Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells.

Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration.

Axonal growth on surfaces with periodic geometrical patterns.

The formation of neuron networks is a complex phenomenon of fundamental importance for understanding the development of the nervous system, and for creating novel bioinspired materials for tissue engineering and neuronal repair. The basic process underlying the network formation is axonal growth, a process involving the extension of axons from the cell body towards target neurons. Axonal growth is guided by environmental stimuli that include intercellular interactions, biochemical cues, and the mechanical and geometrical features of the growth substrate. The dynamics of the growing axon and its biomechanical interactions with the growing substrate remains poorly understood. In this paper, we develop a model of axonal motility which incorporates mechanical interactions between the axon and the growth substrate. We combine experimental data with theoretical analysis to measure the parameters that describe axonal growth on surfaces with micropatterned periodic geometrical features: diffusion (cell motility) coefficients, speed and angular distributions, and axon bending rigidities. Experiments performed on neurons treated Taxol (inhibitor of microtubule dynamics) and Blebbistatin (disruptor of actin filaments) show that the dynamics of the cytoskeleton plays a critical role in the axon steering mechanism. Our results demonstrate that axons follow geometrical patterns through a contact-guidance mechanism, in which high-curvature geometrical features impart high traction forces to the growth cone. These results have important implications for our fundamental understanding of axonal growth as well as for bioengineering novel substrates that promote neuronal growth and nerve repair.

Vinaxanthone inhibits Semaphorin3A induced axonal growth cone collapse in embryonic neurons but fails to block its growth promoting effects on adult neurons.

Semaphorin3A is considered a classical repellent molecule for developing neurons and a potent inhibitor of regeneration after nervous system trauma. Vinaxanthone and other Sema3A inhibitors are currently being tested as possible therapeutics to promote nervous system regeneration from injury. Our previous study on Sema3A demonstrated a switch in Sema3A's function toward induction of nerve regeneration in adult murine corneas and in culture of adult peripheral neurons. The aim of the current study is to determine the direct effects of Vinaxanthone on the Sema3A induced adult neuronal growth. We first demonstrate that Vinaxanthone maintains its anti-Sema3A activity in embryonic dorsal root ganglia neurons by inhibiting Sema3A-induced growth cone collapse. However, at concentrations approximating its IC50 Vinaxanthone treatment does not significantly inhibit neurite formation of adult peripheral neurons induced by Sema3A treatment. Furthermore, Vinaxanthone has off target effects when used at concentrations above its IC50, and inhibits neurite growth of adult neurons treated with either Sema3A or NGF. Our results suggest that Vinaxanthone's pro-regenerative effects seen in multiple in vivo models of neuronal injury in adult animals need further investigation due to the pleiotropic effect of Sema3A on various non-neuronal cell types and the possible effect of Vinaxanthone on other neuroregenerative signals.

A monolayer hiPSC culture system for autophagy/mitophagy studies in human dopaminergic neurons.

Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions in vitro. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) - the predominant neuronal sub-type afflicted in PD - have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.Abbreviations: AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4',6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco's modified eagle's medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.

Serotonin functions as a bidirectional guidance molecule regulating growth cone motility.

The neurotransmitter serotonin has been implicated in a range of complex neurological disorders linked to alterations of neuronal circuitry. Serotonin is synthesized in the developing brain before most neuronal circuits become fully functional, suggesting that serotonin might play a distinct regulatory role in shaping circuits prior to its function as a classical neurotransmitter. In this study, we asked if serotonin acts as a guidance cue by examining how serotonin alters growth cone motility of rodent sensory neurons in vitro. Using a growth cone motility assay, we found that serotonin acted as both an attractive and repulsive guidance cue through a narrow concentration range. Extracellular gradients of 50 µM serotonin elicited attraction, mediated by the serotonin 5-HT2a receptor while 100 µM serotonin elicited repulsion mediated by the 5-HT1b receptor. Importantly, high resolution imaging of growth cones indicated that these receptors signalled through their canonical pathways of endoplasmic reticulum-mediated calcium release and cAMP depletion, respectively. This novel characterisation of growth cone motility in response to serotonin gradients provides compelling evidence that secreted serotonin acts at the molecular level as an axon guidance cue to shape neuronal circuit formation during development.

Covering the proximal nerve stump with chondroitin sulfate proteoglycans prevents traumatic painful neuroma formation by blocking axon regeneration after neurotomy in Sprague Dawley rats.

