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Growth-associated protein 43 (GAP43) is a membrane-associated phosphoprotein predominantly expressed in the nervous systems, and controls axonal growth, branching, and pathfinding. While the association between GAP43 and human neurological disorders have been reported, the underlying mechanisms remain largely unknown. We performed whole exome sequencing on a patient with intellectual disability (ID), neurodevelopmental disorders, short stature, and skeletal abnormalities such as left-right difference in legs and digital deformities, and identified a heterozygous missense variation in the GAP43 gene [NM_001130064.2: c.436G > A/p.(E146K)]. The variant GAP43 protein was unstable in primary cultured cortical neurons and hippocampal neurons in vitro. In utero electroporation of the variant protein also confirmed its instability in vivo, suggesting that the variant led to a condition similar with haploinsufficiency in the patient. Silencing of GAP43 via in utero electroporation of RNAi vectors demonstrated that loss of GAP43 suppressed axon elongation into the contralateral hemisphere and impaired the dendritic arbor formation as shown by decreased dendritic branch points and shortened total dendritic lengths. Collectively, these findings confirmed the critical roles of GAP43 in brain development and the pathological basis of GAP43-associated diseases. Our study will contribute to a better understanding of how dysregulation of GAP43 leads to human diseases., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Spinal cord injury (SCI) is a debilitating central nervous system (CNS) disorder that leads to significant motor and sensory impairments. Given the limited regenerative capacity of adult mammalian neurons, this study presents an innovative strategy to enhance axonal regeneration and functional recovery by identifying a novel factor that markedly promotes axonal regeneration. Employing a zebrafish model with targeted single axon injury in Mauthner cells (M-cells) and utilizing the Tg (Tol056: EGFP) transgenic line for in vivo monitoring, we investigate the intrinsic mechanisms underlying axonal regeneration. This research specifically examines the role of amino acid transport, emphasizing the role of the solute carrier 1A4 amino acid transporter in axonal regeneration. Our findings demonstrate that Slc1a4 overexpression significantly enhances axonal regeneration in M-cells, whereas Slc1a4 deficiency impedes this process, which is concomitant with the downregulation of the P53/Gap43 signaling pathway. By elucidating the fundamental role of Slc1a4 in axonal regeneration and uncovering its underlying mechanisms, this study thus provides novel insights into therapeutic strategies for SCI.

Growth-associated protein of 43 kDa (GAP43) is a key cytoskeleton-associated component of the presynaptic terminal that facilitates neuroplasticity. Downregulation of GAP43 expression has been associated to various psychiatric conditions in humans and evokes hippocampus-dependent memory impairments in mice. Despite the extensive studies conducted on hippocampal GAP43 in past decades, however, very little is known about its roles in modulating the excitatory versus inhibitory balance in other brain regions. We recently generated conditional knock-out mice in which the Gap43 gene was selectively inactivated in either telencephalic glutamatergic neurons ( Gap43 fl/fl ; Nex1 Cre mice, hereafter Glu-GAP43 -/- mice) or forebrain GABAergic neurons ( Gap43 fl/fl ; Dlx5/6 Cre mice, hereafter GABA-GAP43 -/- mice). Here, we show that Glu-GAP43 -/- but not GABA-GAP43 -/- mice of either sex show a striking hyperactive phenotype when exposed to a novel environment. This behavioral alteration of Glu-GAP43 -/- mice was linked to a selective activation of dorsal-striatum neurons, as well as to an enhanced corticostriatal glutamatergic transmission and an abrogation of corticostriatal endocannabinoid-mediated long-term depression. In line with these observations, GAP43 was abundantly expressed in corticostriatal glutamatergic terminals of wild-type mice. The novelty-induced hyperactive phenotype of Glu-GAP43 -/- mice was abrogated by chemogenetically inhibiting corticostriatal afferences with a G i -coupled "designer receptor exclusively activated by designer drugs" (DREADDs), thus further supporting that novelty-induced activity is controlled by GAP43 at corticostriatal excitatory projections. Taken together, these findings show an unprecedented regulatory role of GAP43 in the corticostriatal circuitry and provide a new mouse model with a delimited neuronal-circuit alteration for studying novelty-induced hyperactivity, a phenotypic shortfall that occurs in diverse psychiatric diseases., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Maroto et al.)

Age-related reduction in spine density, synaptic marker expression, and synaptic efficiency are frequently reported. These changes provide the cellular and molecular basis for the cognitive decline characteristic for old age. Nevertheless, there are several approaches that have the potential to ameliorate these processes and improve cognition, caloric restriction being one of the most promising and widely studied. While lifelong caloric restriction is known for its numerous beneficial effects, including improved cognitive abilities and increased expression of proteins essential for synaptic structure and function, the effects of late-onset and/or short-term CR on synaptic plasticity have yet to be investigated. We have previously documented that the effects of CR are strongly dependent on whether CR is initiated in young or old subjects. With this in mind, we conducted a long-term study in aging Wistar rats to examine changes in the expression of several key synaptic markers under the regimen of CR started at different time points in life. We found a significant increase in the expression of both presynaptic and postsynaptic markers. However, taking into account previously reported changes in the behavior detected in these animals, we consider that this increase cannot represent beneficial effect of CR., (© 2024 International Union of Biochemistry and Molecular Biology.)

