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How dystrophic neurites form around amyloid plaques is a key aspect of understanding the early pathophysiology of Alzheimer's disease. At present, three hypotheses prevail: (1) dystrophies result from extracellular amyloid-beta (Aβ) toxicity; (2) dystrophies results from accumulation of Aβ into distal neurites; and (3) dystrophies represent blebbing of the somatic membrane of a neuron with high Aβ load. We utilized a unique feature of the common 5xFAD AD mouse model to test these hypotheses. Cortical layer 5 pyramidal neurons show intracellular APP and Aβ accumulation before amyloid plaque formation while dentate granule cells in these mice show no APP accumulation at any age. However, the dentate gyrus shows amyloid plaques by 3 months of age. By a careful confocal microscopic analysis we found no evidence of severe degeneration in amyloid laden layer 5 pyramidal neurons in contrast to hypothesis 3. Using injecting red fluorescent marker into lateral entorhinal projection neurons in 5xFAD mice with endogenous green fluorescent protein (GFP) in dentate granule cells we could demonstrate that all dystrophies is outer molecular layer originate from the axon terminal of entorhinal projection neurons. Immunostaining with vesicular glutamate transporter supported the axonal nature of the dystrophies in the acellular dentate molecular layer. We observed few small dystrophies in the GFP labeled granule cell dendrites. In general GFP labeled dendrites appear normal around the amyloid plaques. These findings favor hypothesis 2 as the most likely mechanism of dystrophic neurite formation., (Copyright © 2023. Published by Elsevier Inc.)

The concept of spatial confinement is the basis of cell positioning and guidance in in vitro studies. In vivo, it reflects many situations faced during embryonic development. In vitro, spatial confinement of neurons is achieved using different technological approaches: adhesive patterning, topographical structuring, microfluidics and the use of hydrogels. The notion of chemical or physical frontiers is particularly central to the behaviors of growth cones and neuronal processes under confinement. They encompass phenomena of cell spreading, boundary crossing, and path finding on surfaces with different adhesive properties. However, the most universal phenomenon related to confinement, regardless of how it is implemented, is the acceleration of neuronal growth. Overall, a bi-directional causal link emerges between the shape of the growth cone and neuronal elongation dynamics, both in vivo and in vitro. The sensing of adhesion discontinuities by filopodia and the subsequent spatial redistribution and size adaptation of these actin-rich filaments seem critical for the growth rate in conditions in which adhesive contacts and actin-associated clutching forces dominate. On the other hand, the involvement of microtubules, specifically demonstrated in 3D hydrogel environments and leading to ameboid-like locomotion, could be relevant in a wider range of growth situations. This review brings together a literature collected in distinct scientific fields such as development, mechanobiology and bioengineering that highlight the consequences of confinement and raise new questions at different cellular scales. Its ambition is to stimulate new research that could lead to a better understanding of what gives neurons their ability to establish and regulate their exceptional size., Competing Interests: Conflict of interest The author declare no conflict of interest and competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

TDP-43 is pathologically and genetically with associated amyotrophic lateral sclerosis and frontotemporal lobar degeneration. These diseases are characterized by significant neurite defects, including cytoskeletal pathology. The involvement of TDP-43 in the degeneration of neurons in these diseases are not yet well understood, however accumulating evidence shows involvement in neurite outgrowth, remodelling and in regulation of many components of the neuronal cytoskeleton. In order to investigate how alterations to TDP-43 expression levels may exert effects on the neuronal cytoskeleton, primary cortical neurons from transgenic mice overexpressing one or two copies of human wildtype TDP-43 under the prion promoter were examined. Label-free quantitative proteomic analysis, followed by functional annotation clustering to identify protein families that clustered together within up- or down-regulated protein groups, revealed that actin-binding proteins were significantly more abundant in neurons overexpressing TDP-43 compared to wildtype neurons. Morphological analysis demonstrated that during early development neurons expressing one copy of human TDP-43 had an increased number of neurite branches and alterations to growth cone morphology, while no changes were observed in neurons expressing two copies of TDP-43. These developmental processes require specific expression and organization of the cytoskeleton. The results from these studies provide further insight into the normal function of TDP-43 and how alterations in TDP-43 expression may impact the cytoskeleton., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Neonicotinoids are one of most widely used pesticides targeting nicotinic acetylcholine receptors (nAChRs) of insects. Recent epidemiological evidence revealed increasing amounts of neonicotinoids detected in human samples, raising the critical question of whether neonicotinoids affect human health. We investigated the effects of a neonicotinoid pesticide clothianidin (CTD) on human neuroblastoma SH-SY5Y cells as in vitro models of human neuronal cells. Cellular and functional effects of micromolar doses of CTD were evaluated by changes in cell growth, intracellular signaling activities and gene expression profiles. We examined further the effects of CTD on neuronal differentiation by measuring neurite outgrowth. Exposure to CTD (1-100 μM) significantly increased the number of cells within 24 h of culture. The nAChRs antagonists, mecamylamine and SR16584, inhibited this effect, suggesting human α3β4 nAChRs could be targets of neonicotinoids. We observed a transient intracellular calcium influx and increased phosphorylation of extracellular signal-regulated kinase 1/2 shortly after exposure to CTD. Transcriptome analysis revealed that CTD down-regulated genes involved in neuronal function (e.g., formation of filopodia and calcium ion influx) and morphology (e.g., axon guidance signaling and cytoskeleton signaling); these changes were reflected by a finding of increased neurite length during neuronal differentiation. These findings provide novel insight into the potential risks of neonicotinoids to the human nervous system., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Recently, our laboratory identified sensory innervation within head and neck squamous cell carcinomas (HNSCCs) and subsequently defined a mechanism whereby HNSCCs promote their own innervation via the release of exosomes that stimulate neurite outgrowth. Interestingly, we noted that exosomes from human papillomavirus (HPV)-positive cell lines were more effective at promoting neurite outgrowth than those from HPV-negative cell lines. As nearly all cervical tumors are HPV-positive, we hypothesized that these findings would extend to cervical cancer., Methods: We use an in vitro assay with PC12 cells to quantify the axonogenic potential of cervical cancer exosomes. PC12 cells are treated with cancer-derived exosomes, stained with the pan-neuronal marker (β-III tubulin) and the number of neurites quantified. To assess innervation in cervical cancer, we immunohistochemically stained cervical cancer patient samples for β-III tubulin and TRPV1 (sensory marker) and compared the staining to normal cervix., Results: Here, we show the presence of sensory nerves within human cervical tumors. Additionally, we show that exosomes derived from HPV-positive cervical cancer cell lines effectively stimulate neurite outgrowth., Conclusions: These data identify sensory nerves as components of the cervical cancer microenvironment and suggest that tumor- derived exosomes promote their recruitment., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The neuronal pathways of the circadian clock in the brain of R. prolixus have been described in detail previously, but there is no information concerning the cells or their pathways which relay either inputs to the clock (e.g. for light entrainment), or outputs from it to driven rhythms. Here, we employ antisera to three neuropeptides (type A allatostatin-7, crustacean cardioactive peptide and FMRFamide), and serotonin in confocal laser scanning immunohistochemistry to analyze the distribution of cell bodies and their projections in relation to the principle circadian clock cells (lateral cells, LNs) for all four neuron types. LNs are revealed following labelling with anti- pigment dispersing factor in double labelled preparations. Regions of potential communication between ramifications of the LNs and each of the four other neuron types is described (identified by close superposition of their neurites in various brain regions), as is their detailed projections within the brain. Neuromodulation is sometimes suggested by close, but not intimate, proximity of varicosities of neurites. We infer that some neuron types comprise input pathways to the LNs, some are outputs to neuroendocrine or behavioral rhythms, and others participate in both input and output pathways, sometimes by the same neuron type but in different locations. For example, one retinula cell in each ommatidium is immunoreactive for allatostatin A; its axon projects to the medulla making superpositions with LNs, as do serotonin cells in the optic lobe, indicating roles of both neuron types in light input (entrainment) to the clock. But in other brain areas, these same types appear to mediate outputs from the clock. The accessory medulla has been widely reported as the principle center of integration in other insects; but we found sparse evidence of this in R. prolixus as it contains few neurites other than those from the clock cells. Rather, the importance of neural pathways involving the medulla and the superior protocerebrum is emphasized. We conclude that there is a vast and complex web of interactions in the brain with the LNs, which potentially receive multiple pathways of inputs and outputs that could drive rhythmicity in a multitude of downstream cells, rendering a host of output pathways rhythmic, notably hormone release from neurosecretory cells and behaviors., (Copyright © 2018. Published by Elsevier Inc.)

