extension, thus resulting in the formation of functional neurons.

GPR3 accelerates neurite outgrowth and neuronal polarity formation via PI3 kinase-mediating signaling pathway in cultured primary neurons

During neuronal development, immature neurons extend neurites and subsequently polarize to form an axon and dendrites. We have previously reported that G protein-coupled receptor 3 (GPR3) levels increase during neuronal development, and that GPR3 has functions in neurite outgrowth and neuronal differentiation in cerebellar granular neurons. Moreover, GPR3 is transported and concentrated at the tips of neurite, thereby contributing to the local activation of protein kinase A (PKA). However, the signaling pathways for GPR3-mediated neurite outgrowth and its subsequent effects on neuronal polarization have not yet been elucidated. We therefore analyzed the signaling pathways related to GPR3-mediated neurite outgrowth, and also focused on the possible roles of GPR3 in axon polarization. We demonstrated that, in cerebellar granular neurons, GPR3-mediated neurite outgrowth was mediated by multiple signaling pathways, including those of PKA, extracellular signal-regulated kinases (ERKs), and most strongly phosphatidylinositol 3-kinase (PI3K). In addition, the GPR3-mediated activation of neurite outgrowth was associated with G protein-coupled receptor kinase 2 (GRK2)-mediated signaling and phosphorylation of the C-terminus serine/threonine residues of GPR3, which affected downstream protein kinase B (Akt) signaling. We further demonstrated that GPR3 was transiently increased early in the development of rodent hippocampal neurons. It was subsequently concentrated at the tip of the longest neurite, and was thus associated with accelerated polarity formation in a PI3K-dependent manner in rat hippocampal neurons. In addition, GPR3 knockout in mouse hippocampal neurons led to delayed neuronal polarity formation, thereby affecting the dephosphorylation of collapsing response mediator protein 2 (CRMP2), which is downstream of the PI3K signaling pathway. Taken together, these findings suggest that the intrinsic expression of GPR3 in differentiated neurons constitutively activates PI3K-mediated signaling pathway predominantly, thus accelerating neurite outgrowth and further augmenting polarity formation in primary cultured neurons.

Differential adhesion regulates neurite placement via a retrograde zippering mechanism.

During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the C. elegans brain neuropil, and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers. Differential adhesion is orchestrated via developmentally regulated expression of the IgCAM SYG-1, and its partner ligand SYG-2. Changes in SYG-1 expression across neuropil layers result in changes in adhesive forces, which sort SYG-2-expressing neurons. Sorting to layers occurs, not via outgrowth from the neurite tip, but via an alternate mechanism of retrograde zippering, involving interactions between neurite shafts. Our study indicates that biophysical principles from differential adhesion govern neurite placement and synaptic specificity in vivo in developing neuropil bundles.

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance.

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses ofsingle speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

Kinesin-1-dependent transport of the βPIX/GIT complex in neuronal cells

Proper targeting of the βPAK-interacting exchange factor (βPIX)/G protein-coupled receptor kinase-interacting target protein (GIT) complex into distinct cellular compartments is essential for its diverse functions including neurite extension and synaptogenesis. However, the mechanism for translocation of this complex is still unknown. In the present study, we reported that the conventional kinesin, called kinesin-1, can transport the βPIX/GIT complex. Additionally, βPIX bind to KIF5A, a neuronal isoform of kinesin-1 heavy chain, but not KIF1 and KIF3. Mapping analysis revealed that the tail of KIF5s and LZ domain of βPIX were the respective binding domains. Silencing KIF5A or the expression of a variety of mutant forms of KIF5A inhibited βPIX targeting the neurite tips in PC12 cells. Fur-thermore, truncated mutants of βPIX without LZ domain did not interact with KIF5A, and were unable to target the neurite tips in PC12 cells. These results defined kinesin-1 as a motor protein of βPIX, and may provide new insights into βPIX/GIT complex-dependent neuronal pathophysiology.

Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons-New Insights into the Role of the C-Terminal Region of Tpm3.1.

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

Genotype and defects in microtubule-based motility correlate with clinical severity in KIF1A-associated neurological disorder.