Schizophrenia is associated with altered cortical circuitry. Although the schizophrenia risk gene NRG1 is known to affect the wiring of inhibitory interneurons, its role in excitatory neurons and axonal development is unclear. Here, we investigated the role of Nrg1 in the development of the corpus callosum, the major interhemispheric connection formed by cortical excitatory neurons. We found that deletion of Nrg1 impaired callosal axon development in vivo. Experiments in vitro and in vivo demonstrated that Nrg1 is cell-autonomously required for axonal outgrowth and that intracellular signaling of Nrg1 is sufficient to promote axonal development in cortical neurons and specifically in callosal axons. Furthermore, our data suggest that Nrg1 signaling regulates the expression of Growth Associated Protein 43, a key regulator of axonal growth. In conclusion, our study demonstrates that NRG1 is involved in the formation of interhemispheric callosal connections and provides a novel perspective on the relevance of NRG1 in excitatory neurons and in the etiology of schizophrenia., (© 2024 Rodríguez-Prieto et al.)

Introduction: Self-repair of spinal cord injury (SCI) has been found in humans and experimental animals with partial recovery of neurological functions. However, the regulatory mechanisms underlying the spontaneous locomotion recovery after SCI are elusive., Aims: This study was aimed at evaluating the pathological changes in injured spinal cord and exploring the possible mechanism related to the spontaneous recovery., Results: Immunofluorescence staining was performed to detect GAP43 expression in lesion site after spinal cord transection (SCT) in rats. Then RNA sequencing and gene ontology (GO) analysis were employed to predict lncRNA that correlates with GAP43. LncRNA smart-silencing was applied to verify the function of lncRNA vof16 in vitro, and knockout rats were used to evaluate its role in neurobehavioral functions after SCT. MicroRNA sequencing, target scan, and RNA22 prediction were performed to further explore the underlying regulatory mechanisms, and miR-185-5p stands out. A miR-185-5p site-regulated relationship with GAP43 and vof16 was determined by luciferase activity analysis. GAP43-silencing, miR-185-5p-mimic/inhibitor, and miR-185-5p knockout rats were also applied to elucidate their effects on spinal cord neurite growth and neurobehavioral function after SCT. We found that a time-dependent increase of GAP43 corresponded with the limited neurological recovery in rats with SCT. CRNA chip and GO analysis revealed lncRNA vof16 was the most functional in targeting GAP43 in SCT rats. Additionally, silencing vof16 suppressed neurite growth and attenuated the motor dysfunction in SCT rats. Luciferase reporter assay showed that miR-185-5p competitively bound the same regulatory region of vof16 and GAP43., Conclusions: Our data indicated miR-185-5p could be a detrimental factor in SCT, and vof16 may function as a ceRNA by competitively binding miR-185-5p to modulate GAP43 in the process of self-recovery after SCT. Our study revealed a novel vof16-miR-185-5p-GAP43 regulatory network in neurological self-repair after SCT and may underlie the potential treatment target for SCI., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

In Alzheimer's disease, amyloid-beta (Aβ) triggers the trans-synaptic spread of tau pathology, and aberrant synaptic activity has been shown to promote tau spreading. Aβ induces aberrant synaptic activity, manifesting in increases in the presynaptic growth-associated protein 43 (GAP-43), which is closely involved in synaptic activity and plasticity. We therefore tested whether Aβ-related GAP-43 increases, as a marker of synaptic changes, drive tau spreading in 93 patients across the aging and Alzheimer's spectrum with available CSF GAP-43, amyloid-PET and longitudinal tau-PET assessments. We found that (1) higher GAP-43 was associated with faster Aβ-related tau accumulation, specifically in brain regions connected closest to subject-specific tau epicenters and (2) that higher GAP-43 strengthened the association between Aβ and connectivity-associated tau spread. This suggests that GAP-43-related synaptic changes are linked to faster Aβ-related tau spread across connected regions and that synapses could be key targets for preventing tau spreading in Alzheimer's disease., (© 2024. The Author(s).)