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of polyglutamine region in the androgen receptor. To gain insights into mechanisms of SBMA, four wild-type and five SBMA iPSC lines were differentiated to spinal motor neurons (sMNs) with high efficiency. SBMA sMNs showed neurite defects, reduced sMN survival and decreased protein synthesis levels. Microarray analysis revealed a dysregulation in various neuronal-related signalling pathways in SBMA sMNs. Strikingly, FAM135B a novel gene of unknown function, was found drastically downregulated in SBMA sMNs. Knockdown of FAM135B in wild-type sMNs reduced their survival and contributed to neurite defects, similar to SBMA sMNs, suggesting a functional role of FAM135B in SBMA. The degenerative phenotypes and dysregulated genes revealed could be potential therapeutic targets for SBMA., (Copyright © 2018 Elsevier Inc. All rights reserved.)

CaBP1 is a Ca 2+ binding protein that is widely expressed in neurons in the brain, retina, and cochlea. In heterologous expression systems, CaBP1 interacts with and regulates voltage-gated Ca v Ca 2+ channels but whether this is the case in neurons is unknown. Here, we investigated the cellular functions of CaBP1 in cochlear spiral ganglion neurons (SGNs), which express high levels of CaBP1. Consistent with the role of CaBP1 as a suppressor of Ca 2+ -dependent inactivation (CDI) of Ca v 1 (L-type) channels, Ca v 1 currents underwent greater CDI in SGNs from mice lacking CaBP1 (C-KO) than in wild-type (WT) SGNs. The coupling of Ca v 1 channels to downstream signaling pathways was also disrupted in C-KO SGNs. Activity-dependent repression of neurite growth was significantly blunted and unresponsive to Ca v 1 antagonists in C-KO SGNs in contrast to WT SGNs. Moreover, Ca v 1-mediated Ca 2+ signals and phosphorylation of cAMP-response element binding protein were reduced in C-KO SGNs compared to WT SGNs. Our findings establish a role for CaBP1 as an essential regulator of Ca v 1 channels in SGNs and their coupling to downstream pathways controlling activity-dependent transcription and neurite growth., (Copyright © 2018. Published by Elsevier Inc.)

The mechanisms that lead to neuronal death in neurodegenerative diseases are poorly understood. Prion diseases, like many more common disorders such as Alzheimer's and Parkinson's diseases, are characterized by the progressive accumulation of misfolded disease-specific proteins. The earliest changes observed in brain tissue include a reduction in synaptic number and retraction of dendritic spines, followed by reduced length and branching of neurites. These pathologies are observable during presymptomatic stages of disease and are accompanied by altered expression of transcripts that include miRNAs. Here we report that miR-16 localized within hippocampal CA1 neurons is increased during early prion disease. Modulating miR-16 expression in mature murine hippocampal neurons by expression from a lentivirus, thus mimicking the modest increase seen in vivo, was found to induce neurodegeneration. This was characterized by retraction of neurites and reduced branching. We performed immunoprecipitation of the miR-16 enriched RISC complex, and identified associated transcripts from the co-immunoprecipitated RNA (Ago2 RIP-Chip). These transcripts were enriched with predicted binding sites for miR-16, including the validated miR-16 targets APP and BCL2, as well as numerous novel targets. In particular, genes within the neurotrophin receptor mediated MAPK/ERK pathway were potentially regulated by miR-16; including TrkB (NTRK2), MEK1 (MAP2K1) and c-Raf (RAF). Increased miR-16 expression in neurons during presymptomatic prion disease and reduction in proteins involved in MAPK/ERK signaling represents a possible mechanism by which neurite length and branching are decreased during early stages of disease., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