OBJECTIVE: Neuropathic pain caused by traumatic neuromas is an extremely intractable clinical problem. Disorderly scar tissue accumulation and irregular and immature axon regeneration around the injury site mainly contribute to traumatic painful neuroma formation. Therefore, successfully preventing traumatic painful neuroma formation requires the effective inhibition of irregular axon regeneration and disorderly accumulation of scar tissue. Considering that chondroitin sulfate proteoglycans (CSPGs) can act on the growth cone and effectively inhibit axon regeneration, the authors designed and manufactured a CSPG-gelatin blocker to regulate the CSPGs' spatial distribution artificially and applied it in a rat model after sciatic nerve neurectomy to evaluate its effects in preventing traumatic painful neuroma formation. METHODS: Sixty female Sprague Dawley rats were randomly divided into three groups (positive group: no covering; blank group: covering with gelatin blocker; and CSPG group: covering with the CSPG-gelatin blocker). Pain-related factors were evaluated 2 and 8 weeks postoperatively (n = 30). Neuroma growth, autotomy behavior, and histological features of the neuromas were assessed 8 weeks postoperatively (n = 30). RESULTS: Eight weeks postoperatively, typical bulb-shaped neuromas did not form in the CSPG group, and autotomy behavior was obviously better in the CSPG group (p < 0.01) than in the other two groups. Also, in the CSPG group the regenerated axons showed a lower density and more regular and improved myelination (p < 0.01). Additionally, the distribution and density of collagenous fibers and the expression of α-smooth muscle actin were significantly lower in the CSPG group than in the positive group (p < 0.01). Regarding pain-related factors, c-fos, substance P, interleukin (IL)-17, and IL-1ß levels were significantly lower in the CSPG group than those in the positive and blank groups 2 weeks postoperatively (p < 0.05), while substance P and IL-17 remained lower in the CSPG group 8 weeks postoperatively (p < 0.05). CONCLUSIONS: The authors found that CSPGs loaded in a gelatin blocker can prevent traumatic neuroma formation and effectively relieve pain symptoms after sciatic nerve neurotomy by blocking irregular axon regeneration and disorderly collagenous fiber accumulation in the proximal nerve stump. These results indicate that covering the proximal nerve stump with CSPGs may be a new and promising strategy to prevent traumatic painful neuroma formation in the clinical setting.

Evaluating the effectiveness of anti-Nogo treatment in spinal cord injuries.

As humans, we cannot regenerate axons within the central nervous system (CNS), therefore, making any damage to it permanent. This leads to the loss of sensory and motor function below the site of injury and can be crippling to a person's health. Spontaneous recovery can occur from plastic changes, but it is minimal. The absence of regeneration is due to the inhibitory environment of the CNS as well as the inherent inability of CNS axons to form growth cones. Amongst many factors, one of the major inhibitory signals of the CNS environment is the myelin-associated Nogo pathway. Nogo-A, Nogo-B and Nogo-C (Nogo), stimulate the Nogo receptor, inhibiting neurite outgrowth by causing growth cones to collapse through activation of Rho Kinase (ROCK). Antibodies can be used to target this signalling pathway by binding to Nogo and thus promote the outgrowth of neuronal axons in the CNS. This use of anti-Nogo antibodies has been shown to upregulate CNS regeneration as well as drastically improve sensory and motor function in both rats and primates when coupled with adequate training. Here, we evaluate whether the experimental success of anti-Nogo at improving CNS regeneration can be carried over into the clinical setting to treat spinal cord injuries (SCI) and their symptoms successfully. Furthermore, we also discuss potential methods to improve the current treatment and any developmental obstacles.

Pregnenolone and pregnenolone-methyl-ether rescue neuronal defects caused by dysfunctional CLIP170 in a neuronal model of CDKL5 Deficiency Disorder.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene are responsible for the onset of CDKL5 Deficiency Disorder (CDD), a neurological pathology characterised by severe infantile seizures, intellectual disability, impairment of gross motor skills, sleep and gastrointestinal disturbances. CDKL5 is a serine/threonine kinase the molecular network of which is not yet fully understood. Loss of CDKL5 both in vitro and in vivo leads to altered neuronal morphology including axon specification and outgrowth, dendritic arborisation and spine morphology suggesting a link between CDKL5 and the regulation of proper cytoskeleton functioning. Recently, we found that CDKL5 regulates the binding of CLIP170 to microtubules (MT). CLIP170 is a MT-plus end tracking protein (+TIP) that associates with MTs when present in its open, active conformation. Here we present evidence suggesting CLIP170 contributes to neuronal CDKL5-dependent defects and that it represents an important novel druggable target for CDD; indeed, CLIP170 is directly targeted by the neuroactive steroid pregnenolone (PREG), which induces the active conformation of the protein thus promoting MT-dynamics. We here show that PREG and a synthetic derivative pregnenolone-methyl-ether (PME) can restore the MT association of CLIP170 and revert morphological and molecular defects in Cdkl5-KO neurons at different stages of maturation. All together, these findings identify CLIP170 as possible novel druggable target for CDKL5 related disorders providing an intriguing prospective for future disease-modifying drug-based therapies.