Objectives: In this study, we employed an in vitro culturing technique to investigate the impact of p53 on the modulation of growth-associated protein-43 (GAP-43) within the primary cortical neurons of rat specimens., Methods: (1) Within the first 24 hours after birth, the bilateral cortex was extracted from newborn Wistar rats and primary cortical neurons were cultured and identified. (2) The changes in the mRNA and protein expressions of GAP-43 induced by p53 in rat primary cortical neurons cultured in vitro were identified utilizing real-time polymerase chain reaction and western blot techniques., Results: (1) Lentiviral transfection of p53 within primary cortical neurons of rats elicited elevated levels of both mRNA and protein expressions of GAP-43, consequently culminating in a noteworthy augmentation of p53 expression. (2) The introduction of a p53 inhibitor in rat primary cortical neurons resulted in a reduction in both mRNA and protein expressions of GAP-43., Conclusion: Within primary rat cortical neurons, p53 has the potential to prompt an augmentation in both the transcriptional and protein expression levels of the GAP-43 protein., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Growth-associated protein 43 (GAP-43) has long been used as a marker for nerve regeneration following nerve injury, with numerous in vitro and animal studies showing its upregulation in regenerating neurons. In humans, expression of GAP-43 has predominantly been examined in skin biopsies from patients with peripheral neuropathies; with several studies showing a reduction in GAP-43 immunoreactive cutaneous nerve fibres. However, it remains elusive whether cutaneous GAP-43 is a valid marker for human nerve regeneration. Here, we present a cohort of 22 patients with electrodiagnostically confirmed carpal tunnel syndrome (CTS), used as a model system for focal nerve injury and neural regeneration after decompression surgery. We evaluate GAP-43 immunoreactivity and RNA expression levels in finger skin biopsies taken before and 6 months after surgery, relative to healthy controls. We further classify patients as 'regenerators' or 'non-regenerators' based on post-surgical epidermal re-innervation. We demonstrate that patients with CTS have lower GAP-43 positive intra-epidermal nerve fibre density (IENFD) before surgery than healthy controls. However, this difference disappears when normalising for total IENFD. Of note, we found surgery did not change GAP-43 expression in IENF, with no differences both in patients who were classified as regenerators and non-regenerators. We also did not identify pre-post surgical differences in cutaneous GAP-43 gene expression or associations with regeneration. These findings suggest cutaneous GAP-43 may not be a compelling marker for nerve regeneration in humans., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Carroll et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: Radiation-induced heart disease (RIHD) is a common sequela of thoracic irradiation. At the same time, nerve remodeling is involved in the progression of heart disease. However, the activation of the nerve remodeling related genes in radiation-induced heart disease is still lacking., Methods: In this study, C57BL/J mice was anesthetized by intraperitoneal injection with pentobarbital sodium (2%, 40 mg/kg), and radiation was delivered using a cobalt-60 ( 60 Co) teletherapy unit (Cirus). When the mice were anesthetized, none of them showed the signs of peritonitis, pain, or discomfort. The mice hearts were exposed to a γ -radiation field of 5 mm × 5 mm. The total dose of γ -radiation was 3 Gy/day for each animal for 5 consecutive days. The mice were executed by severed neck, and its limbs were weak. Quantitative Polymerase Chain Reaction (qPCR) and immunohistochemistry were used to explore the possible mechanism of arrhythmia in patients with RIHD., Results: Our results demonstrated that Growth-Associated Protein 43 (GAP43) was increased significantly after radioactive heart injury compared with the control group. Moreover, the protein expression of Tyrosine hydroxylase (TH) and Choline acetyl-transferase (CHAT) was significantly decreased compared with the control group and gradually increased with time rend. The nerve growth factor (NGF) was remarkably increased after radiation-induced heart injury compared with the control group. Immunohistochemistry results indicated that the nerve growth factors GAP43 and NGF were significantly increased after radiation-induced heart injury., Conclusions: Chest radiotherapy could activate the neural modeling related genes in RIHD. This may provide a new treatment plan for the future treatment of heart problems caused by chest radiotherapy., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2021 Zhiyong Liu et al.)

Objective: To observe the effect of acupuncture serum on the expression of microtubule associated protein-2 (MAP-2) and nerve growth associated protein-43 (GAP-43) in cultured hippocampal neurons of convulsive rats., Methods: The acute convulsion model was induced by intraperitoneal injection of pentylenetetrazol in SD rats who were then randomized into model group and acupuncture group. Rats of the acupuncture group received manual acupuncture stimulation of "Baihui" (GV20) and "Dazhui" (GV14) for 30 min, once daily for 7 days. Then, the blood samples taken from the abdominal aorta of rats in the convulsion model and acupuncture groups were processed into serum samples, i.e. non-acupuncture serum and acupuncture se-rum. The primary-cultured hippocampal neurons of fetal rats were cultured for 10 days and then divided into normal extracellular fluid (normal) group, magnesium (Mg 2+ ) free extracellular fluid group, acupuncture serum group and non-acupuncture serum group. At the 10th day, the neurons in the normal group were cultured continuously in extracellular fluid for 3 h, and then cultured in DMEM/F12(1∶1) medium (planting fluid); neurons in the Mg 2+ free group were cultured in magnesium-free fluid medium to induce epileptic-like discharge; neurons in the acupuncture serum group were cultured in the mixed medium of planting fluid and 10% acupuncture serum; and neurons in the non-acupuncture serum were cultured in the mixed culture medium of planting fluid and non-acupuncture serum (10%). At last, these neurons in the above-mentioned groups were cultured in the magnesium-free extracellular fluid continuously for 2, 12 and 48 h, respectively, followed by detecting the expression levels of MAP-2 and GAP-43 proteins at the 3 time points by using immunofluorescence and Western blot, separately., Results: The rate of MAP-2 positive cells and protein expression at 2, 12 and 48 h, and the rate of GAP-43 positive cells and protein expression at 12 and 48 h in the hippocampal neurons were significantly down-regulated in the Mg 2+ free group in contrast to the normal group ( P <0.05， P <0.01). Compared to the Mg 2+ free group, the rates of MAP-2 and GAP-43 positive cells and protein expression at 2, 12 and 48 h were considerably up-regulated in the acupuncture serum group ( P <0.05， P <0.01), but not in the non-acupuncture serum group (P>0.05)., Conclusion: Acupuncture serum can significantly up-regulate the expression of MAP-2 and GAP-43 proteins in hip-pocampal neurons, which may play a positive role in improving synaptic plasticity and neuronal damage in convulsion rats.

