Arachidonic acid-containing phosphatidylcholine species are increased in selected brain regions of a depressive animal model: Implications for pathophysiology

The Flinders Sensitive Line (FSL) rat is a genetic animal model of depression. Following recent findings that the brain fatty acid composition of FSL is characterised by increased arachidonic acid (AA), we used electrospray tandem mass spectrometry and ¹H-NMR to examine lipid species in different brain areas. Cholesterol and sphingolipids were increased in the hypothalamus of the FSL rats. Furthermore, arachidonic acid-containing phosphatidylcholine (AA-PC) species were elevated with PC16:0/20:4, PC18:1/20:4 and PC18:0/20:4 (p<0.003) increased in the hypothalamus and striatum. In contrast, there was a decrease in some docosahexaenoic acid (DHA)-containing species, specifically PC18:1/22:6 (p<0.003) in the striatum and PE18:1/22:6 (p<0.004) in the prefrontal cortex. Since no significant differences were observed in the erythrocyte fatty acid concentrations, dietary or environmental causes for these observations are unlikely. The increase in AA-PC species which in this animal model may be associated with altered neuropathy target esterase activity, an enzyme involved in membrane PC homeostasis, may contribute to the depressive phenotype of the FSL rats.     

Rufy3, a protein specifically expressed in neurons,interacts with actin‐bundling protein Fascin to control the growth of axons

For our nervous system to function properly, each neuron must generate a single axon and elongate the axon to reach its target. It is known that actin filaments and their dynamic interaction with microtubules within growth cones play important roles in inducing axon extension. However, it remains unclear how cytoskeletal dynamics is controlled in growth cones. In this study, we report that Rufy3, a RUN domain‐containing protein, is a neuron‐specific and actin filament‐relevant protein. We find that the appropriate expression of Rufy3 in mouse hippocampal neurons is required for the development of a single axon and axon growth. Our results show that Rufy3 specifically interacts with actin filament‐binding proteins, such as Fascin, and colocalizes with Fascin in growth cones. Knockdown of Rufy3 impairs the distribution of Fascin and actin filaments, accompanied by an increased proportion of neurons with multiple axons and a decrease in the axon length. Therefore, Rufy3 may be particularly important for neuronal axon elongation by interacting with Fascin to control actin filament organization in axonal growth cones.

     

BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin

Bone morphogenic proteins (BMPs) are involved in axon pathfinding, but how they guide growth cones remains elusive. In this study, we report that a BMP7 gradient elicits bidirectional turning responses from nerve growth cones by acting through LIM kinase (LIMK) and Slingshot (SSH) phosphatase to regulate actin-depolymerizing factor (ADF)/cofilin-mediated actin dynamics. Xenopus laevis growth cones from 4-8-h cultured neurons are attracted to BMP7 gradients but become repelled by BMP7 after overnight culture. The attraction and repulsion are mediated by LIMK and SSH, respectively, which oppositely regulate the phosphorylation-dependent asymmetric activity of ADF/cofilin to control the actin dynamics and growth cone steering. The attraction to repulsion switching requires the expression of a transient receptor potential (TRP) channel TRPC1 and involves Ca2+ signaling through calcineurin phosphatase for SSH activation and growth cone repulsion. Together, we show that spatial regulation of ADF/cofilin activity controls the directional responses of the growth cone to BMP7, and Ca2+ influx through TRPC tilts the LIMK-SSH balance toward SSH-mediated repulsion.     

Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination

Actin reorganization is important for regulation of neuronal morphology. Neural Wiskott-Aldrich syndrome protein (N-WASP) is an important regulator of actin polymerization and also known to be strongly expressed in brain. Recently, Toca-1 (transducer of Cdc42-dependent actin assembly) has been shown to be required for Cdc42 to activate N-WASP from biochemical experiments. Toca-1 has three functional domains: an F-BAR/EFC domain at the N terminus, an HR1 at the center, and an SH3 domain at the C terminus. The F-BAR/EFC domain induces tubular invagination of plasma membrane, while Toca-1 binds both N-WASP and Cdc42 through the SH3 domain and the HR1, respectively. However, the physiological role of Toca-1 is completely unknown. Here we have investigated the neural function of Toca-1. Toca-1 is strongly expressed in neurons including hippocampal neurons in developing brain at early times. Knockdown of Toca-1 in PC12 cells significantly enhances neurite elongation. Consistently, overexpression of Toca-1 suppresses neurite elongation through the F-BAR/EFC domain with a membrane invaginating property, suggesting an implication of membrane trafficking in the neural function of Toca-1. In addition, knockdown of N-WASP, to our surprise, also enhances neurite elongation in PC12 cells, which is in clear contrast to the previous report that dominant negative mutants of N-WASP suppress neurite extension in PC12 cells. On the other hand, knockdown of Toca-1 in cultured rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of N-WASP enhances both axon elongation and branching. These results suggest that a vesicle trafficking regulator Toca-1 regulates different aspects of neuronal morphology from N-WASP.     

