Transplantation of dorsal root ganglia overexpressing the NaChBac sodium channel improves locomotion after complete SCI.

Spinal cord injury (SCI) is a debilitating condition currently lacking treatment. Severe SCI causes the loss of most supraspinal inputs and neuronal activity caudal to the injury, which, coupled with the limited endogenous capacity for spontaneous regeneration, can lead to complete functional loss even in anatomically incomplete lesions. We hypothesized that transplantation of mature dorsal root ganglia (DRGs) genetically modified to express the NaChBac sodium channel could serve as a therapeutic option for functionally complete SCI. We found that NaChBac expression increased the intrinsic excitability of DRG neurons and promoted cell survival and neurotrophic factor secretion in vitro. Transplantation of NaChBac-expressing dissociated DRGs improved voluntary locomotion 7 weeks after injury compared to control groups. Animals transplanted with NaChBac-expressing DRGs also possessed higher tubulin-positive neuronal fiber and myelin preservation, although serotonergic descending fibers remained unaffected. We observed early preservation of the corticospinal tract 14 days after injury and transplantation, which was lost 7 weeks after injury. Nevertheless, transplantation of NaChBac-expressing DRGs increased the neuronal excitatory input by an increased number of VGLUT2 contacts immediately caudal to the injury. Our work suggests that the transplantation of NaChBac-expressing dissociated DRGs can rescue significant motor function, retaining an excitatory neuronal relay activity immediately caudal to injury.

Intellectual Disability and Behavioral Deficits Linked to CYFIP1 Missense Variants Disrupting Actin Polymerization.

BACKGROUND: 15q11.2 deletions and duplications have been linked to autism spectrum disorder, schizophrenia, and intellectual disability. Recent evidence suggests that dysfunctional CYFIP1 (cytoplasmic FMR1 interacting protein 1) contributes to the clinical phenotypes observed in individuals with 15q11.2 deletion/duplication syndrome. CYFIP1 plays crucial roles in neuronal development and brain connectivity, promoting actin polymerization and regulating local protein synthesis. However, information about the impact of single nucleotide variants in CYFIP1 on neurodevelopmental disorders is limited. METHODS: Here, we report a family with 2 probands exhibiting intellectual disability, autism spectrum disorder, spastic tetraparesis, and brain morphology defects and who carry biallelic missense point mutations in the CYFIP1 gene. We used skin fibroblasts from one of the probands, the parents, and typically developing individuals to investigate the effect of the variants on the functionality of CYFIP1. In addition, we generated Drosophila knockin mutants to address the effect of the variants in vivo and gain insight into the molecular mechanism that underlies the clinical phenotype. RESULTS: Our study revealed that the 2 missense variants are in protein domains responsible for maintaining the interaction within the wave regulatory complex. Molecular and cellular analyses in skin fibroblasts from one proband showed deficits in actin polymerization. The fly model for these mutations exhibited abnormal brain morphology and F-actin loss and recapitulated the core behavioral symptoms, such as deficits in social interaction and motor coordination. CONCLUSIONS: Our findings suggest that the 2 CYFIP1 variants contribute to the clinical phenotype in the probands that reflects deficits in actin-mediated brain development processes.

The GTPase Rab21 is required for neuronal development and migration in the cerebral cortex.

Development of the mammalian neocortex requires proper inside-out migration of developing cortical neurons from the germinal ventricular zone toward the cortical plate. The mechanics of this migration requires precise coordination of different cellular phenomena including cytoskeleton dynamics, membrane trafficking, and cell adhesion. The small GTPases play a central role in all these events. The small GTPase Rab21 regulates migration and neurite growth in developing neurons. Moreover, regulators and effectors of Rab21 have been implicated in brain pathologies with cortical malformations, suggesting a key function for the Rab21 signaling pathway in cortical development. Mechanistically, it has been posited that Rab21 influences cell migration by controlling the trafficking of endocytic vesicles containing adhesion molecules. However, direct evidence of the participation of Rab21 or its mechanism of action in the regulation of cortical migration is still incomplete. In this study, we demonstrate that Rab21 plays a critical role in the differentiation and migration of pyramidal neurons by regulating the levels of the amyloid precursor protein on the neuronal cell surface. Rab21 loss of function increased the levels of membrane-exposed APP, resulting in impaired cortical neuronal differentiation and migration. These findings further our understanding of the processes governing the development of the cerebral cortex and shed light onto the molecular mechanisms behind cortical development disorders derived from the malfunctioning of Rab21 signaling effectors.

Centrosomal microtubule nucleation regulates radial migration of projection neurons independently of polarization in the developing brain.

Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with in vivo imaging uncovered that dysregulation of centrosomal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regulated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor γ-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in γ-tubulin.

Endothelial PlexinD1 signaling instructs spinal cord vascularization and motor neuron development.

How the vascular and neural compartment cooperate to achieve such a complex and highly specialized structure as the central nervous system is still unclear. Here, we reveal a crosstalk between motor neurons (MNs) and endothelial cells (ECs), necessary for the coordinated development of MNs. By analyzing cell-to-cell interaction profiles of the mouse developing spinal cord, we uncovered semaphorin 3C (Sema3C) and PlexinD1 as a communication axis between MNs and ECs. Using cell-specific knockout mice and in vitro assays, we demonstrate that removal of Sema3C in MNs, or its receptor PlexinD1 in ECs, results in premature and aberrant vascularization of MN columns. Those vascular defects impair MN axon exit from the spinal cord. Impaired PlexinD1 signaling in ECs also causes MN maturation defects at later stages. This study highlights the importance of a timely and spatially controlled communication between MNs and ECs for proper spinal cord development.

Methods for culturing adult CNS neurons reveal a CNS conditioning effect.

Neuronal cultures provide a basis for reductionist insights that rely on molecular and pharmacological manipulation. However, the inability to culture mature adult CNS neurons limits our understanding of adult neuronal physiology. Here, we report methods for culturing adult central nervous system neurons in large numbers and across multiple brain regions for extended time periods. Primary adult neuronal cultures develop polarity; they establish segregated dendritic and axonal compartments, maintain resting membrane potentials, exhibit spontaneous and evoked electrical activity, and form neural networks. Cultured adult neurons isolated from different brain regions such as the hippocampus, cortex, brainstem, and cerebellum exhibit distinct cell morphologies, growth patterns, and spontaneous firing characteristics reflective of their regions of origin. Using adult motor cortex cultures, we identify a CNS "conditioning" effect after spinal cord injury. The ability to culture adult neurons offers a valuable tool for studying basic and therapeutic science of the brain.

An active vesicle priming machinery suppresses axon regeneration upon adult CNS injury.

Axons in the adult mammalian central nervous system fail to regenerate after spinal cord injury. Neurons lose their capacity to regenerate during development, but the intracellular processes underlying this loss are unclear. We found that critical components of the presynaptic active zone prevent axon regeneration in adult mice. Transcriptomic analysis combined with live-cell imaging revealed that adult primary sensory neurons downregulate molecular constituents of the synapse as they acquire the ability to rapidly grow their axons. Pharmacogenetic reduction of neuronal excitability stimulated axon regeneration after adult spinal cord injury. Genetic gain- and loss-of-function experiments uncovered that essential synaptic vesicle priming proteins of the presynaptic active zone, but not clostridial-toxin-sensitive VAMP-family SNARE proteins, inhibit axon regeneration. Systemic administration of Baclofen reduced voltage-dependent Ca2+ influx in primary sensory neurons and promoted their regeneration after spinal cord injury. These findings indicate that functional presynaptic active zones constitute a major barrier to axon regeneration.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures.

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

RhoA drives actin compaction to restrict axon regeneration and astrocyte reactivity after CNS injury.

An inhibitory extracellular milieu and neuron-intrinsic processes prevent axons from regenerating in the adult central nervous system (CNS). Here we show how the two aspects are interwoven. Genetic loss-of-function experiments determine that the small GTPase RhoA relays extracellular inhibitory signals to the cytoskeleton by adapting mechanisms set in place during neuronal polarization. In response to extracellular inhibitors, neuronal RhoA restricts axon regeneration by activating myosin II to compact actin and, thereby, restrain microtubule protrusion. However, astrocytic RhoA restricts injury-induced astrogliosis through myosin II independent of microtubules by activating Yes-activated protein (YAP) signaling. Cell-type-specific deletion in spinal-cord-injured mice shows that neuronal RhoA activation prevents axon regeneration, whereas astrocytic RhoA is beneficial for regenerating axons. These data demonstrate how extracellular inhibitors regulate axon regeneration, shed light on the capacity of reactive astrocytes to be growth inhibitory after CNS injury, and reveal cell-specific RhoA targeting as a promising therapeutic avenue.

RhoA Controls Axon Extension Independent of Specification in the Developing Brain.