Axon regeneration after lesions to the central nervous system (CNS) is largely limited by the presence of growth inhibitory molecules expressed in myelin. Nogo‑A is a principal inhibitor of neurite outgrowth, and blocking the activity of Nogo‑A can induce axonal sprouting and functional recovery. However, there are limited data on the expression of Nogo‑A after CNS lesions, and the mechanism underlying its influences on myelin growth remains unknown. The aim of the present study was to observe the time course of Nogo‑A after cerebral ischemia/reperfusion in rats using immunohistochemistry and western blot techniques, and to test the effect of its inhibitor Nogo extracellular peptide 1‑40 (NEP1‑40) on neural plasticity proteins, growth‑associated binding protein 43 (GAP‑43) and microtubule associated protein 2 (MAP‑2), as a possible mechanism underlying myelin suppression. A classic model of middle cerebral artery occlusion (MCAO) was established in Sprague‑Dawley rats, which were divided into three groups: i) MCAO model group; ii) MCAO + saline group; and iii) MCAO + NEP1‑40 group. Rats of each group were divided into five subgroups by time points as follows: days 1, 3, 7, 14 and 28. Animals that only received sham operation were used as controls. The Nogo‑A immunoreactivity was located primarily in the cytoplasm of oligodendrocytes. The number of Nogo‑A immunoreactive cells significantly increased from day 1 to day 3 after MCAO, nearly returning to the control level at day 7, increased again at day 14 and decreased at day 28. Myelin basic protein (MBP) immunoreactivity in the ipsilateral striatum gradually decreased from day 1 to day 28 after ischemia, indicating myelin loss appeared at early time points and continuously advanced during ischemia. Then, intracerebroventricular infusion of NEP1‑40, which is a Nogo‑66 receptor antagonist peptide, was administered at days 1, 3 and 14 after MCAO. It was observed that GAP‑43 considerably increased from day 1 to day 7 and then decreased to a baseline level at day 28 compared with the control. MAP‑2 expression across days 1‑28 significantly decreased after MCAO. Administration of NEP1‑40 attenuated the reduction of MBP, and upregulated GAP‑43 and MAP‑2 expression at the corresponding time points after MCAO compared with the MCAO + saline group. The present results indicated that NEP1‑40 ameliorated myelin damage and promoted regeneration by upregulating the expression of GAP‑43 and MAP‑2 related to neuronal and axonal plasticity, which may aid with the identification of a novel molecular mechanism of restriction in CNS regeneration mediated by Nogo‑A after ischemia in rats.

Neuroinflammation has been put forward as a mechanism triggering axonal regrowth in the mammalian central nervous system (CNS), yet little is known about the underlying cellular and molecular players connecting these two processes. In this study, we provide evidence that MMP2 is an essential factor linking inflammation to axonal regeneration by using an in vivo mouse model of inflammation-induced axonal regeneration in the optic nerve. We show that infiltrating myeloid cells abundantly express MMP2 and that MMP2 deficiency results in reduced long-distance axonal regeneration. However, this phenotype can be rescued by restoring MMP2 expression in myeloid cells via a heterologous bone marrow transplantation. Furthermore, while MMP2 deficiency does not affect the number of infiltrating myeloid cells, it does determine the coordinated expression of pro- and anti-inflammatory molecules. Altogether, in addition to its role in axonal regeneration via resolution of the glial scar, here, we reveal a new mechanism via which MMP2 facilitates axonal regeneration, namely orchestrating the expression of pro- and anti-inflammatory molecules by infiltrating innate immune cells.

We investigated the effects of intermittent theta-burst stimulation (iTBS) on locomotor function, motor plasticity, and axonal regeneration in an animal model of incomplete spinal cord injury (SCI). Aneurysm clips with different compression forces were applied extradurally around the spinal cord at T10. Motor plasticity was evaluated by examining the motor evoked potentials (MEPs). Long-term iTBS treatment was given at the post-SCI 5th week and continued for 2 weeks (5 consecutive days/week). Time-course changes in locomotor function and the axonal regeneration level were measured by the Basso Beattie Bresnahan (BBB) scale, and growth-associated protein (GAP)-43 expression was detected in brain and spinal cord tissues. iTBS-induced potentiation was reduced at post-1-week SCI lesion and had recovered by 4 weeks post-SCI lesion, except in the severe group. Multiple sessions of iTBS treatment enhanced the motor plasticity in all SCI rats. The locomotor function revealed no significant changes between pre- and post-iTBS treatment in SCI rats. The GAP-43 expression level in the spinal cord increased following 2 weeks of iTBS treatment compared to the sham-treatment group. This preclinical model may provide a translational platform to further investigate therapeutic mechanisms of transcranial magnetic stimulation and enhance the possibility of the potential use of TMS with the iTBS scheme for treating SCIs., Competing Interests: The authors have no conflicts of interest to disclose.

Peripheral nerve injury (PNI) is a major health problem that results in loss of motor and sensory functions. In treatment of PNI, various methods such as anastomosis, nerve grafts, nonneural tissue grafts, and nerve conduits are applied. In the present study, it was aimed to investigate the effects of Theranekron and Alpha-lipoic acid (ALA) combined treatment on nerve healing in experimental PNI by using histomorphometric, electron microscopic, immunohistochemical and molecular biological methods. Sixty-two Wistar rats were divided into six groups; the normal control group, sham operation group, experimental control group having a crush type injury with no treatment, Theranekron treatment group, ALA treatment group and Theranekron+ALA combined treatment group. Sciatic nerve tissue samples were obtained on days 1, 7 and 14 following injury in all groups. GAP-43 expression was upregulated in all PNI received groups compared to the control group. Krox-20 expression was downregulated in all groups that received PNI compared to the control group. While intensely positive TNF-α and IL-6 expressions were observed up to the 1st to the 14th day for the experimental control group, these expressions were seen as "weakly positive" in the treatment groups from the 1st day to the 14th day. The number of myelinated fibers was higher in the control and sham operation groups. Additionally, the number of myelinated nerve fibers increased in the combined treatment group. In conclusion, these findings suggest that combined therapy of Theranekron and ALA promotes structural recovery and it should be considered as an effective treatment protocol following PNI.