Lamellipodia in Schwann cells (SCs) are crucial for myelination, but their other biological functions remain largely uncharacterised. Two types of lamellipodia exist in SCs: axial lamellipodia at the outermost edge of the cell processes, and radial lamellipodia appearing peripherally along the entire cell. We have previously shown that radial lamellipodia on olfactory glia (olfactory ensheathing cells; OECs) promote cell-cell adhesion, contact-mediated migration and phagocytosis. Here we have investigated whether lamellipodia in SCs have similar roles. Using live-cell imaging, we show that the radial lamellipodia in SCs are highly motile, appear at multiple cellular sites and rapidly move in a wave-like manner. We found that axial and radial lamellipodia had strikingly different roles and are regulated by different intracellular pathways. Axial lamellipodia initiated interactions with other SCs and with neurons by contacting radial lamellipodia on SCs, and budding neurites/axons. Most SC-SC interactions resulted in repulsion, and, lamellipodial activity (unlike in OECs) did not promote contact-mediated migration. We show that lamellipodia are crucial for SC-mediated phagocytosis of both axonal debris and bacteria, and demonstrated that inhibition of lamellipodial activity by blocking the Rho/Rac pathways also inhibits phagocytosis. We also show that heregulin, which induces SC differentiation and maturation, alters lamellipodial behaviour but does not affect phagocytic activity. Overall, the results show that SC lamellipodia are important for cell interactions and phagocytosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The current aging of the world population will increase the number of people suffering from brain degenerative diseases such as Alzheimer's disease (AD). There are evidence showing that the use of growth factors such as BMP-9 could restored cognitive function as it acts on many AD hallmarks at the same time. However, BMP-9 is a big protein expensive to produce that can hardly access the central nervous system. We have therefore developed a small peptide, SpBMP-9, derived from the knuckle epitope of BMP-9 and showed its therapeutic potential in a previous study. Since it is known that the native protein, BMP-9, can act in synergy with other growth factors in the context of AD, here we study the potential synergistic effect of various combinations of SpBMP-9 with bFGF, EGF, IGF-2 or NGF on the cholinergic differentiation of human neuroblastoma cells SH-SY5Y. We found that, in opposition to IGF-2 or EGF, the combination of SpBMP-9 with bFGF or NGF can stimulate to a greater extent the neurite outgrowth and neuronal differentiation toward the cholinergic phenotype as shown by expression and localization of the neuronal markers NSE and VAchT and the staining of intracellular calcium. Those results strongly suggest that SpBMP-9 plus NGF or bFGF are promising therapeutic combinations against AD that required further attention., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Microtubule-actin crosslinking factor 1 (MACF1), also known as actin crosslinking factor 7 (ACF7), is essential for proper modulation of actin and microtubule cytoskeletal networks. Most MACF1 isoforms are expressed broadly in the body, but some are exclusively found in the nervous system. Consequentially, MACF1 is integrally involved in multiple neural processes during development and in adulthood, including neurite outgrowth and neuronal migration. Furthermore, MACF1 participates in several signaling pathways, including the Wnt/β-catenin and GSK-3 signaling pathways, which regulate key cellular processes, such as proliferation and cell migration. Genetic mutation or dysregulation of the MACF1 gene has been associated with neurodevelopmental and neurodegenerative diseases, specifically schizophrenia and Parkinson's disease. MACF1 may also play a part in neuromuscular disorders and have a neuroprotective role in the optic nerve. In this review, the authors seek to synthesize recent findings relating to the roles of MACF1 within the nervous system and explore potential novel functions of MACF1 not yet examined., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The c-Jun N-terminal kinases (JNKs) are important mediators of cell viability and structural integrity in postmitotic neurons, which is required for maintaining synaptic connections and neural plasticity. In the present study, we chose differentiated PC12 cells as a well-characterised neuronal model system to selectively examine the regulation of basal JNK activity by extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. We detected a complex interaction between the kinases to prevent cell death and neurite loss. Especially the appropriate level of JNK activation determined cellular survival. Basal activity of ERK1/2 attenuated the potentiation of JNK phosphorylation and thereby the induction of apoptosis. Importantly, when JNK activity was too low, cell viability and the number of neurite-bearing cells also decreased, even though the activation of ERK1/2 was enhanced. In this case, the JNK-mediated survival signals via activating transcription factor-3 (ATF3) were inhibited. Furthermore, the phosphorylation of ERK1/2 induced by the JNK inhibitor SP600125 inhibited the basal activity of Akt, which normally supported cell viability. Thus, controlling JNK activity is crucial to promote survival and neurite stability of differentiated neuronal cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The detection of developmental neurotoxicity (DNT) of chemicals has high relevance for protection of human health. However, DNT of many pesticides is only little known. Furthermore, validated in vitro systems for assessment of DNT are not well established. Here we employed the rat phaeochromocytoma cell line PC-12 to evaluate DNT of 18 frequently used pesticides of different classes, including neonicotinoids, pyrethroids, organophosphates, organochlorines, as well as quaternary ammonium compounds, the organic compound used in pesticides, piperonyl butoxide, as well as the insect repellent diethyltoluamide (DEET). We determined the outgrowth of neurites in PC-12 cells co-treated with nerve growth factor and different concentrations of biocides for 5days. Furthermore, we determined transcriptional alterations of selected genes that may be associated with DNT, such as camk2α and camk2β, gap-43, neurofilament-h, tubulin-α and tubulin-β. Strong and dose- dependent inhibition of neurite outgrowth was induced by azamethiphos and chlorpyrifos, and dieldrin and heptachlor, which was correlated with up-regulation of gap-43. No or only weak effects on neurite outgrowth and transcriptional alterations occurred for neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam, the pyrethroids λ-cyhalothrin, cyfluthrin, deltamethrin, and permethrin, the biocidal disinfectants C12-C14-alkyl(ethylbenzyl)dimethylammonium (BAC), benzalkonium chloride and barquat (dimethyl benzyl ammonium chloride), and piperonyl butoxide and DEET. Our study confirms potential developmental neurotoxicity of some pesticides and provides first evidence that azamethiphos has the potential to act as a developmental neurotoxic compound. We also demonstrate that inhibition of neurite outgrowth and transcriptional alterations of gap-43 expression correlate, which suggests the employment of gap-43 expression as a biomarker for detection and initial evaluation of potential DNT of chemicals., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Estrogen receptors (ERs) α and β are distributed in most tissues of women and men. ERs are bound by estradiol (E2), a natural hormone, and mediate the pleiotropic and tissue-specific effects of E2, such as proliferation of breast epithelial cells or protection and differentiation of neuronal cells. Numerous environmental molecules, called endocrine disrupting compounds, also interact with ERs. Phytoestrogens belong to this large family and are considered potent therapeutic molecules that act through their selective estrogen receptor modulator (SERM) activity. Using breast cancer cell lines as a model of estrogen-dependent proliferation and a stably ER-expressing PC12 cell line as a model of neuronal differentiating cells, we studied the SERM activity of major dietary compounds, such as apigenin, liquiritigenin, daidzein, genistein, coumestrol, resveratrol and zearalenone. The ability of these compounds to induce ER-transactivation and breast cancer cell proliferation and enhance Nerve Growth Factor (NGF) -induced neuritogenesis was assessed. Surprisingly, although all compounds were able to activate the ER through an estrogen responsive element reporter gene, they showed differential activity toward proliferation or differentiation. Apigenin and resveratrol showed a partial or no proliferative effect on breast cancer cells but fully contributed to the neuritogenesis effect of NGF. However, daidzein and zearalenone showed full effects on cellular proliferation but did not induce cellular differentiation. In summary, our results suggest that the therapeutic potential of phytoestrogens can diverge depending on the molecule and the phenotype considered. Hence, apigenin and resveratrol might be used in the development of therapeutics for breast cancer and brain diseases., (Copyright © 2017 Elsevier Inc. All rights reserved.)

ADP-ribosylation factors (ARFs) are small guanosine triphosphatases of the Ras superfamily involved in membrane trafficking and regulation of the actin cytoskeleton. Aplysia Sec7 protein (ApSec7), a guanine nucleotide exchange factor for ARF1 and ARF6, induces neurite outgrowth and plays a key role in 5-hydroxyltryptamine-induced neurite growth and synaptic facilitation in Aplysia sensory-motor synapses. However, the specific role of ARF6 signaling on neurite outgrowth in Aplysia neurons has not been examined. In the present study, we cloned Aplysia ARF6 (ApARF6) and revealed that an overexpression of enhanced green fluorescent protein (EGFP)-fused constitutively active ApARF6 (ApARF6-Q67L-EGFP) could induce neurite outgrowth in Aplysia sensory neurons. Further, we observed that ApARF6-induced neurite outgrowth was inhibited by the co-expression of a Sec7 activity-deficient mutant of ApSec7 (ApSec7-E159K). The pleckstrin homology domain of ApSec7 may bind to active ApARF6 at the plasma membrane and prevent active ApARF6-induced functions, including intracellular vacuole formation in HEK293T cells. The results of the present study suggest that activation of ARF6 signaling could induce neurite outgrowth in Aplysia neurons and may be involved in downstream signaling of ApSec7-induced neurite outgrowth in Aplysia neurons., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The proper organization and function of the mammalian nervous system relies on neuronal processes or "neurites," extended morphological projections that include axons and dendrites. Tau is a structural microtubule-associated protein that is widely expressed in the nervous system that mediates the establishment of cell polarity, neurite outgrowth, and axonal transport. A useful model for studying the establishment and maintenance of these neuronal structures are rat neuronal PC12 cells, which can be induced to express tau and project neurites by treating the cells with nerve growth factor. Here, we present a simple method for continuously measuring the rate of neurite outgrowth and retraction over time by neurite length and neurite area analyses. This method uses freely available ImageJ software and widely available phase-contrast imaging., (© 2017 Elsevier Inc. All rights reserved.)