The specification of an axon and its subsequent outgrowth are key steps during neuronal polarization, a prerequisite to wire the brain. The Rho-guanosine triphosphatase (GTPase) RhoA is believed to be a central player in these processes. However, its physiological role has remained undefined. Here, genetic loss- and gain-of-function experiments combined with time-lapse microscopy, cell culture, and in vivo analysis show that RhoA is not involved in axon specification but confines the initiation of neuronal polarization and axon outgrowth during development. Biochemical analysis and super-resolution microscopy together with molecular and pharmacological manipulations reveal that RhoA restrains axon growth by activating myosin-II-mediated actin arc formation in the growth cone to prevent microtubules from protruding toward the leading edge. Through this mechanism, RhoA regulates the duration of axon growth and pause phases, thus controlling the tightly timed extension of developing axons. Thereby, this work unravels physiologically relevant players coordinating actin-microtubule interactions during axon growth.

ADF/Cofilin-Mediated Actin Turnover Promotes Axon Regeneration in the Adult CNS.

Injured axons fail to regenerate in the adult CNS, which contrasts with their vigorous growth during embryonic development. We explored the potential of re-initiating axon extension after injury by reactivating the molecular mechanisms that drive morphogenetic transformation of neurons during development. Genetic loss- and gain-of-function experiments followed by time-lapse microscopy, in vivo imaging, and whole-mount analysis show that axon regeneration is fueled by elevated actin turnover. Actin depolymerizing factor (ADF)/cofilin controls actin turnover to sustain axon regeneration after spinal cord injury through its actin-severing activity. This pinpoints ADF/cofilin as a key regulator of axon growth competence, irrespective of developmental stage. These findings reveal the central role of actin dynamics regulation in this process and elucidate a core mechanism underlying axon growth after CNS trauma. Thereby, neurons maintain the capacity to stimulate developmental programs during adult life, expanding their potential for plasticity. Thus, actin turnover is a key process for future regenerative interventions.

The physiological role of the amyloid precursor protein as an adhesion molecule in the developing nervous system.

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein better known for its participation in the physiopathology of Alzheimer disease as the source of the beta amyloid fragment. However, the physiological functions of the full length protein and its proteolytic fragments have remained elusive. APP was first described as a cell-surface receptor; nevertheless, increasing evidence highlighted APP as a cell adhesion molecule. In this review, we will focus on the current knowledge of the physiological role of APP as a cell adhesion molecule and its involvement in key events of neuronal development, such as migration, neurite outgrowth, growth cone pathfinding, and synaptogenesis. Finally, since APP is over-expressed in Down syndrome individuals because of the extra copy of chromosome 21, in the last section of the review, we discuss the potential contribution of APP to the neuronal and synaptic defects described in this genetic condition. Read the Editorial Highlight for this article on page 9. Cover Image for this issue: doi. 10.1111/jnc.13817.

The Calcium Channel Subunit Alpha2delta2 Suppresses Axon Regeneration in the Adult CNS.

Injuries to the adult CNS often result in permanent disabilities because neurons lose the ability to regenerate their axon during development. Here, whole transcriptome sequencing and bioinformatics analysis followed by gain- and loss-of-function experiments identified Cacna2d2, the gene encoding the Alpha2delta2 subunit of voltage-gated calcium channels (VGCCs), as a developmental switch that limits axon growth and regeneration. Cacna2d2 gene deletion or silencing promoted axon growth in vitro. In vivo, Alpha2delta2 pharmacological blockade through Pregabalin (PGB) administration enhanced axon regeneration in adult mice after spinal cord injury (SCI). As PGB is already an established treatment for a wide range of neurological disorders, our findings suggest that targeting Alpha2delta2 may be a novel treatment strategy to promote structural plasticity and regeneration following CNS trauma.

Axonal regeneration. Systemic administration of epothilone B promotes axon regeneration after spinal cord injury.

After central nervous system (CNS) injury, inhibitory factors in the lesion scar and poor axon growth potential prevent axon regeneration. Microtubule stabilization reduces scarring and promotes axon growth. However, the cellular mechanisms of this dual effect remain unclear. Here, delayed systemic administration of a blood-brain barrier-permeable microtubule-stabilizing drug, epothilone B (epoB), decreased scarring after rodent spinal cord injury (SCI) by abrogating polarization and directed migration of scar-forming fibroblasts. Conversely, epothilone B reactivated neuronal polarization by inducing concerted microtubule polymerization into the axon tip, which propelled axon growth through an inhibitory environment. Together, these drug-elicited effects promoted axon regeneration and improved motor function after SCI. With recent clinical approval, epothilones hold promise for clinical use after CNS injury.

Wingless-type family member 3A triggers neuronal polarization via cross-activation of the insulin-like growth factor-1 receptor pathway.