Objective: To observe the effect of moxibustion at "Huantiao" (GB 30) on the expression of growth-associated protein-43 (GAP-43) in the sciatic nerve trunk and ventral horn of spinal cord (L 4 -L 6 ) in rats with primary sciatica, and to explore the mechanism of moxibustion in improving primary sciatica., Methods: A total of 48 healthy male SD rats were randomly divided into a normal group, a sham operation group, a model group and a moxibustion group, 12 rats in each group. The rat model of primary sciatic pain was established by chronic constriction injury (CCI) of the sciatic nerve in the model group and the moxibustion group. On the 8th day of the experiment, moxibustion was adopted at "Huantiao" (GB 30) in the moxibustion group for 5-10 min, once a day for 14 consecutive days. Sciatic nerve function index (SFI) was measured and compared in each group at day 1, 7, 14 and 21. On the 21st day of the experiment, HE staining was used to observe the morphology of ventral horn of rat spinal cord and sciatic nerve trunk. Immunohistochemical method and real-time PCR were used to detect mRNA and protein expressions of GAP-43 in the spinal cord and sciatic nerve trunk of rats., Results: On day 7, 14 and 21, there was no statistical difference in SFI between the sham operation group and the normal group ( P >0.05); compared with the sham operation group on day 7, 14 and 21, the SFI of the model group was reduced ( P <0.01); compared with the model group on day 14 and 21, SFI in the moxibustion group was increased ( P <0.01). In the normal group and the sham operation group, neuronal cells were in order in the ventral horn of the spinal cord, nissl bodies were spaced regularly, the myelin sheath structure of sciatic nerve axon was clearly visible. In the model group, neuronal cells were deformed and ruptured in the ventral horn of the spinal cord, the number of nissl bodies was less, and the demyelination of sciatic axons appeared. In the moxibustion group, neuronal cells were found in the ventral horn of spinal cord, and the number of nissl bodies was increased, and less demyelinating changes of axons appeared in sciatic nerve. Compared with the normal group, the expressions of GAP-43 mRNA and GAP-43 protein in the sciatic nerve trunk and GAP-43 protein in the ventral horn of spinal cord were increased in the sham operation group ( P <0.01). Compared with the sham operation group, the expression of GAP-43 mRNA and GAP-43 protein in the spinal cord and sciatic nerve trunk of rats in the model group was increased. Compared with the model group, the expression of GAP-43 mRNA and GAP-43 protein in the spinal cord and sciatic nerve trunk of rats in the moxibustion group was increased ( P <0.01)., Conclusion: Moxibustion at "Huantiao" (GB 30) could improve the sciatic nerve function in rats with primary sciatica and its mechanism may be related to improving the expression of GAP-43 and enhancing the self-repair ability of the sciatic nerve after injury.

Neurite outgrowth is the basis for wiring during the development of the nervous system. Dl-3-n-butylphthalide (NBP) has been recognized as a promising treatment to improve behavioral, neurological and cognitive outcomes in ischemic stroke. However, little is known about the effect and mechanism of NBP on the neurite outgrowth. In this study, we used different methods to investigate the potential effects of NBP on the neurite extension and plasticity of immature and mature primary cortical neurons and explored the underlying mechanisms. Our results demonstrated that in immature and mature cortical neurons, NBP promoted the neurite length and intersections, increased neuritic arborization, elevated numbers of neurite branch and terminal points and improved neurite complexity and plasticity of neuronal development processes. Besides, our data revealed that NBP promoted neurite extension and branching partly by activating Shh signaling pathway via increasing Gap43 expression both in immature and mature primary cortical neurons. The present study provided new insights into the contribution of NBP in neuronal plasticity and unveiled a novel pathway to induce Gap43 expression in primary cortical neurons., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The growth- and plasticity-associated protein-43 (GAP43) is biasedly expressed in indigestive system and nervous system. Recent study has shown that GAP43 is responsible for the development of neuronal growth and axonal regeneration in normal nervous tissue, while serves as a specific biomarker of relapsed or refractory neuroblastoma. However, its expression pattern and function in digestive system cancer remains to be clarified., Methods: In this study, we examined the GAP43 status with qRT-PCR and bisulfite genomic sequencing in colorectal cancer (CRC). We investigated the effect of overexpressed GAP43 in CRC cells with RNA-seq. The RNA-seq data was analyzed with DAVID and IPA., Results: GAP43 was downregulated in CRC compared to the adjacent tissues. DNA methylase inhibitor 5-Aza-CdR treatment could significantly induce GAP43, indicated that the silencing of GAP43 gene in CRC is closely related to DNA methylation. Bisulfite genomic sequencing confirmed the promoter methylation of GAP43 in CRC. To explore the transcriptional alterations by overexpressed GAP43 in CRC, we performed RNA-seq and found that upregulated genes were significantly enriched in the signaling pathways of ABC transporters and ECM-receptor interaction, while downregulated genes were significantly enriched in Ribosome signaling pathway. Further Ingenuity Pathway Analysis (IPA) showed that EIF2 signaling pathway was significantly repressed by overexpression of GAP43., Conclusion: Our findings provide a novel mechanistic insight of GAP43 in CRC. Transcriptome profiling of overexpressed GAP43 in CRC uncovered the functional roles of GAP43 in the development of human CRC.