Several reports have linked the presence of high titers of anti-Gg Abs with delayed recovery/poor prognosis in GBS. In most cases, failure to recover is associated with halted/deficient axon regeneration. Previous work identified that monoclonal and patient-derived anti-Gg Abs can act as inhibitory factors in an animal model of axon regeneration. Further studies using primary dorsal root ganglion neuron (DRGn) cultures demonstrated that anti-Gg Abs can inhibit neurite outgrowth by targeting gangliosides via activation of the small GTPase RhoA and its associated kinase (ROCK), a signaling pathway common to other established inhibitors of axon regeneration. We aimed to study the molecular basis of the inhibitory effect of anti-Gg abs on neurite outgrowth by dissecting the molecular dynamics of growth cones (GC) cytoskeleton in relation to the spatial-temporal analysis of RhoA activity. We now report that axon growth inhibition in DRGn induced by a well characterized mAb targeting gangliosides GD1a/GT1b involves: i) an early RhoA/ROCK-independent collapse of lamellipodia; ii) a RhoA/ROCK-dependent shrinking of filopodia; and iii) alteration of GC microtubule organization/and presumably dynamics via RhoA/ROCK-dependent phosphorylation of CRMP-2 at threonine 555. Our results also show that mAb 1B7 inhibits peripheral axon regeneration in an animal model via phosphorylation/inactivation of CRMP-2 at threonine 555. Overall, our data may help to explain the molecular mechanisms underlying impaired nerve repair in GBS. Future work should define RhoA-independent pathway/s and effectors regulating actin cytoskeleton, thus providing an opportunity for the design of a successful therapy to guarantee an efficient target reinnervation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The microtubule-associated protein MAP1B plays a key role in axon regeneration. We investigated the role of GSK3-mediated MAP1B phosphorylation in local fine-tuning of neurite branching and the underlying microtubule (MT) dynamics. In wildtype adult dorsal root ganglia (DRG) neurons, MAP1B phosphorylation is locally reduced at branching points, and branching dynamics from growth cones and distal neurite shafts is increased upon GSK3 inhibition. While map1b-/- neurites, that display increased branching, are not affected by GSK3 inhibition, transfection of map1b-/- neurons with full-length map1b-cDNA restores the wildtype branching phenotype, demonstrating that MAP1B is a key effector downstream of GSK3. Experiments in mutant mice lacking tyrosinated MTs indicate a preferential association of phospho-MAP1B with tyrosinated MTs. Interestingly, inhibition of GSK3-mediated MAP1B phosphorylation in map1b-cDNA-transfected fibroblasts protects both tyrosinated and acetylated MTs from nocodazole-induced depolymerization, while detyrosinated MTs are less abundant in the presence of MAP1B. Our data thus provide new insight into the molecular link between GSK3, MAP1B, neurite branching and MT stability regulation. We suggest that, at branching points, MAP1B undergoes a fine regulation of both its phosphorylation and sub-cellular amounts, in order to modulate the local balance between acetylated, detyrosinated, and tyrosinated microtubule pools., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The runt-related transcription factor Runx1 regulates cell-type specification and axonal projections of nociceptive dorsal root ganglion (DRG) neurons, whereas bone morphogenetic protein 4 (BMP4) is required for axonal growth during neuronal development. Although Runx1 has been shown to be involved in BMP4 signaling in non-neural tissues, the Runx1 function in BMP4-dependent regulation of neuronal development is unclear. To investigate interactions between Runx1 and BMP4 in neurite outgrowth, we cultured DRGs from wild-type and Runx1-deficient mouse embryos in the presence or absence of BMP4. Neurite outgrowth was decreased in BMP4-treated wild-type DRGs and untreated Runx1-deficient DRGs, suggesting the inhibitory effect of BMP4 and facilitatory effect of Runx1 on neurite outgrowth. In addition, the combination of BMP4 treatment and Runx1 deficiency increased neurite outgrowth, suggesting that Runx1 is required for BMP4-induced suppression of neurite outgrowth and that the loss of Runx1 results in a functional switch of BMP4 from neurite growth suppressing to neurite growth promoting. Both BMP4 treatment and Runx1 deficiency increased calcitonin gene-related peptide (CGRP)-positive neurons, and CGRP expression was not increased by BMP4 treatment in Runx1-deficient mice, suggesting that Runx1 contributes to BMP4-induced CGRP expression in DRG neurons. Thus, Runx1 contributes to BMP4 regulation of neurite outgrowth and CGRP expression in DRG and may control BMP4 functional switching during embryogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Nogo-66 receptor (NgR1), a receptor for Nogo-A, contributes to the inhibition of axonal regeneration in the adult central nervous system after traumatic injuries. Thus, NgR1 has been considered a critical target in axon regeneration therapy. Here, we identified a specific NgR1 antagonist peptide (HIYTALV, named NAP2) which promotes neurite regeneration in vitro from a phage display heptapeptide library. NAP2 was co-localized with NgR1 on the surface of PC12 cells and cerebellar granule cells (CGCs) by immunofluorescence assay. Horseradish peroxidase (HRP)-streptavidin-biotin assay further showed that NAP2 binds to NgR1 and the dissociation constant (Kd) was 0.45 μM Functional analyses indicated that NAP2 could reduce the inhibitory effects of Nogo-66 on neurite outgrowth in differentiated PC12 cells and CGCs by blocking the Nogo-66-induced activation of Rho-associated coiled coil-containing protein kinase (ROCK), collapsin response mediator protein 2 (CRMP2) and myosin light chain (MLC). Taken together, the small molecule NgR1 antagonist peptide NAP2 (MW: 815.98Da) has a potential ability in crossing blood brain barrier and will be a promising therapeutic agent for the treatment of spinal cord injury and neurodegenerative diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Neuroinflammation plays a critical role in the regeneration of peripheral nerves following axotomy. An injury to the sciatic nerve leads to significant macrophage accumulation in the L5 DRG, an effect not seen when the dorsal root is injured. We recently demonstrated that this accumulation around axotomized cell bodies is necessary for a peripheral conditioning lesion response to occur. Here we asked whether overexpression of the monocyte chemokine CCL2 specifically in DRG neurons of uninjured mice is sufficient to cause macrophage accumulation and to enhance regeneration or whether other injury-derived signals are required. AAV5-EF1α-CCL2 was injected intrathecally, and this injection led to a time-dependent increase in CCL2 mRNA expression and macrophage accumulation in L5 DRG, with a maximal response at 3 weeks post-injection. These changes led to a conditioning-like increase in neurite outgrowth in DRG explant and dissociated cell cultures. This increase in regeneration was dependent upon CCL2 acting through its primary receptor CCR2. When CCL2 was overexpressed in CCR2-/- mice, macrophage accumulation and enhanced regeneration were not observed. To address the mechanism by which CCL2 overexpression enhances regeneration, we tested for elevated expression of regeneration-associated genes in these animals. Surprisingly, we found that CCL2 overexpression led to a selective increase in LIF mRNA and neuronal phosphorylated STAT3 (pSTAT3) in L5 DRGs, with no change in expression seen in other RAGs such as GAP-43. Blockade of STAT3 phosphorylation by each of two different inhibitors prevented the increase in neurite outgrowth. Thus, CCL2 overexpression is sufficient to induce macrophage accumulation in uninjured L5 DRGs and increase the regenerative capacity of DRG neurons via a STAT3-dependent mechanism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Peripheral nerve regrowth is less robust than commonly assumed, particularly when it accompanies common clinical scenarios such as diabetes mellitus. Brief extracellular electrical stimulation (ES) facilitates the regeneration of peripheral nerves in part through early activation of the conditioning injury response and BDNF. Here, we explored intrinsic neuronal responses to ES to identify whether ES might impact experimental diabetes, where regeneration is attenuated. ES altered several regeneration related molecules including rises in tubulin, Shh (Sonic hedgehog) and GAP43 mRNAs. ES was associated with rises in neuronal intracellular calcium but its strict linkage to regrowth was not confirmed. In contrast, we identified PI3K-PTEN involvement, an association previously linked to diabetic regenerative impairment. Following ES there were declines in PTEN protein and mRNA both in vitro and in vivo and a PI3K inhibitor blocked its action. In vitro, isolated diabetic neurons were capable of mounting robust responsiveness to ES. In vivo, ES improved electrophysiological and behavioral indices of nerve regrowth in a chronic diabetic model of mice with pre-existing neuropathy. Regrowth of myelinated axons and reinnervation of the epidermis were greater following ES than sham stimulation. Taken together, these findings identify a role for ES in supporting regeneration during the challenges of diabetes mellitus., (Copyright © 2015. Published by Elsevier Inc.)