Inhibition of Rho GTPases in Invertebrate Growth Cones Induces a Switch in Responsiveness to Retinoic Acid.

During development, growth cones are essential for axon pathfinding by sensing numerous guidance cues in their environment. Retinoic acid, the metabolite of vitamin A, is important for neurite outgrowth during vertebrate development, but may also play a role in axon guidance, though little is known of the cellular mechanisms involved. Our previous studies showed that retinoid-induced growth cone turning of invertebrate motorneurons requires local protein synthesis and calcium influx. However, the signalling pathways that link calcium influx to cytoskeletal dynamics involved in retinoid-mediated growth cone turning are not currently known. The Rho GTPases, Cdc42 and Rac, are known regulators of the growth cone cytoskeleton. Here, we demonstrated that inhibition of Cdc42 or Rac not only prevented growth cone turning toward retinoic acid but could also induce a switch in growth cone responsiveness to chemorepulsion or growth cone collapse. However, the effects of Cdc42 or Rac inhibition on growth cone responsiveness differed, depending on whether the turning was induced by the all-trans or 9-cis retinoid isomer. The effects also differed depending on whether the growth cones maintained communication with the cell body. These data strongly suggest that Cdc42 and Rac are downstream effectors of retinoic acid during growth cone guidance.

The Roles of Microtubules and Membrane Tension in Axonal Beading, Retraction, and Atrophy.

Axonal beading, or the formation of a series of swellings along the axon, and retraction are commonly observed shape transformations that precede axonal atrophy in Alzheimer's disease, Parkinson's disease, and other neurodegenerative conditions. The mechanisms driving these morphological transformations are poorly understood. Here, we report controlled experiments that can induce either beading or retraction and follow the time evolution of these responses. By making quantitative analysis of the shape modes under different conditions, measurement of membrane tension, and using theoretical considerations, we argue that membrane tension is the main driving force that pushes cytosol out of the axon when microtubules are degraded, causing axonal thinning. Under pharmacological perturbation, atrophy is always retrograde, and this is set by a gradient in the microtubule stability. The nature of microtubule depolymerization dictates the type of shape transformation, vis-à-vis beading or retraction. Elucidating the mechanisms of these shape transformations may facilitate development of strategies to prevent or arrest axonal atrophy due to neurodegenerative conditions.

Anesthetics disrupt growth cone guidance cue sensing through actions on the GABAA α2 receptor mediated by the immature chloride gradient.

BACKGROUND: General anesthetics (GAs) may exert harmful effects on the developing brain by disrupting neuronal circuit formation. Anesthetics that act on γ-aminobutyric acid (GABA) receptors can interfere with axonal growth cone guidance, a critical process in the assembly of neuronal circuitry. Here we investigate the mechanism by which isoflurane prevents sensing of the repulsive guidance cue, Semaphorin 3A (Sema3A). METHODS: Growth cone sensing was assayed by measuring growth cone collapse in dissociated neocortical cultures exposed to recombinant Sema3A in the presence or absence of isoflurane and/or a panel of reagents with specific actions on components of the GABA receptor and chloride ion systems. RESULTS: Isoflurane exposure prevents Sema3A induced growth cone collapse. A GABAA α2 specific agonist replicates this effect (36.83 ±â€¯3.417% vs 70.82 ±â€¯2.941%, in the Sema3A induced control group, p < 0.0001), but an α1-specific agonist does not. Both a Na-K-Cl cotransporter 1 antagonism (bumetanide, BUM) and a chloride ionophore (IONO) prevent isoflurane from disrupting growth cone sensing of Sema3A. (65.67 ±â€¯3.775% in Iso + BUM group vs 67.45 ±â€¯3.624% in Sema3A induced control group, 65.34 ±â€¯1.678% in Iso + IONO group vs 68.71 ±â€¯2.071% in Sema3A induced control group, no significant difference) (n = 96 growth cones per group). CONCLUSION: Our data suggest that the effects of isoflurane on growth cone sensing are mediated by the α2 subunit of the GABAA receptor and also that they are dependent on the developmental chloride gradient, in which Cl- exhibits a depolarizing effect. These findings provide a rationale for why immature neurons are particularly susceptible to anesthetic toxicity.