Antagonistic forces generated by cytoplasmic dynein and myosin-II during growth cone turning and axonal retraction

Cytoplasmic dynein transports short microtubules down the axon in part by pushing against the actin cytoskeleton. Recent studies have suggested that comparable dynein-driven forces may impinge upon the longer microtubules within the axon. Here, we examined a potential role for these forces on axonal retraction and growth cone turning in neurons partially depleted of dynein heavy chain (DHC) by small interfering RNA. While DHC-depleted axons grew at normal rates, they retracted far more robustly in response to donors of nitric oxide than control axons, and their growth cones failed to efficiently turn in response to substrate borders. Live cell imaging of dynamic microtubule tips showed that microtubules in DHC-depleted growth cones were largely confined to the central zone, with very few extending into filopodia. Even under conditions of suppressed microtubule dynamics, DHC depletion impaired the capacity of microtubules to advance into the peripheral zone of the growth cone, indicating a direct role for dynein-driven forces on the distribution of the microtubules. These effects were all reversed by inhibition of myosin-II forces, which are known to underlie the retrograde flow of actin in the growth cone and the contractility of the cortical actin during axonal retraction. Our results are consistent with a model whereby dynein-driven forces enable microtubules to overcome myosin-II-driven forces, both in the axonal shaft and within the growth cone. These dynein-driven forces oppose the tendency of the axon to retract and permit microtubules to advance into the peripheral zone of the growth cone so that they can invade filopodia.     

CSPGs inhibit axon branching by impairing mitochondria‐dependent regulation of actin dynamics and axonal translation

Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The regulation of the axonal cytoskeleton and mitochondria are important components of the mechanism of branching. Actin‐dependent axonal plasticity, reflected in the dynamics of axonal actin patches and filopodia, is greatest along segments of the axon populated by mitochondria. It is reported that CSPGs partially depolarize the membrane potential of axonal mitochondria, which impairs the dynamics of the axonal actin cytoskeleton and decreases the formation and duration of axonal filopodia, the first steps in the mechanism of branching. The effects of CSPGs on actin cytoskeletal dynamics are specific to axon segments populated by mitochondria. In contrast, CSPGs do not affect the microtubule content of axons, or the localization of microtubules into axonal filopodia, a required step in the mechanism of branch formation. It is also reported that CSPGs decrease the mitochondria‐dependent axonal translation of cortactin, an actin associated protein involved in branching. Finally, the inhibitory effects of CSPGs on axon branching, actin cytoskeletal dynamics and the axonal translation of cortactin are reversed by culturing neurons with acetyl‐l ‐carnitine, which promotes mitochondrial respiration. Collectively these data indicate that CSPGs impair mitochondrial function in axons, an effect which contributes to the inhibition of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Collapsin Response Mediator Protein 4 Regulates Growth Cone Dynamics through the Actin and Microtubule Cytoskeleton

Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4−/− mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4−/− mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton.     

Scapinin-induced inhibition of axon elongation is attenuated by phosphorylation and translocation to the cytoplasm

Scapinin is an actin- and PP1-binding protein that is exclusively expressed in the brain; however, its function in neurons has not been investigated. Here we show that expression of scapinin in primary rat cortical neurons inhibits axon elongation without affecting axon branching, dendritic outgrowth, or polarity. This inhibitory effect was dependent on its ability to bind actin because a mutant form that does not bind actin had no effect on axon elongation. Immunofluorescence analysis showed that scapinin is predominantly located in the distal axon shaft, cell body, and nucleus of neurons and displays a reciprocal staining pattern to phalloidin, consistent with previous reports that it binds actin monomers to inhibit polymerization. We show that scapinin is phosphorylated at a highly conserved site in the central region of the protein (Ser-277) by Cdk5 in vitro. Expression of a scapinin phospho-mimetic mutant (S277D) restored normal axon elongation without affecting actin binding. Instead, phosphorylated scapinin was sequestered in the cytoplasm of neurons and away from the axon. Because its expression is highest in relatively plastic regions of the adult brain (cortex, hippocampus), scapinin is a new regulator of neurite outgrowth and neuroplasticity in the brain.     