Understanding the intra- and extracellular proteins involved in the development of the corticospinal tract (CST) may offer insights into how the pathway could be regenerated following traumatic spinal cord injury. Currently, however, little is known about the proteome of the developing corticospinal system. The present study, therefore, has used quantitative proteomics and bioinformatics to detail the protein profile of the rat CST during its formation in the spinal cord. This analysis identified increased expression of 65 proteins during the early ingrowth of corticospinal axons into the spinal cord, and 36 proteins at the period of heightened CST growth. A majority of these proteins were involved in cellular assembly and organization, with annotations being most highly associated with cytoskeletal organization, microtubule dynamics, neurite outgrowth, and the formation, polymerization and quantity of microtubules. In addition, 22 proteins were more highly expressed within the developing CST in comparison to other developing white matter tracts of the spinal cord of age-matched animals. Of these differentially expressed proteins, only one, stathmin 1 (a protein known to be involved in microtubule dynamics), was both highly enriched in the developing CST and relatively sparse in other developing descending and ascending spinal tracts. Immunohistochemical analyses of the developing rat spinal cord and fetal human brain stem confirmed the enriched pattern of stathmin expression along the developing CST, and in vitro growth assays of rat corticospinal neurons showed a reduced length of neurite processes in response to pharmacological perturbation of stathmin activity. Combined, these findings suggest that stathmin activity may modulate axonal growth during development of the corticospinal projection, and reinforces the notion that microtubule dynamics could play an important role in the generation and regeneration of the CST., (Copyright © 2015 Elsevier Inc. All rights reserved.)

As a widely used anti-bacterial agent and a metabolic inhibitor as well as AMP-activated protein kinase (AMPK) activator, berberine (BBR) has been shown to cross the blood-brain barrier. Its efficacy has been investigated in various disease models of the central nervous system. Neurite outgrowth is critical for nervous system development and is a highly energy-dependent process regulated by AMPK-related pathways. In the present study, we aimed to investigate the effects of BBR on AMPK activation and neurite outgrowth in neurons. The neurite outgrowth of primary rat cortical neurons at different stages of polarization was monitored after exposure of BBR. Intracellular energy level, AMPK activation and polarity-related pathways were also inspected. The results showed that BBR suppressed neurite outgrowth and affected cytoskeleton stability in the early stages of neuronal polarization, which was mediated by lowered energy status and AMPK activation. Liver kinase B1 and PI3K-Akt-GSK3β signaling pathways were also involved. In addition, mitochondrial dysfunction and endoplasmic reticulum stress contributed to the lowered energy status induced by BBR. This study highlighted the knowledge of the complex activities of BBR in neurons and corroborated the significance of energy status during the neuronal polarization., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Complement control protein (CCP) domains have adhesion properties and are commonly found in proteins that control the complement immune system. However, an increasing number of proteins containing CCP domains have been reported to display neuronal functions. Susd2 is a transmembrane protein containing one CCP domain. It was previously identified as a tumor-reversing protein, but has no characterized function in the CNS. The present study investigates the expression and function of Susd2 in the rat hippocampus. Characterization of Susd2 during development showed a peak in mRNA expression two weeks after birth. In hippocampal neuronal cultures, the same expression profile was observed at 15days in vitro for both mRNA and protein, a time consistent with synaptogenesis in our model. At the subcellular level, Susd2 was located on the soma, axons and dendrites, and appeared to associate preferentially with excitatory synapses. Inhibition of Susd2 by shRNAs led to decreased numbers of excitatory synaptic profiles, exclusively. Also, morphological parameters were studied on young (5DIV) developing neurons. After Susd2 inhibition, an increase in dendritic tree length but a decrease in axon elongation were observed, suggesting changes in adhesion properties. Our results demonstrate a dual role for Susd2 at different developmental stages, and raise the question whether Susd2 and other CCP-containing proteins expressed in the CNS could be function-related., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Neurotrophic factors have been intensively studied as potential therapeutic agents for promoting neural regeneration and functional recovery after nerve injury. Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) that forms a signalling complex with GFRα3 and the tyrosine kinase Ret. Systemic administration of artemin in rodents is reported to facilitate regeneration of primary sensory neurons following axotomy, improve recovery of sensory function, and reduce sensory hypersensitivity that is a cause of pain. However, the biological mechanisms that underlie these effects are mostly unknown. This study has investigated the biological significance of the colocalisation of GFRα3 with TrkA (neurotrophin receptor for nerve growth factor [NGF]) in the peptidergic type of unmyelinated (C-fibre) sensory neurons in rat dorsal root ganglia (DRG). In vitro neurite outgrowth assays were used to study the effects of artemin and NGF by comparing DRG neurons that were previously uninjured, or were axotomised in vivo by transecting a visceral or somatic peripheral nerve. We found that artemin could facilitate neurite initiation but in comparison to NGF had low efficacy for facilitating neurite elongation and branching. This low efficacy was not increased when a preconditioning in vivo nerve injury was used to induce a pro-regenerative state. Neurite initiation was unaffected by artemin when PI3 kinase and Src family kinase signalling were blocked, but NGF had a reduced effect., (Copyright © 2015. Published by Elsevier Inc.)

Adipose derived stem cells (ADSC) can be differentiated into Schwann cell-like cells which enhance nerve function and regeneration. However, the signalling mechanisms underlying the neurotrophic potential of ADSC remain largely unknown. In this study, we hypothesised that ADSC, upon stimulation with a combination of growth factors, could rapidly produce brain derived neurotrophic factor (BDNF) with a similar molecular mechanism to that functioning in the nervous system. Within 48 h of stimulation, ADSC demonstrated potent neurotrophic effects on dorsal root ganglion neurons, at a magnitude equivalent to that of the longer term differentiated Schwann cell-like cells. Stimulated ADSC showed rapid up-regulation of the neuronal activity dependent promoter BDNF exon IV along with an augmented expression of full length protein encoding BDNF exon IX. BDNF protein was secreted at a concentration similar to that produced by differentiated Schwann cell-like cells. Stimulation also activated the BDNF expression gating transcription factor, cAMP responsive element binding (CREB) protein. However, blocking phosphorylation of CREB with the protein kinase A small molecule inhibitor H89 did not suppress secretion of BDNF protein. These results suggest rapid BDNF production in ADSC is mediated through multiple compensatory pathways independent of, or in addition to, the CREB neuronal activation cascade., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sulf1 and Sulf2 are endosulfatases that cleave 6-O-sulphate groups from Heparan Sulphate Proteoglycans (HSPGs). Sulfation levels of HSPGs are critical for their role in modulating the activity of various growth factor receptors. Sulf1 and Sulf2 mRNAs were found to be widely expressed in the rodent nervous system and their full-length proteins were found in many types of neuronal perikarya and axons in the cerebral cortex, cerebellum, spinal cord and dorsal root ganglia (DRG) of adult rats. Sulf1/2 were also strongly expressed by cultured DRG neurons. To determine if blocking Sulf1 or Sulf2 activity affected neurite outgrowth in vitro, cultured DRG neurons were treated with neutralising antibodies to Sulf1 or Sulf2. Blocking Sulf1 and Sulf2 activity did not affect neurite outgrowth from cultured DRG neurons grown on a laminin/polylysine substrate but ameliorated the inhibitory effects of chondroitin sulphate proteoglycans (CSPGs) on neurite outgrowth. Blocking epidermal growth factor receptor (ErbB1) activity also improved neurite outgrowth in the presence of CSPGs, but the effects of ErbB1 antagonists and blocking SULFs were not additive. It is proposed that Sulf1, Sulf2 and ErbB1 are involved in the signalling pathway from CSPGs that leads to inhibition of neurite outgrowth and may regulate structural plasticity and regeneration in the nervous system., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL)--a proteolytic peptide fraction--were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons., (Published by Elsevier Inc.)