Initial axonal elongation is essential for neuronal polarization and requires polarized activation of IGF-1 receptors (IGF-1r) and the phosphatidylinositol 3 kinase (PI3k) pathway. Wingless-type family growth factors (Wnts) have also been implied in the regulation of axonal development. It is not known, however, if Wnts have any participation in the regulation of initial axonal outgrowth and the establishment of neuronal polarity. We used cultured hippocampal neurons and growth cone particles (GCPs) isolated from fetal rat brain to show that stimulation with the wingless family factor 3A (Wnt3a) was sufficient to promote neuronal polarization in the absence of IGF-1 or high insulin. We also show that Wnt3a triggered a strong activation of IGF-1r, PI3k, and Akt in developmental Stage 2 neurons and that the presence of activatable IGF-1r and PI3k activation were necessary for Wnt3a polarizing effects. Surface plasmon resonance (SPR) experiments show that Wnt3a did not bind specifically to the IGF-1r. Using crosslinking and immuno-precipitation experiments, we show that stimulation with Wnt3a triggered the formation of a complex including IGF-1r-Wnt3a-Frizzled-7. We conclude that Wnt3a triggers polarization of neurons via cross-activation of the IGF-1r/PI3k pathway upon binding to Fz7.

The insulin-like growth factor 1 receptor is essential for axonal regeneration in adult central nervous system neurons.

Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the ßgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both ßgc and the ß subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, ßgc was localised to distal regions and leading growth cones in RGC. IGF-1R-ßgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor LY294002, all significantly reduced axon regeneration from adult RGC in vitro (∼40% RGC possessed axons in controls vs 2-8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma.

ADF/cofilin-mediated actin retrograde flow directs neurite formation in the developing brain.

Neurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons.

The TC10-Exo70 complex is essential for membrane expansion and axonal specification in developing neurons.

Axonal elongation is one of the hallmarks of neuronal polarization. This phenomenon requires axonal membrane growth by exocytosis of plasmalemmal precursor vesicles (PPVs) at the nerve growth cone, a process regulated by IGF-1 activation of the PI3K (phosphatidylinositol-3 kinase) pathway. Few details are known, however, about the targeting mechanisms for PPVs. Here, we show, in cultured hippocampal pyramidal neurons and growth cones isolated from fetal rat brain, that IGF-1 activates the GTP-binding protein TC10, which triggers translocation to the plasma membrane of the exocyst component exo70 in the distal axon and growth cone. We also show that TC10 and exo70 function are necessary for addition of new membrane and, thus, axon elongation stimulated by IGF-1. Moreover, expression silencing of either TC10 or exo70 inhibit the establishment of neuronal polarity by hindering the insertion of IGF-1 receptor in one of the undifferentiated neurites. We conclude that, in hippocampal pyramidal neurons in culture, (1) membrane expansion at the axonal growth cone is regulated by IGF-1 via a cascade involving TC10 and the exocyst complex, (2) TC10 and exo70 are essential for the polarized externalization of IGF-1 receptor, and (3) this process is necessary for axon specification.

IGF-1 receptor is essential for the establishment of hippocampal neuronal polarity.

How a neuron becomes polarized remains largely unknown. Results obtained with a function-blocking antibody and an siRNA targeting the insulin-like growth factor-1 (IGF-1) receptor suggest that an essential step in the establishment of hippocampal neuronal polarity and the initiation of axonal outgrowth is the activation of the phosphatidylinositol 3-kinase (PI3k)-Cdc42 pathway by the IGF-1 receptor, but not by the TrkA or TrkB receptors.

PI3K activation by IGF-1 is essential for the regulation of membrane expansion at the nerve growth cone.

Exocytotic incorporation of plasmalemmal precursor vesicles (PPVs) into the cell surface is necessary for axonal outgrowth and is known to occur mainly at the nerve growth cone. We have demonstrated recently that plasmalemmal expansion is regulated at the growth cone by IGF-1, but not by BDNF, in a manner that is quasi independent of the neuron's perikaryon. To begin elucidating the signaling pathway by which exocytosis of the plasmalemmal precursor is regulated, we studied activation of the IRS/PI3K/Akt pathway in isolated growth cones and hippocampal neurons in culture stimulated with IGF-1 or BDNF. Our results show that IGF-1, but not BDNF, significantly and rapidly stimulates IRS/PI3K/Akt and membrane expansion. Inhibition of PI3K with Wortmannin or LY294002 blocked IGF-1-stimulated plasmalemmal expansion at the growth cones of cultured neurons. Finally, our results show that, upon stimulation with IGF-1, most active PI3K becomes associated with distal microtubules in the proximal or central domain of the growth cone. Taken together, our results suggest a critical role for IGF-1 and the IRS/PI3K/Akt pathway in the process of membrane assembly at the axonal growth cone.