Comparing the effects of uncoated nanostructured surfaces on primary neurons and astrocytes.

The long-term application of central nervous system implants is currently limited by the negative response of the brain tissue, affecting both the performance of the device and the survival of nearby cells. Topographical modification of implant surfaces mimicking the structure and dimensions of the extracellular matrix may provide a solution to this negative tissue response and has been shown to affect the attachment and behavior of both neurons and astrocytes. In our study, commonly used neural implant materials, silicon, and platinum were tested with or without nanoscale surface modifications. No biological coatings were used in order to only examine the effect of the nanostructuring. We seeded primary mouse astrocytes and hippocampal neurons onto four different surfaces: flat polysilicon, nanostructured polysilicon, and platinum-coated versions of these surfaces. Fluorescent wide-field, confocal, and scanning electron microscopy were used to characterize the attachment, spreading and proliferation of these cell types. In case of astrocytes, we found that both cell number and average cell spreading was significantly larger on platinum, compared to silicon surfaces, while silicon surfaces impeded glial proliferation. Nanostructuring did not have a significant effect on either parameter in astrocytes but influenced the orientation of actin filaments and glial fibrillary acidic protein fibers. Neuronal soma attachment was impaired on metal surfaces while nanostructuring seemed to influence neuronal growth cone morphology, regardless of surface material. Taken together, the type of metals tested had a profound influence on cellular responses, which was only slightly modified by nanopatterning.

Pharmacological evidence for the role of RAR in axon guidance and embryonic development of a protostome species.

Retinoic acid (RA), the active metabolite of vitamin A, functions through nuclear receptors, one of which is the retinoic acid receptor (RAR). Though the RAR is essential for various aspects of vertebrate development, little is known about the role of RAR in nonchordate invertebrates. Here, we examined the potential role of an invertebrate RAR in mediating chemotropic effects of retinoic acid. The RAR of the protostome Lymnaea stagnalis is present in the growth cones of regenerating cultured motorneurons, and a synthetic RAR agonist (EC23), was able to mimic the effects of retinoic acid in inducing growth cone turning. We also examined the ability of the natural retinoids, all-trans RA and 9-cis RA, as well as the synthetic RAR agonists, to disrupt embryonic development in Lymnaea. Developmental defects included delays in embryo hatching, arrested eye, and shell development, as well as more severe abnormalities such as halted development. Developmental defects induced by some (but not all) synthetic RAR agonists were found to mimic those induced by addition of high concentrations of the natural retinoid isomers. These pharmacological data support a possible physiological role for the RAR in axon guidance and embryonic development of an invertebrate protostome species.

Visualizing Axonal Growth Cone Collapse and Early Amyloid ß Effects in Cultured Mouse Neurons.

Amyloid-ß (Aß) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce Aß levels in the brains of AD patients, these do not improve memory functions. Since Aß aggregates in the brain before the appearance of memory impairments, targeting Aß may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to Aß deposition should be blocked before AD development. Aß induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of Aß toxicity, the source of Aß toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by Aß in axonal growth cones of cultured neurons. This protocol revealed that Aß induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents Aß toxicity. This protocol will be useful in studying the early effects of Aß and may lead to more efficient and preventative AD treatment.

Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System.

Retinoic acid (RA) is the biologically active metabolite of vitamin A and has become a well-established factor that induces neurite outgrowth and regeneration in both vertebrates and invertebrates. However, the underlying regulatory mechanisms that may mediate RA-induced neurite sprouting remain unclear. In the past decade, microRNAs have emerged as important regulators of nervous system development and regeneration, and have been shown to contribute to processes such as neurite sprouting. However, few studies have demonstrated the role of miRNAs in RA-induced neurite sprouting. By miRNA sequencing analysis, we identify 482 miRNAs in the regenerating central nervous system (CNS) of the mollusc Lymnaeastagnalis, 219 of which represent potentially novel miRNAs. Of the remaining conserved miRNAs, 38 show a statistically significant up- or downregulation in regenerating CNS as a result of RA treatment. We further characterized the expression of one neuronally-enriched miRNA upregulated by RA, miR-124. We demonstrate, for the first time, that miR-124 is expressed within the cell bodies and neurites of regenerating motorneurons. Moreover, we identify miR-124 expression within the growth cones of cultured ciliary motorneurons (pedal A), whereas expression in the growth cones of another class of respiratory motorneurons (right parietal A) was absent in vitro. These findings support our hypothesis that miRNAs are important regulators of retinoic acid-induced neuronal outgrowth and regeneration in regeneration-competent species.