The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aβ) residues 1-42 (oAβ1-42) using our donor-acceptor 3-D co-culture model. We now show that different Aβ-isoforms (fluorescently labeled 1-42, 3(pE)-40, 1-40 and 11-42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aβ-isoform. Although different Aβ isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aβ can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aβ1-42 isoform, which further promotes cell-to-cell transfer; thus, oAβ1-42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The unique architecture of neurons requires the establishment and maintenance of polarity, which relies in part on microtubule-based kinesin motor transport to deliver essential cargo into axons and dendrites. In developing neurons, kinesin trafficking is essential for delivering organelles and molecules that are crucial for elongation and guidance of the growing axonal and dendritic termini. In mature neurons, kinesin cargo delivery is essential for neuron dynamic physiological functions which are critical in brain development. In this work, we followed Spatial (Tbata) gene expression during primary hippocampal neuron development and showed that it is highly expressed during dendrite formation. Spatial protein exhibits a somatodendritic distribution and we show that the kinesin motor Kif17, among other dendrite specific kinesins, is crucial for Spatial localization to dendrites of hippocampal neurons. Furthermore, Spatial down regulation in primary hippocampal cells revealed a role for Spatial in maintaining neurons' polarity by ensuring proper neurite outgrowth. This polarity is specified by intrinsic and extracellular signals that allow neurons to determine axon and dendrite fate during development. Neurotrophic factors, such as the Nerve Growth Factor (NGF), are candidate extracellular polarity-regulating cues which are proposed to accelerate neuronal polarization by enhancing dendrite growth. Here, we show that NGF treatment increases Spatial expression in hippocampal neurons. Altogether, these data suggest that Spatial, in response to NGF and through its transport by Kif17, is crucial for neuronal polarization and can be a key regulator of neurite outgrowth., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is characterized by accumulation of two misfolded and aggregated proteins, β-amyloid and hyperphosphorylated tau. Both cellular systems responsible for clearance of misfolded and aggregated proteins, the lysosomal and the proteasomal, have been shown to be malfunctioning in the aged brain and more so in patients with neurodegenerative diseases, including AD. This malfunction could be contributing to β-amyloid and tau accumulation, eventually aggregating in plaques and tangles. We have investigated the impact of decreased proteasome activity on tau phosphorylation as well as on microtubule stability and transport. To do this, we used our recently developed neuronal model where human SH-SY5Y cells obtain neuronal morphology and function through differentiation. We found that exposure to low doses of the proteasome inhibitor MG-115 caused tau phosphorylation, microtubule destabilization and disturbed neuritic transport. Furthermore, reduced proteasome activity activated several proteins implicated in tau phosphorylation and AD pathology, including c-Jun N-terminal kinase, c-Jun and extracellular signal-regulated protein kinase (ERK) 1/2. Restoration of the microtubule transport was achieved by inhibiting ERK 1/2 activation, and simultaneous inhibition of both ERK 1/2 and c-Jun reversed the proteasome inhibition-induced tau phosphorylation. Taken together, this study suggests that a decrease in proteasome activity can, through activation of c-Jun and ERK 1/2, result in several events related to neurodegenerative diseases. Restoration of proteasome activity or modulation of ERK 1/2 and c-Jun function can open new treatment possibilities against neurodegenerative diseases such as AD., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The actin cytoskeleton is critically involved in the regulation of neurite outgrowth., Results: The actin cytoskeleton-associated protein tropomyosin induces neurite outgrowth in B35 neuroblastoma cells and regulates neurite branching in an isoform-dependent manner., Conclusions: Our data indicate that tropomyosins are key regulators of the actin cytoskeleton during neurite outgrowth., Significance: Revealing the molecular machinery that regulates the actin cytoskeleton during neurite outgrowth may provide new therapeutic strategies to promote neurite regeneration after nerve injury., Summary: The formation of a branched network of neurites between communicating neurons is required for all higher functions in the nervous system. The dynamics of the actin cytoskeleton is fundamental to morphological changes in cell shape and the establishment of these branched networks. The actin-associated proteins tropomyosins have previously been shown to impact on different aspects of neurite formation. Here we demonstrate that an increased expression of tropomyosins is sufficient to induce the formation of neurites in B35 neuroblastoma cells. Furthermore, our data highlight the functional diversity of different tropomyosin isoforms during neuritogenesis. Tropomyosins differentially impact on the expression levels of the actin filament bundling protein fascin and increase the formation of filopodia along the length of neurites. Our data suggest that tropomyosins are central regulators of actin filament populations which drive distinct aspects of neuronal morphogenesis., (© 2013.)

HspB1 is a small heat shock protein implicated in neuronal survival and neurite growth; mutations in HspB1 have been identified in hereditary motor neuronopathies and Charcot Marie Tooth Type 2 neuropathies. In cortical neurons we found that expression of HspB1 decreased RhoA activity and RhoA-GTP protein, and reversed the inhibition of neurite extension induced by NogoA. HspB1 decreased PDZ-RhoGEF, a RhoA specific guanine nucleotide exchange factor, while other regulators of RhoA activity were unchanged. The decrease in PDZ-RhoGEF was independent of proteasomal or lysosomal degradation pathways and was not associated with changes in PDZ-RhoGEF mRNA. We sequenced the 3'UTR of rat PDZ-RhoGEF and found binding sites for miRNAs miR-20a, miR-128 and miR-132. Expression of these microRNAs was substantially increased in cortical neurons transfected with HspB1. Co-transfection of HspB1 with specific inhibitors of miR-20a or miR-128 prevented the decrease in PDZ-RhoGEF and blocked the neurite growth promoting effects of HspB1. Using the 3'UTR of PDZ-RhoGEF mRNA in a luciferase reporter construct we observed that HspB1, miR-20a and miR-128 each inhibited luciferase expression. We conclude that HspB1 regulates RhoA activity through modulation of PDZ-RhoGEF levels achieved by translational control through enhanced expression of specific miRNAs (miR-20a and miR-128). Regulation of RhoA activity by translational silencing of PDZ-RhoGEF may be the mechanism through which HspB1 is involved in regulation of neurite growth. As RhoA-GTPase plays a regulatory role in the organization and stability of cytoskeletal networks through its downstream effectors, the results suggest a possible mechanism linking HspB1 mutations and axonal cytoskeletal pathology., (© 2013.)