Effective nose-to-brain delivery needs to be developed to treat neurodegenerative diseases. Regulating miR-124 can effectively improve the symptoms of ischemic brain injury and provide a certain protective effect from brain damage after cerebral ischemia. We used rat models of middle cerebral artery occlusion (t-MCAO) with ischemic brain injury, and we delivered RVG29-NPs-miR124 intranasally to treat neurological damage after cerebral ischemia. Rhoa and neurological scores in rats treated by intranasal administration of RVG29-PEG-PLGA/miRNA-124 were significantly lower than those in PEG-PLGA/miRNA-124 nasal administration and RVG29-PLGA/miRNA-124 nasal administration group treated rats. These results indicate that the nose-to-brain delivery of PLGA/miRNA-124 conjugated with PEG and RVG29 alleviated the symptoms of cerebral ischemia-reperfusion injury. Thus, nasal delivery of RVG29-PEG-PLGA/miRNA-124 could be a new method for treating neurodegenerative diseases.

Aims: Neurodegeneration of the optic nerve and retinal ganglion cells (RGCs) leads to progressive vision loss. As part of the central nervous system, RGCs show limited ability to regenerate and there is extensive search for neuroprotective agents for optic nerve damage. Methylene blue (MB) exhibits beneficial effects against various neurodegenerative diseases of the central nervous system. However, the mechanisms associated with its putative protection on neuronal survival and regeneration remain obscure. This study used the optic nerve transection model to examine the effects of MB on RGC survival, the expression of regenerative marker GAP-43 in RGCs and microglial activation., Main Methods: Axons of RGCs were injured by cutting the optic nerve. MB was injected intravitreally either immediately post-injury or delayed to 3 days post-injury. Using immunohistochemical staining, surviving RGCs, GAP-43-positive RGCs and microglial cells were quantified in wholemount retinas 7 days post-injury., Key Findings: Both immediate and delayed (a more clinically realistic situation) intravitreal injection of MB promoted RGC survival. MB also increased the number of GAP-43-positive RGCs, suggesting an enhanced ability of RGCs to regenerate. This was exemplified by the regenerative sprouting of axon-like processes from injured RGCs after MB treatment. The increase in RGC survival and GAP-43 expression correlated with an increase in the number of microglial cells., Significance: These results reveal that MB has survival-promoting and growth-promoting effects on RGCs after optic nerve injury. Together with the established safety profile of MB in humans, MB is a promising treatment for neurodegeneration and injury of the optic nerve., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Spinal cord injury (SCI) is a disabling central nervous system disorder. This study aimed to explore the effects of repetitive trans-spinal magnetic stimulation (rTSMS) of different spinal cord segments on movement function and growth-associated protein-43 (GAP43) and 5-hydroxytryptamine (5-HT) expression in rats after acute SCI and to preliminarily discuss the optimal rTSMS treatment site to provide a theoretical foundation and experimental evidence for clinical application of rTSMS in SCI., Methods: A rat T10 laminectomy SCI model produced by transient application of an aneurysm clip was used in the study. The rats were divided into group A (sham surgery), group B (acute SCI without stimulation), group C (T6 segment stimulation), group D (T10 segment stimulation), and group E (L2 segment stimulation)., Results: In vivo magnetic stimulation protected motor function, alleviated myelin sheath damage, decreased NgR and Nogo-A expression levels, increased GAP43 and 5-HT expression levels, and inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and apoptosis-related protein expression in rats at 8 weeks after the surgery., Conclusions: This study suggests that rTSMS can promote GAP43 and 5-HT expression and axonal regeneration in the spinal cord, which is beneficial to motor function recovery after acute SCI.

The evaluation of human epidermal innervation and its impact by disease has largely focused on rigorous immunohistochemical counts of PGP 9.5 labelled axons. In this brief and preliminary report, we expand the repertoire of epidermal axon markers to include those with an influence on their regenerative plasticity. We studied human lower limb punch skin samples with tandem analyses of their mRNA content using qRT-PCR. Normal human subjects (n = 11) and two patients with newly diagnosed CIDP were sampled with the latter undergoing serial tandem biopsies before and after 3 months of immunotherapy. Controls expressed regeneration proteins within dermal and epidermal axons: GAP43 (growth associated protein 43), Shh (sonic hedgehog) and SCG (superior cervical ganglion-10; stathmin 2). Moreover, this expression accompanied intraepidermal nerve fiber density (IENF) within normal established values. CIDP patients had lower IENF but also expressed GAP43, Shh, and SCG. Tandem qRT-PCR identified confirmed the presence not only of these plasticity markers but of additional regeneration related mRNAs. CIDP patients had marked elevation of several mRNAs, with improvement following treatment. The findings support the concept of dynamic skin axon plasticity in humans is relevant toward consideration of newer therapeutic approaches., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This work tested the role of carbamazepine in alleviating alloxan-induced diabetic neuropathy and the enhancement of spinal plasticity. Mice were randomized into four groups: normal, control, carbamazepine (25-mg/kg) and carbamazepine (50-mg/kg). Nine weeks after induction of diabetes, symptoms of neuropathy were confirmed and carbamazepine (or vehicle) was given every other day for five weeks. After completing the treatment period, mice were sacrificed and the pathologic features in the spinal cord and the sciatic nerves were determined. The spinal cords were evaluated for synaptic plasticity (growth associated protein-43, GAP43), microglia cell expression (by CD11b) and astrocyte expression (glial fibrillary acidic protein, GFAP). Further, sciatic nerve expression of Nav1.5 was measured. Results revealed that carbamazepine 50 mg/kg prolonged the withdrawal threshold of von-Frey filaments and increased the hot plate jumping time. Carbamazepine improved the histopathologic pictures of the sciatic nerves and spinal cords. Spinal cord of carbamazepine-treated groups had enhanced expression of GAP43 but lower content of CD11b and GFAP. Furthermore, specimens from the sciatic nerve indicated low expression of Nav1.5. In conclusion, this work provided evidence, for the first time, that the preventive effect of carbamazepine against diabetic neuropathy involves correction of spinal neuronal plasticity and glia cell expression.