Polarized Dock Activity Drives Shh-Mediated Axon Guidance.

In the developing spinal cord, Sonic hedgehog (Shh) attracts commissural axons toward the floorplate. How Shh regulates the cytoskeletal remodeling that underlies growth cone turning is unknown. We found that Shh-mediated growth cone turning requires the activity of Docks, which are unconventional GEFs. Knockdown of Dock3 and 4, or their binding partner ELMO1 and 2, abolished commissural axon attraction by Shh in vitro. Dock3/4 and ELMO1/2 were also required for correct commissural axon guidance in vivo. Polarized Dock activity was sufficient to induce axon turning, indicating that Docks are instructive for axon guidance. Mechanistically, we show that Dock and ELMO interact with Boc, the Shh receptor, and that this interaction is reduced upon Shh stimulation. Furthermore, Shh stimulation translocates ELMO to the growth cone periphery and activates Rac1. This identifies Dock/ELMO as an effector complex of non-canonical Shh signaling and demonstrates the instructive role of GEFs in axon guidance.

Alpha 7 nicotinic receptors attenuate neurite development through calcium activation of calpain at the growth cone.

The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that plays an important role in cellular calcium signaling contributing to synaptic development and plasticity, and is a key drug target for the treatment of neurodegenerative conditions such as Alzheimer's disease. Here we show that α7 nAChR mediated calcium signals in differentiating PC12 cells activate the proteolytic enzyme calpain leading to spectrin breakdown, microtubule retraction, and attenuation in neurite growth. Imaging in growth cones confirms that α7 activation decreases EB3 comet motility in a calcium dependent manner as demonstrated by the ability of α7 nAChR, ryanodine, or IP3 receptor antagonists to block the effect of α7 nAChR on growth. α7 nAChR mediated EB3 comet motility, spectrin breakdown, and neurite growth was also inhibited by the addition of the selective calpain blocker calpeptin and attenuated by the expression of an α7 subunit unable to bind Gαq and activate calcium store release. The findings indicate that α7 nAChRs regulate cytoskeletal dynamics through local calcium signals for calpain protease activity.

Inhibition of RhoA reduces propofol-mediated growth cone collapse, axonal transport impairment, loss of synaptic connectivity, and behavioural deficits.

BACKGROUND: Exposure of the developing brain to propofol results in cognitive deficits. Recent data suggest that inhibition of neuronal apoptosis does not prevent cognitive defects, suggesting mechanisms other than neuronal apoptosis play a role in anaesthetic neurotoxicity. Proper neuronal growth during development is dependent upon growth cone morphology and axonal transport. Propofol modulates actin dynamics in developing neurones, causes RhoA-dependent depolymerisation of actin, and reduces dendritic spines and synapses. We hypothesised that RhoA inhibition prevents synaptic loss and subsequent cognitive deficits. The present study tested whether RhoA inhibition with the botulinum toxin C3 (TAT-C3) prevents propofol-induced synapse and neurite loss, and preserves cognitive function. METHODS: RhoA activation, growth cone morphology, and axonal transport were measured in neonatal rat neurones (5-7 days in vitro) exposed to propofol. Synapse counts (electron microscopy), dendritic arborisation (Golgi-Cox), and network connectivity were measured in mice (age 28 days) previously exposed to propofol at postnatal day 5-7. Memory was assessed in adult mice (age 3 months) previously exposed to propofol at postnatal day 5-7. RESULTS: Propofol increased RhoA activation, collapsed growth cones, and impaired retrograde axonal transport of quantum dot-labelled brain-derived neurotrophic factor, all of which were prevented with TAT-C3. Adult mice previously treated with propofol had decreased numbers of total hippocampal synapses and presynaptic vesicles, reduced hippocampal dendritic arborisation, and infrapyramidal mossy fibres. These mice also exhibited decreased hippocampal-dependent contextual fear memory recall. All anatomical and behavioural changes were prevented with TAT-C3 pre-treatment. CONCLUSION: Inhibition of RhoA prevents propofol-mediated hippocampal neurotoxicity and associated cognitive deficits.