Rnd1 regulates axon extension by enhancing the microtubule destabilizing activity of SCG10

The GTPase Rnd1 affects actin dynamics antagonistically to Rho and has been implicated in the regulation of neurite outgrowth, dendrite development, and axon guidance. Here we show that Rnd1 interacts with the microtubule regulator SCG10. This interaction requires a central domain of SCG10 comprising about 40 amino acids located within the N-terminal-half of a putative alpha-helical domain and is independent of phosphorylation at the four identified phosphorylation sites that regulate SCG10 activity. Rnd1 enhances the microtubule destabilizing activity of SCG10 and both proteins colocalize in neurons. Knockdown of Rnd1 or SCG10 by RNAi suppressed axon extension, indicating a critical role for both proteins during neuronal differentiation. Overexpression of Rnd1 in neurons induces the formation of multiple axons. The effect of Rnd1 on axon extension depends on SCG10. These results indicate that SCG10 acts as an effector downstream of Rnd1 to regulate axon extensions by modulating microtubule organization.     

Growth cone‐like waves transport actin and promote axonogenesis and neurite branching

Axonogenesis involves a shift from uniform delivery of materials to all neurites to preferential delivery to the putative axon, supporting its more rapid extension. Waves, growth cone‐like structures that propagate down the length of neurites, were shown previously to correlate with neurite growth in dissociated cultured hippocampal neurons. Waves are similar to growth cones in their structure, composition and dynamics. Here, we report that waves form in all undifferentiated neurites, but occur more frequently in the future axon during initial neuronal polarization. Moreover, wave frequency and their impact on neurite growth are altered in neurons treated with stimuli that enhance axonogenesis. Coincident with wave arrival, growth cones enlarge and undergo a marked increase in dynamics. Through their engorgement of filopodia along the neurite shaft, waves can induce de novo neurite branching. Actin in waves maintains much of its cohesiveness during transport whereas actin in nonwave regions of the neurite rapidly diffuses as measured by live cell imaging of photoactivated GFP‐actin and photoconversion of Dendra‐actin. Thus, waves represent an alternative axonal transport mechanism for actin. Waves also occur in neurons in organotypic hippocampal slices where they propagate along neurites in the dentate gyrus and the CA regions and induce branching. Taken together, our results indicate that waves are physiologically relevant and contribute to axon growth and branching via the transport of actin and by increasing growth cone dynamics. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Characterization of CLP36/Elfin/PDLIM1 in the nervous system

CLP36, one of the α-Actinin Associated LIM Protein (ALP)/Enigma family proteins, has a wide tissue distribution, but little is known about its expression and role in the nervous system. We show here that CLP36 is expressed in sensory ganglia but not in the CNS of adult rats. In primary dorsal root ganglion (DRG) neurons, CLP36 is distributed in the soma and neurites with enrichment in the growth cones. CLP36 forms a complex with α-actinin and is localized to actin cytoskeleton. To examine the role of CLP36 in neuronal cells, we transfected PC12 cells with a series of CLP36 deletion mutants and found that over-expression of CLP36 PDZ domain affects neurite outgrowth. Reduction of CLP36 function in PC12 cells by RNA interference (RNAi) induced lamellipodial protrusions around cell periphery and activated growth-cone movements, resulting in an increase in the length and number of neurites. Similarly, inhibition of CLP36 in primary DRG neurons increased the rate of neurite-bearing cells. We also found that CLP36 is up-regulated in DRG neurons and facial motoneurons after nerve injury. These findings suggest that CLP36 serves as a scaffold to form a multiprotein complex that regulates actin cytoskeleton dynamics and plays a role in controlling neurite outgrowth.     

Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.     

Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration

Microtubule dynamics are important for axon growth during development as well as axon regeneration after injury. We have previously identified HDAC5 as an injury-regulated tubulin deacetylase that functions at the injury site to promote axon regeneration. However, the mechanisms involved in the spatial control of HDAC5 activity remain poorly understood. Here we reveal that HDAC5 interacts with the actin binding protein filamin A via its C-terminal domain. Filamin A plays critical roles in HDAC5-dependent tubulin deacetylation because, in cells lacking filamin A, the levels of acetylated tubulin are elevated markedly. We found that nerve injury increases filamin A axonal expression in a protein synthesis-dependent manner. Reducing filamin A levels or interfering with the interaction between HDAC5 and filamin A prevents injury-induced tubulin deacetylation as well as HDAC5 localization at the injured axon tips. In addition, neurons lacking filamin A display reduced axon regeneration. Our findings suggest a model in which filamin A local translation following axon injury controls localized HDAC5 activity to promote axon regeneration.     

The role of the cytoskeleton during neuronal polarization

The formation of an axon and dendrites, neuronal polarization, is a prerequisite for neurons to integrate and propagate information within the brain. During the past years progress has been made toward understanding the initial stage of neuronal polarization, axon formation. First, the physiological role of some candidate regulators of neuronal polarity has been affirmed, including Sad kinases, the Rho-GTPase Cdc42, and the actin regulators Ena/VASP proteins. Second, recent studies have revealed microtubule stabilization as a mechanism complementary to actin dynamics underlying neuronal polarization. Moreover, stable microtubules in the axon may form a landmark to confer identity to the axon. This review highlights the recent advances in understanding the intracellular mechanisms underlying neuronal polarization and discusses them in the context of putative cytoskeletal effectors.     