Epigenetic changes associated with aging have been linked to functional and cognitive deficits in the adult CNS. Histone acetylation is involved in the control of the transcription of plasticity and regeneration-associated genes. The intrinsic axon growth capacity in the CNS is negatively regulated by phosphatase and tensin homolog (Pten). Inhibition of Pten is an effective method to stimulate axon growth following an injury to the optic nerve, corticospinal tract (CST), and rubrospinal tract (RST). Our laboratory has previously demonstrated that the deletion of Pten in aged animals diminishes the regenerative capacity in rubrospinal neurons. We hypothesize that changes in the chromatin structure might contribute to this age-associated decline. Here, we assessed whether Trichostatin A (TSA), a histone deacetylases (HDACs) inhibitor, reverses the decline in regeneration in aged Pten f/f mice. We demonstrate that HDAC inhibition induces changes in the expression of GAP43 in both young and aged Pten f/f mice. The regenerative capacity of the RST did not improve significantly in young mice, neither their motor function on the horizontal ladder or cylinder test after TSA treatment for 7 days. Interestingly, TSA treatment in the aged mice worsened their motor function deficits, suggesting that the systemic treatment with TSA might have an overall adverse effect on motor recovery after SCI in aged animals., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protein S-acylation is a reversible post-translational modification involving the addition of fatty acids to cysteines and is catalyzed by transmembrane protein acyltransferases (PATs) mainly expressed at the Golgi complex. In case of soluble proteins, S-acylation confers stable membrane attachment. Myristoylation or farnesylation of many soluble proteins constitutes the initial transient membrane adsorption step prior to S-acylation. However, some S-acylated soluble proteins, such as the neuronal growth-associated protein Growth-associated protein-43 (GAP-43), lack the hydrophobic modifications required for this initial membrane interaction. The signals for GAP-43 S-acylation are confined to the first 13 amino acids, including the S-acylatable cysteines 3 and 4 embedded in a hydrophobic region, followed by a cluster of basic amino acids. We found that mutation of critical basic amino acids drastically reduced membrane interaction and hence S-acylation of GAP-43. Interestingly, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) at the Golgi complex reduced GAP-43 membrane binding, highlighting a new, pivotal role for this anionic lipid and supporting the idea that basic amino acid residues are involved in the electrostatic interactions between GAP-43 and membranes of the Golgi complex where they are S-acylated., (© 2020 The Author(s).)

Environmental pollutant, Lead (Pb) is known to induce neurotoxicity in human. The central nervous system is the most vulnerable to the minute levels of Pb induced toxicity. Pb has been linked to Alzheimer's disease (AD) as a probable risk factor, as it shows epigenetic and developmental link associated with Alzheimer's disease-like pathology. Beta amyloid peptides were considered as the crucial factors in the beta amyloid plaque formation in Alzheimer's disease brain. In this context, we investigated the molecular mechanism involved in the development of Pb induced Alzheimer's disease in in vitro. Previous data from our studies have reported that Pb in the presence of beta Amyloid peptide (1-40) and (25-35) induces more apoptosis than individual exposures. Here, to further evaluate the molecular mechanism underlying Pb induced Alzheimer's disease; we focussed on the involvement of calcium signalling in inducing cell death. Our experimental observations suggesting that Pb in the presence of beta amyloid peptide alters intracellular calcium levels, which leads to the increased beta-secretase activity, which further promotes the generation of beta amyloid peptides. It also showed depression in the levels of GAP-43 expression, inhibition of PKC activity and altering synaptic activity further leads to cell death., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The telomerase reverse transcriptase protein, TERT, is expressed in the adult brain and its exogenic expression protects neurons from oxidative stress and from the cytotoxicity of amyloid beta (Aβ). We previously showed that telomerase increasing compounds (AGS) protected neurons from oxidative stress. Therefore, we suggest that increasing TERT by AGS may protect neurons from the Aβ-induced neurotoxicity by influencing genes and factors that participate in neuronal survival and plasticity. Here we used a primary hippocampal cell culture exposed to aggregated Aβ and hippocampi from adult mice. AGS treatment transiently increased TERT gene expression in hippocampal primary cell cultures in the presence or absence of Aβ and protected neurons from Aβ induced neuronal degradation. An increase in the expression of Growth associated protein 43 (GAP43), and Feminizing locus on X-3 genes (NeuN), in the presence or absence of Aβ, and Synaptophysin (SYP) in the presence of Aβ was observed. GAP43, NeuN, SYP, Neurotrophic factors (NGF, BDNF), beta-catenin and cyclin-D1 expression were increased in the hippocampus of AGS treated mice. This data suggests that increasing TERT by pharmaceutical compounds partially exerts its neuroprotective effect by enhancing the expression of neurotrophic factors and neuronal plasticity genes in a mechanism that involved Wnt/beta-catenin pathway.