This study assessed the potential of highly purified (Stro-1(+)) human mesenchymal precursor cells (hMPCs) in combination with the anti-scarring protein decorin to repair the injured spinal cord (SC). Donor hMPCs isolated from spinal cord injury (SCI) patients were transplanted into athymic rats as a suspension graft, alone or after previous treatment with, core (decorin(core)) and proteoglycan (decorin(pro)) isoforms of purified human recombinant decorin. Decorin was delivered via mini-osmotic pumps for 14 days following sub-acute (7 day) or chronic (1 month) SCI. hMPCs were delivered to the spinal cord at 3 weeks or 6 weeks after the initial injury at T9 level. Behavioral and anatomical analysis in this study showed statistically significant improvement in functional recovery, tissue sparing and cyst volume reduction following hMPC therapy. The combination of decorin infusion followed by hMPC therapy did not improve these measured outcomes over the use of cell therapy alone, in either sub-acute or chronic SCI regimes. However, decorin infusion did improve tissue sparing, reduce spinal tissue cavitation and increase transplanted cell survivability as compared to controls. Immunohistochemical analysis of spinal cord sections revealed differences in glial, neuronal and extracellular matrix molecule expression within each experimental group. hMPC transplanted spinal cords showed the increased presence of serotonergic (5-HT) and sensory (CGRP) axonal growth within and surrounding transplanted hMPCs for up to 2 months; however, no evidence of hMPC transdifferentiation into neuronal or glial phenotypes. The number of hMPCs was dramatically reduced overall, and no transplanted cells were detected at 8 weeks post-injection using lentiviral GFP labeling and human nuclear antigen antibody labeling. The presence of recombinant decorin in the cell transplantation regimes delayed in part the loss of donor cells, with small numbers remaining at 2 months after transplantation. In vitro co-culture experiments with embryonic dorsal root ganglion explants revealed the growth promoting properties of hMPCs. Decorin did not increase axonal outgrowth from that achieved by hMPCs. We provide evidence for the first time that (Stro-1(+)) hMPCs provide: i) an advantageous source of allografts for stem cell transplantation for sub-acute and chronic spinal cord therapy, and (ii) a positive host microenvironment that promotes tissue sparing/repair that subsequently improves behavioral outcomes after SCI. This was not measurably improved by recombinant decorin treatment, but does provide important information for the future development and potential use of decorin in contusive SCI therapy., (© 2013 Elsevier Inc. All rights reserved.)

Rho GTPases are molecular switches that modulate multiple intracellular signaling processes by means of various effector proteins. As a result, Rho GTPase activities are tightly spatiotemporally regulated in order to ensure homeostasis within the cell. Though the roles of Rho GTPases during neural development have been well documented, their participation during neurodegeneration has been far less characterized. Herein we discuss our current knowledge of the role and function of Rho GTPases and regulators during neurodegeneration, and highlight their potential as targets for therapeutic intervention in common neurodegenerative disorders., (© 2013 Elsevier Inc. All rights reserved.)

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family and a ligand for the tropomyosin-receptor kinase B (TrkB), mediates neuronal survival, differentiation, and synaptic plasticity. However, BDNF is not used to treat neurodegenerative diseases because of its poor pharmacokinetic profile, side effects, and absence of survival properties in clinical trials. Consequently, alternative approaches such as TrkB receptor agonist application are gaining importance. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, has been described as a robust TrkB receptor agonist in hippocampal neurons. Nevertheless, the influence of 7,8-DHF on motoneurons, one of the main targets of BDNF in vivo, is so far unknown. Therefore, we investigated the impact of 7,8-DHF treatment on primary cultured mouse motoneurons. Indeed, we found an activation of the TrkB receptor. Moreover, 7,8-DHF application promotes survival and neurite growth of cultured motoneurons and these effects appear dose-dependent. To investigate the PI3K/AKT and MAPK pathway activation in 7,8-DHF treated motoneurons, we developed a high-density culture system of primary mouse motoneurons. Analysis of both pathways demonstrated a PI3K/AKT but not MAPK pathway activation in cultured motoneurons. This is in contrast to previously published reports about BDNF-mediated activation of TrkB. The lack of MAPK pathway activation is also in contrast to what has been found for hippocampal neurons that indeed show MAPK activation after 7,8-DHF treatment. The ability of 7,8-DHF to imitate BDNF function in motoneurons by using Trk receptor signaling would provide a new approach for the treatment of motoneuron diseases, but needs a more detailed analysis of the activation profile of 7,8-DHF., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The successful outcome of peripheral neuronal regeneration is attributed both to the growth permissive milieu and the intrinsic ability of the neuron to initiate appropriate cellular responses such as changes in gene expression and cytoskeletal rearrangements. Even though numerous studies have shown the importance of interactions between the neuron and the extracellular matrix (ECM) in axonal outgrowth, the molecular mechanisms underlying the contact between ECM receptors and the cellular cytoskeleton remain largely unknown. Unconventional myosins constitute an important group of cytoskeletal-associated motor proteins. One member of this family is the recently described myosin-X. This protein interacts with several members of the axon growth-associated ECM receptor family of integrins and could therefore be important in neuronal outgrowth. In this study, using radioactive in situ hybridization, we found that expression of myosin-X mRNA is upregulated in adult rat sensory neurons and spinal motoneurons after peripheral nerve injury, but not after central injury. Thus, myosin-X was upregulated after injuries that can be followed by axonal regeneration. We also found that the protein is localized to neuronal growth cones and that silencing of myosin-X using RNA interference impairs the integrin-mediated growth of neurites on laminin, but has no effect on non-integrin mediated growth on N-cadherin., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Tar-DNA binding protein of 43kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A balanced chromosomal translocation segregating with schizophrenia and affective disorders in a large Scottish family disrupting DISC1 implicated this gene as a susceptibility gene for major mental illness. Here we study neurons derived from a genetically engineered mouse strain with a truncating lesion disrupting the endogenous Disc1 ortholog. We provide a detailed account of the consequences of this mutation on axonal and dendritic morphogenesis as well as dendritic spine development in cultured hippocampal and cortical neurons. We show that the mutation has distinct effects on these two types of neurons, supporting a cell-type specific role of Disc1 in establishing structural connections among neurons. Moreover, using a validated antibody we provide evidence indicating that Disc1 localizes primarily to Golgi apparatus-related vesicles. Our results support the notion that in vitro cultures derived from Disc1(Tm1Kara) mice provide a valuable model for future mechanistic analysis of the cellular and biochemical effects of this mutation, and can thus serve as a platform for drug discovery efforts., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Glial cell line-derived neurotrophic factor (GDNF) increases survival and neurite extension of spiral ganglion neurons (SGNs), the primary neurons of the auditory system, via yet unknown signaling mechanisms. In other cell types, signaling is achieved by the GPI-linked GDNF family receptor α1 (GFRα1) via recruitment of transmembrane receptors: Ret (re-arranged during transformation) and/or NCAM (neural cell adhesion molecule). Here we show that GDNF enhances neuritogenesis in organotypic cultures of spiral ganglia from 5-day-old rats and mice. Addition of GFRα1-Fc increases this effect. GDNF/GFRα1-Fc stimulation activates intracellular PI3K/Akt and MEK/Erk signaling cascades as detected by Western blot analysis of cultures prepared from rats at postnatal days 5 (P5, before the onset of hearing) and 20 (P20, after the onset of hearing). Both cascades mediate GDNF stimulation of neuritogenesis, since application of the Akt inhibitor Wortmannin or the Erk inhibitor U0126 abolished GDNF/GFRα1-Fc stimulated neuritogenesis in P5 rats. Since cultures of P5 NCAM-deficient mice failed to respond by neuritogenesis to GDNF/GFRα1-Fc, we conclude that NCAM serves as a receptor for GDNF signaling responsible for neuritogenesis in early postnatal spiral ganglion., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The olfactory system continuously incorporates new neurons into functional circuits throughout life. Axons from olfactory sensory neurons (OSNs) in the nasal cavity synapse on mitral, tufted and periglomerular (PG) cells in the main olfactory bulb, and low levels of turnover within the OSN population results in ingrowth of new axons under normal physiological conditions. Subpopulations of bulb interneurons are continually eliminated by apoptosis, and are replaced by new neurons derived from progenitors in the adult forebrain subventricular zone. Integration of new neurons, including PG cells that are contacted by sensory axons, leads to ongoing reorganization of adult olfactory bulb circuits. The mechanisms regulating this adaptive structural plasticity are not all known, but the process is reminiscent of early nervous system development. Neurotrophic factors have well-established roles in controlling neuronal survival and connectivity during development, leading to speculation that trophic interactions between OSNs and their target bulb neurons may mediate some of these same processes in adults. A number of different trophic factors and their cognate receptors are expressed in the adult olfactory pathway. Neurotrophin-3 (NT3) is among these, as reflected by beta-galactosidase expression in transgenic reporter mice expressing lacZ under the NT3 promoter. Using a combination of approaches, including immunocytochemistry, real-time PCR of laser-captured RNA, and adenovirus-mediated gene transfer of NT3 fusion peptides in vivo, we demonstrate that OSNs express and anterogradely transport NT3 to the olfactory bulb. We additionally observe that in mice treated with adenovirus encoding NT3 tagged with hemagglutinin (HA), a subset of bulb neurons expressing the TrkC neurotrophin receptor are immunoreactive for HA, suggesting their acquisition of the fusion peptide from infected sensory neurons. Our results therefore provide evidence that OSNs may serve as an afferent source of trophic signals for the adult mouse olfactory bulb., (Published by Elsevier Inc.)