HDAC5 is a novel injury-regulated tubulin deacetylase controlling axon regeneration

Axon regeneration is an essential process to rebuild functional connections between injured neurons and their targets. Regenerative axonal growth requires alterations in axonal microtubule dynamics, but the signalling mechanisms involved remain incompletely understood. Our results reveal that axon injury induces a gradient of tubulin deacetylation, which is required for axon regeneration both in vitro and in vivo. This injury-induced tubulin deacetylation is specific to peripheral neurons and fails to occur in central neurons. We found that tubulin deacetylation is initiated by calcium influx at the site of injury, and requires protein kinase C-mediated activation of the histone deacetylase 5 (HDAC5). Our findings identify HDAC5 as a novel injury-regulated tubulin deacetylase that plays an essential role in growth cone dynamics and axon regeneration. In addition, our results suggest a mechanism for the spatial control of tubulin modifications that is required for axon regeneration.     

EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.     

Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development

Members of the Cas family of Src homology 3 (SH3)-domain-containing cytosolic signaling proteins are crucial regulators of actin cytoskeletal dynamics in non-neuronal cells; however, their neuronal functions are poorly understood. Here, we identify a Drosophila Cas (DCas), find that Cas proteins are highly expressed in neurons and show that DCas is required for correct axon guidance during development. Functional analyses reveal that Cas specifies axon guidance by regulating the degree of fasciculation among axons. These guidance defects are similar to those observed in integrin mutants, and genetic analysis shows that integrins function together with Cas to facilitate axonal defasciculation. These results strongly support Cas proteins working together with integrins in vivo to direct axon guidance events.     

DSCR1 is required for both axonal growth cone extension and steering

Local information processing in the growth cone is essential for correct wiring of the nervous system. As an axon navigates through the developing nervous system, the growth cone responds to extrinsic guidance cues by coordinating axon outgrowth with growth cone steering. It has become increasingly clear that axon extension requires proper actin polymerization dynamics, whereas growth cone steering involves local protein synthesis. However, molecular components integrating these two processes have not been identified. Here, we show that Down syndrome critical region 1 protein (DSCR1) controls axon outgrowth by modulating growth cone actin dynamics through regulation of cofilin activity (phospho/dephospho-cofilin). Additionally, DSCR1 mediates brain-derived neurotrophic factor–induced local protein synthesis and growth cone turning. Our study identifies DSCR1 as a key protein that couples axon growth and pathfinding by dually regulating actin dynamics and local protein synthesis.     

Differential trafficking of transport vesicles contributes to the localization of dendritic proteins

In neurons, transmembrane proteins are targeted to dendrites in vesicles that traffic solely within the somatodendritic compartment. How these vesicles are retained within the somatodendritic domain is unknown. Here, we use a novel pulse-chase system, which allows synchronous release of exogenous transmembrane proteins from the endoplasmic reticulum to follow movements of post-Golgi transport vesicles. Surprisingly, we found that post-Golgi vesicles carrying dendritic proteins were equally likely to enter axons and dendrites. However, once such vesicles entered the axon, they very rarely moved beyond the axon initial segment but instead either halted or reversed direction in an actin and Myosin Va-dependent manner. In contrast, vesicles carrying either an axonal or a nonspecifically localized protein only rarely halted or reversed and instead generally proceeded to the distal axon. Thus, our results are consistent with the axon initial segment behaving as a vesicle filter that mediates the differential trafficking of transport vesicles.     

Axon initial segment cytoskeleton comprises a multiprotein submembranous coat containing sparse actin filaments

The axon initial segment (AIS) of differentiated neurons regulates action potential initiation and axon–dendritic polarity. The latter function depends on actin dynamics, but actin structure and functions at the AIS remain unclear. Using platinum replica electron microscopy (PREM), we have characterized the architecture of the AIS cytoskeleton in mature and developing hippocampal neurons. The AIS cytoskeleton assembly begins with bundling of microtubules and culminates in formation of a dense, fibrillar–globular coat over microtubule bundles. Immunogold PREM revealed that the coat contains a network of known AIS proteins, including ankyrin G, spectrin βIV, neurofascin, neuronal cell adhesion molecule, voltage-gated sodium channels, and actin filaments. Contrary to existing models, we find neither polarized actin arrays, nor dense actin meshworks in the AIS. Instead, the AIS contains two populations of sparse actin filaments: short, stable filaments and slightly longer dynamic filaments. We propose that stable actin filaments play a structural role for formation of the AIS diffusion barrier, whereas dynamic actin may promote AIS coat remodeling.