SummaryKIF1A-associated neurological disorder (KAND) encompasses a group of rare neurodegenerative conditions caused by variants in KIF1A,a gene that encodes an anterograde neuronal microtubule (MT) motor protein. Here we characterize the natural history of KAND in 117 individuals using a combination of caregiver or self-reported medical history, a standardized measure of adaptive behavior, clinical records, and neuropathology. We developed a heuristic severity score using a weighted sum of common symptoms to assess disease severity. Focusing on 100 individuals, we compared the average clinical severity score for each variant with in silico predictions of deleteriousness and location in the protein. We found increased severity is strongly associated with variants occurring in protein regions involved with ATP and MT binding: the P loop, switch I, and switch II. For a subset of variants, we generated recombinant proteins, which we used to assess transport in vivo by assessing neurite tip accumulation and to assess MT binding, motor velocity, and processivity using total internal reflection fluorescence microscopy. We find all modeled variants result in defects in protein transport, and we describe three classes of protein dysfunction: reduced MT binding, reduced velocity and processivity, and increased non-motile rigor MT binding. The rigor phenotype is consistently associated with the most severe clinical phenotype, while reduced MT binding is associated with milder clinical phenotypes. Our findings suggest the clinical phenotypic heterogeneity in KAND likely reflects and parallels diverse molecular phenotypes. We propose a different way to describe KAND subtypes to better capture the breadth of disease severity.

PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.

Detailed neuronal distribution of GPR3 and its co-expression with EF-hand calcium-binding proteins in the mouse central nervous system

The G-protein coupled receptor 3 (GPR3), a member of the class A rhodopsin-type GPR family, constitutively activates Gαs proteins without any ligands. Although there have been several reports concerning the functions of GPR3 in neurons, the physiological roles of GPR3 have not been fully elucidated. To address this issue, we analyzed GPR3 distribution in detail using fluorescence-based X-gal staining in heterozygous GPR3 knockout/LacZ knock-in mice, and further investigated the types of GPR3-expressing neurons using fluorescent double labeling with various EF-hand Ca2+-binding proteins. In addition to the previously reported GPR3-expressing areas, we identified GPR3 expression in the basal ganglia and in many nuclei of the cranial nerves, in regions related to olfactory, auditory, emotional, and motor functions. In addition, GPR3 was not only observed in excitatory neurons in layer V of the cerebral cortex, the CA2 region of the hippocampus, and the lateral nucleus of the thalamus, but also in γ-aminobutyric acid (GABA)-ergic interneurons in the cortex, hippocampus, thalamus, striatum, and cerebellum. GPR3 was frequently co-expressed with neuronal Ca2+-binding protein 2 (NECAB2) in neurons in various regions of the central nervous system, especially in the hippocampal CA2, medial habenular nucleus, lateral thalamic nucleus, dorsolateral striatum, brainstem, and spinal cord anterior horn. Furthermore, GPR3 also co-localized with NECAB2 at the tips of neurites in differentiated PC12 cells. These results suggest that GPR3 and NECAB2 are highly co-expressed in specific neurons, and that GPR3 may modulate Ca2+ signaling by interacting with NECAB2 in specific areas of the central nervous system.

Coronin 2B regulates dendrite outgrowth by modulating actin dynamics.

Cytoskeletal remodeling is indispensable for the development and maintenance of neuronal structures and functions. However, the molecular machinery that controls the balance between actin polymerization and depolymerization during these processes is incompletely understood. Here, we report that coronin 2B, a conserved actin-binding protein, is concentrated at the tips of developing dendrites and that knockdown of coronin 2B inhibits the growth of dendrites. Importantly, coronin 2B interacts with actin and reduces the F-actin/G-actin ratio. Furthermore, the coiled-coil domain of coronin 2B is required for its oligomerization, thus confining coronin 2B to neurite tips. Our findings collectively suggest that coronin 2B is important for promoting dendrite outgrowth by limiting the speed of actin polymerization at growth cones.

Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A).

Defects in the motor domain of kinesin family member 1A (KIF1A), a neuron-specific ATP-dependent anterograde axonal transporter of synaptic cargo, are well-recognized to cause a spectrum of neurological conditions, commonly known as KIF1A-associated neurological disorders (KAND). Here, we report one mutation-negative female with classic Rett syndrome (RTT) harboring a de novo heterozygous novel variant [NP_001230937.1:p.(Asp248Glu)] in the highly conserved motor domain of KIF1A. In addition, three individuals with severe neurodevelopmental disorder along with clinical features overlapping with KAND are also reported carrying de novo heterozygous novel [NP_001230937.1:p.(Cys92Arg) and p.(Pro305Leu)] or previously reported [NP_001230937.1:p.(Thr99Met)] variants in KIF1A. In silico tools predicted these variants to be likely pathogenic, and 3D molecular modeling predicted defective ATP hydrolysis and/or microtubule binding. Using the neurite tip accumulation assay, we demonstrated that all novel KIF1A variants significantly reduced the ability of the motor domain of KIF1A to accumulate along the neurite lengths of differentiated SH-SY5Y cells. In vitro microtubule gliding assays showed significantly reduced velocities for the variant p.(Asp248Glu) and reduced microtubule binding for the p.(Cys92Arg) and p.(Pro305Leu) variants, suggesting a decreased ability of KIF1A to move along microtubules. Thus, this study further expanded the phenotypic characteristics of KAND individuals with pathogenic variants in the KIF1A motor domain to include clinical features commonly seen in RTT individuals.