The ability of fish retinal ganglion cells (RGCs) to regenerate their axons was shown to require the re-expression and function of the two proteins reggie-1 and -2. RGCs in mammals fail to upregulate reggie expression and to regenerate axons after lesion suggesting the possibility that induced upregulation might promote regeneration. In the present study, RGCs in adult rats were induced to express reggie-1 by intravitreal injection of adeno-associated viral vectors (AAV2/1) expressing reggie-1 (AAV.R1-EGFP) 14d prior to optic nerve crush. Four weeks later, GAP-43-positive regenerating axons had crossed the lesion and grown into the nerve at significantly higher numbers and length (up to 5mm) than the control transduced with AAV.EGFP. Consistently, after transduction with AAV.R1-EGFP as opposed to AAV.EGFP, primary RGCs in vitro grew long axons on chondroitin sulfate proteoglycan (CSPG) and Nogo-A, both glial cell-derived inhibitors of neurite growth, suggesting that reggie-1 can provide neurons with the ability to override inhibitors of neurite growth. This reggie-1-mediated enhancement of growth was reproduced in mouse hippocampal and N2a neurons which generated axons 40-60% longer than their control counterparts. This correlates with the reggie-1-dependent activation of Src and PI3 kinase (PI3K), of the Rho family GTPase Rac1 and downstream effectors such as cofilin. This increased growth also depends on TC10, the GTPase involved in cargo delivery to the growth cone. Thus, the upregulation of reggie-1 in mammalian neurons provides nerve cells with neuron-intrinsic properties required for axon growth and successful regeneration in the adult mammalian CNS., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The neural cell adhesion molecule L1 (L1CAM) promotes neurite outgrowth via mechanisms that are not completely understood, but are known to involve the cytoskeleton. Here, we show that L1 binds directly to the microtubule associated protein 2c (MAP2c). This isoform of MAP2 is predominantly expressed in developing neurons. We found that the mRNA and protein levels of MAP2c, but not of MAP2a/b, are reduced in brains of young adult L1-deficient transgenic mice. We show via ELISA, that MAP2c, but not MAP2a/b, binds directly to the intracellular domain of L1. Remarkably, all these MAP2 isoforms co-immunoprecipitate with L1, suggesting that MAP2a/b associates with L1 via intermediate binding partners. The expression levels of MAP2a/b/c correlate with those of L1 in different brain regions of early postnatal mice, while expression levels of heat shock cognate protein 70 (Hsc70) or actin do not. L1 enhances the expression of MAP2a/b/c in cultured hippocampal neurons depending on activation of the mitogen-activated protein kinase (MAPK) pathway. Deficiency in both L1 and MAP2a/b/c expression results in reduced neurite outgrowth in vitro. We propose that the L1-triggered increase in MAP2a/b/c expression is required to promote neurite outgrowth., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Although neurite branching is crucial for neuronal network formation after birth, its underlying mechanisms remain unclear. Here, we demonstrate that lysophosphatidic acid (LPA) stimulates neurite branching through a novel signaling pathway. Treatment of neuronal cell lines with LPA resulted in neurite branch formation when LPA(3) receptor was introduced. The effects of LPA were blocked by inhibition of G(q) signaling. Furthermore, expression of inhibitory mutants of the small GTPase Rnd2/Rho7 or an Rnd2 effector rapostlin abolished LPA(3)-mediated neurite branching. The LPA(3) agonist 2(S)-OMPT or LPA also induced axonal branch formation in hippocampal neurons, which was blocked by G(q) and Rnd2 pathway inhibition or LPA(3) knockdown. These findings suggest that the novel signaling pathway involving LPA(3), G(q), and Rnd2 may play an important role in neuronal network formation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neuregulin1 is a family of growth and differentiation factors involved in various functions of both peripheral and central nervous system including the regenerative processes that underlie regeneration of damaged peripheral nerves. In the present study we tested in vitro the effect of Neuregulin1 administration on dissociated rat dorsal root ganglion (DRG). Activity of neuregulin1 was compared to the activity of nerve growth factor in the same in vitro experimental model. Results showed that neurite outgrowth is enhanced by the addition of both neuregulin1 and nerve growth factor to the culture medium. While neuregulin1 was responsible for the growth of longer neurites, DRG neurons incubated with nerve growth factor showed shorter and more branched axons. Using enzyme-linked immunosorbent assay we also showed that the release of nerve growth factor, but not of brain derived neurotrophic factor is improved in DRG neuron treated with neuregulin1. On the other hand, the assay with growth factor blocking antibody, showed that effects exerted by neuregulin1 on neurite outgrowth is only partially due to the release of nerve growth factor. Taken together the results of this study provide a better understanding on the role of neuregulin1 in sensory neurons., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neurite outgrowth, an essential process for constructing nervous system connectivity, requires molecular cues which promote neurite extension and guide growing neurites. The neural cell adhesion molecule L1 is one of the molecules involved in this process. Growth of neurites depends on actin remodeling, but actin-remodeling proteins which act downstream of L1 signaling are not known. In this study, we investigated whether the actin-remodeling protein cofilin, which can be activated by dephosphorylation, is involved in neurite outgrowth stimulated by L1. Upon stimulation with an L1 monoclonal antibody which specifically triggers L1-dependent neurite outgrowth, cofilin phosphorylation in cultured cerebellar granule neurons and isolated growth cones was reduced to 47 ± 13% or 58 ± 9% of IgG control levels, respectively. We therefore investigated whether cofilin phosphorylation plays a role in L1-stimulated neurite outgrowth. Inhibition of calcineurin, a phosphatase acting upstream of cofilin dephosphorylation, impaired L1-dependent neurite extension in cultures of cerebellar granule neurons and led to an increase in cofilin phosphorylation. Moreover, when peptide S3, a competitive inhibitor of cofilin phosphorylation, or peptide pS3, a competitive inhibitor of cofilin dephosphorylation, were transferred into cerebellar neurons in culture, L1-stimulated neurite outgrowth was reduced from 173 ± 15% to 103 ± 4% of poly-L-lysine control levels in the presence of either peptide. Our findings suggest that both activation of cofilin by dephosphorylation and inactivation of cofilin by phosphorylation are essential for L1-stimulated neurite outgrowth. These results are in accordance with a cofilin activity cycle recently proposed for invasive tumor cells and inflammatory cells, indicating that a similar regulatory mechanism might be involved in neurite outgrowth. As L1 is expressed by invasive tumor cells, cofilin might also be a downstream actor of L1 in metastasis., (Copyright © 2011 Elsevier Inc. All rights reserved.)