The Soluble Form of LOTUS inhibits Nogo Receptor-Mediated Signaling by Interfering with the Interaction Between Nogo Receptor Type 1 and p75 Neurotrophin Receptor.

Nogo receptor type 1 (NgR1) is known to inhibit neuronal regeneration in the CNS. Previously, we have shown that lateral olfactory tract usher substance (LOTUS) interacts with NgR1 and inhibits its function by blocking its ligand binding. Therefore, LOTUS is expected to have therapeutic potential for the promotion of neuronal regeneration. However, it remains unknown whether the soluble form of LOTUS (s-LOTUS) also has an inhibitory action on NgR1 function as a candidate for therapeutic agents. Here, we show that s-LOTUS inhibits NgR1-mediated signaling by inhibiting the molecular interaction between NgR1 and its coreceptor, p75 neurotrophin receptor (p75NTR). In contrast to the membrane-bound form of LOTUS, s-LOTUS did not block ligand binding to NgR1. However, we identified p75NTR as a novel LOTUS binding partner and found that s-LOTUS suppressed the interaction between p75NTR and NgR1. s-LOTUS inhibited myelin-associated inhibitor (MAI)-induced RhoA activation in murine cortical neurons. Functional analyses revealed that s-LOTUS inhibited MAI-induced growth cone collapse and neurite outgrowth inhibition in chick DRG neurons. In addition, whereas olfactory bulb neurons of lotus-KO mice are sensitive to MAI due to a lack of LOTUS expression, treatment with s-LOTUS inhibited MAI-induced growth cone collapse in these neurons. Finally, we observed that s-LOTUS promoted axonal regeneration in optic nerve crush injury of mice (either sex). These findings suggest that s-LOTUS inhibits NgR1-mediated signaling, possibly by interfering with the interaction between NgR1 and p75NTR Therefore, s-LOTUS may have potential as a therapeutic agent for neuronal regeneration in the damaged CNS.SIGNIFICANCE STATEMENT Nogo receptor type 1 (NgR1) is a receptor well known to inhibit neuronal regeneration in the CNS. Because the membrane-bound form of lateral olfactory tract usher substance (LOTUS) antagonizes NgR1 through a cis-type molecular interaction between LOTUS and NgR1, the soluble form of LOTUS (s-LOTUS) is expected to be a therapeutic agent for neuronal regeneration. In our present study, we show that s-LOTUS inhibits the interaction between NgR1 and p75NTR, NgR1 ligand-induced RhoA activation, growth cone collapse, and neurite outgrowth inhibition and promotes axonal regeneration. Our results indicate that s-LOTUS inhibits NgR1-mediated signaling through a trans-type molecular interaction between LOTUS and NgR1 and, therefore, s-LOTUS may have therapeutic potential for neuronal regeneration.

VEGF Triggers the Activation of Cofilin and the Arp2/3 Complex within the Growth Cone.

A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor (VEGF), which was identified as a positive effector for growth cone movement. These positive effects are mainly mediated by a reorganization of the actin network. This study shows that VEGF triggers a tight colocalization of cofilin and the Arp2/3 complex to the actin cytoskeleton within chicken dorsal root ganglia (DRG). Live cell imaging after microinjection of GFP (green fluorescent protein)-cofilin and RFP (red fluorescent protein)-LifeAct revealed that both labeled proteins rapidly redistributed within growth cones, and showed a congruent distribution pattern after VEGF supplementation. Disruption of signaling upstream of cofilin via blocking LIM-kinase (LIMK) activity resulted in growth cones displaying regressive growth behavior. Microinjection of GFP-p16b (a subunit of the Arp2/3 complex) and RFP-LifeAct revealed that both proteins redistributed into lamellipodia of the growth cone within minutes after VEGF stimulation. Disruption of the signaling to the Arp2/3 complex in the presence of VEGF by inhibition of N-WASP (neuronal Wiskott-Aldrich-Scott protein) caused retraction of growth cones. Hence, cofilin and the Arp2/3 complex appear to be downstream effector proteins of VEGF signaling to the actin cytoskeleton of DRG growth cones. Our data suggest that VEGF simultaneously affects different pathways for signaling to the actin cytoskeleton, since activation of cofilin occurs via inhibition of LIMK, whereas activation of Arp2/3 is achieved by stimulation of N-WASP.