Actin waves transport RanGTP to the neurite tip to regulate non-centrosomal microtubules in neurons.

Microtubules (MTs) are the most abundant cytoskeleton in neurons, and control multiple facets of their development. While the MT-organizing center (MTOC) in mitotic cells is typically located at the centrosome, the MTOC in neurons switches to non-centrosomal sites. A handful of cellular components have been shown to promote non-centrosomal MT (ncMT) formation in neurons, yet the regulation mechanism remains unknown. Here, we demonstrate that the small GTPase Ran is a key regulator of ncMTs in neurons. Using an optogenetic tool that enables light-induced local production of RanGTP, we demonstrate that RanGTP promotes ncMT plus-end growth along the neurite. Additionally, we discovered that actin waves drive the anterograde transport of RanGTP. Pharmacological disruption of actin waves abolishes the enrichment of RanGTP and reduces growing ncMT plus-ends at the neurite tip. These observations identify a novel regulation mechanism for ncMTs and pinpoint an indirect connection between the actin and MT cytoskeletons in neurons.

Neurite regrowth stimulation by a red-light spot focused on the neuronal cell soma following blue light-induced retraction

The interaction of light with biological tissues has been considered for various therapeutic applications. Light-induced neurite growth has the potential to be a clinically useful technique for neuron repair. However, most previous studies used either a large illumination area to accelerate overall neurite growth or employed a light spot to guide a growing neurite. It is not clear if optical stimulation can induce the regrowth of a retracted neurite. In the present work, we used blue light (wavelength: 473 nm) to cause neurite retraction, and we proved that using a red-light (wavelength: 650 nm) spot to illuminate the soma near the junction of the retracted neurite could induce neurite regrowth. As a comparison, we found that green light (wavelength 550 nm) had a 62% probability of inducing neurite regrowth, while red light had a 75% probability of inducing neurite regrowth at the same power level. Furthermore, the neurite regrowth length induced by red light was increased by the pre-treatment with inhibitors of myosin functions. We also observed actin propagation from the soma to the tip of the re-growing neurite following red-light stimulation of the soma. The red light-induced extension and regrowth were abrogated in the calcium-free medium. These results suggest that illumination with a red-light spot on the soma may trigger the regrowth of a neurite after the retraction caused by blue-light illumination.

Vitronectin is Involved in the Morphological Transition of Neurites in Retinoic Acid-Induced Neurogenesis of Neuroblastoma Cell Line Neuro2a.

Vitronectin (Vtn), one of the extracellular matrix proteins, has been reported to result in cell cycle exit, neurite formation, and polarization of neural progenitor cells during neurogenesis. The underlying mechanism, however, has not been fully understood. In this study, we investigated the roles of Vtn and its integrin receptors, during the transition of neurites from multipolar to bipolar morphology, accompanying the cell cycle exit in neural progenitor cells. We used mouse neuroblastoma cell line Neuro2a as a model of neural progenitor cells which can induce cell cycle exit and the morphological transition of neurites by retinoic acid (RA)-stimulation. Treatment with an antibody for Vtn suppressed the RA-induced cell cycle exit and multipolar-to-bipolar transition. Furthermore, immunostaining results showed that in the cells displaying multipolar morphology Vtn was partially localized at the tips of neurites and in cells displaying bipolar morphology at both tips. This Vtn localization and multipolar-to-bipolar transition was perturbed by the transfection of a dominant negative mutant of cell polarity regulator Par6. In addition, a knockdown of β5 integrin, which is a receptor candidate for Vtn, affected the multipolar-to-bipolar transition. Taken together, these results suggest that Vtn regulates the multipolar-to-bipolar morphological transition via αvβ5 integrin.