Emerging evidence indicates that microRNAs play an important role in neural remodeling, including neurite growth, after acute spinal cord injury (ASCI). This study aims to identify the mechanism by which miR-92b-3p regulates neurite growth in vivo and in vitro. Adult Sprague-Dawley rats were selected to establish the ASCI model, and the expressions of miR-92b-3p and phosphate and tensin homolog deleted on chromosome ten (PTEN) were quantified at different time points. The interaction between miR-92b-3p and PTEN was further detected in the PC12 cell line and dual-luciferase reporter assay. Neurite growth proteins (GAP43 and NF-200) were assessed by western blotting after miR-92b-3p mimics treatment. The PTEN/AKT pathway-related proteins and their roles in miR-92b-3p regulation were also identified using western blotting and immunofluorescence in vitro through LY294002, an AKT inhibitor. The effect of miR-92b-3p was further determined in vivo according to the Basso-Beattie-Bresnahan (BBB) Scale and GAP43 and NF-200 expressions. miR-92b-3p was downregulated after ASCI, while PTEN showed a simultaneous opposing trend. Overexpression of miR-92b-3p downregulated PTEN expression and promoted phosphorylation of AKT, as well as the expression of GAP43 and NF-200 in PC12 cells. Furthermore, the dual-luciferase reporter assay revealed that miR-92b-3p exerted its effect by targeting PTEN's 3'-untranslated regions and that this effect could be counteracted by AKT phosphorylation blocker LY294002 through western blotting and immunofluorescence. Moreover, miR-92b-3p could also improve the BBB scale as well as GAP43 and NF-200 expression levels in vivo. Collectively, these results indicate that miR-92b-3p promotes neurite growth and functional recovery through the PTEN/AKT pathway in ASCI., (© 2019 Wiley Periodicals, Inc.)

Thyroid cancer is maintaining at a high incidence level and its carcinogenesis is mainly affected by a complex gene interaction. By analysis of the next-generation resequencing of paired papillary thyroid cancer (PTC) and adjacent thyroid tissues, we found that Growth Associated Protein 43 (GAP43), a phosphoprotein activated by protein kinase C, might be novel markers associated with PTC. However, its function in thyroid carcinoma has been poorly understood. We discovered that GAP43 was significantly overexpressed in thyroid carcinoma and these results were consistent with that in The Cancer Genome Atlas (TCGA) cohort. In addition, some clinicopathological features of GAP43 in TCGA database showed that up-regulated GAP43 is significantly connected to lymph node metastasis (P < 0.001) and tumour size (P = 0.038). In vitro experiments, loss of function experiments was performed to investigate GAP43 in PTC cell lines (TPC-1 and BCPAP). The results proved that GAP43 knockdown in PTC cell significantly decreased the function of cell proliferation, colony formation, migration, and invasion and induced cell apoptosis. Furthermore, we also indicated that GAP43 could modulate the expression of epithelial-mesenchymal transition-related proteins, which could influence invasion and migration. Put those results together, GAP43 is a gene which was associated with PTC and might be a potential therapeutic target., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Aims: Retinopathy is a neurodegenerative complication associating diabetes mellitus. Diabetic retinopathy (DR) is the primary reason of visual loss during early adulthood. DR has a complicated multifactorial pathophysiology initiated by hyperglycaemia-induced ischaemic neurodegenerative retinal changes, followed by vision-threatening consequences. The main therapeutic modalities for DR involve invasive delivery of intravitreal antiangiogenic agents as well as surgical interventions. The current work aimed to explore the potential anti-inflammatory and retinal neuroprotective effects of levetiracetam., Main Methods: This study was performed on alloxan-induced diabetes in mice (n: 21). After 10 weeks, a group of diabetic animals (n: 7) was treated with levetiracetam (25 mg/kg) for six weeks. Retinal tissues were dissected and paraffin-fixed for examination using (1) morphometric analysis with haematoxylin and eosin (HE), (2) immunohistochemistry (GLUT1, GFAP and GAP43), and (3) RT-PCR-detected expression of retinal inflammatory and apoptotic mediators (TNF-α, IL6, iNOS, NF-κB and Tp53)., Key Findings: Diabetic mice developed disorganized and debilitated retinal layers with upregulation of the gliosis marker GFAP and downregulation of the neuronal plasticity marker GAP43. Additionally, diabetic retinae showed increased transcription of NF-κB, TNF-α, IL6, iNOS and Tp53. Levetiracetam-treated mice showed downregulation of retinal GLUT1 with relief and regression of retinal inflammation and improved retinal structural organization., Significance: Levetiracetam may represent a potential neuroprotective agent in DR. The data presented herein supported an anti-inflammatory role of levetiracetam. However, further clinical studies may be warranted to confirm the effectiveness and safety of levetiracetam in DR patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The reparative process following spinal cord injury (SCI) is extremely complicated. Cells in the microenvironment express multiple inhibitory factors that affect axonal regeneration over a prolonged period of time. The axon growth inhibitory factor glycogen synthase kinase-3 (GSK-3) is an important factor during these processes. TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) is the most effective and specific non-ATP-competitive inhibitor of GSK-3. Here, we show that administering TDZD-8 after SCI was associated with significantly inhibited neuronal apoptosis, upregulated GAP-43 expression, increased density of cortical spinal tract fibers around areas of injury, and increased Basso, Beattie, and Bresnahan (BBB) scores in the lower limbs. These findings support the notion that GSK-3 inhibitors promote neuronal cell regeneration and lower limb functional recovery., Competing Interests: All authors declare no conflicts of interest.

Objective: To investigate the effect of astaxanthin on the neurological function of the middle cerebral artery occlusion (MCAO) mice and its possible mechanism., Materials and Methods: The male C57BL/6 mice were selected to establish the model of MCAO via electrocoagulation, and they were randomly divided into 4 groups: the sham operation group (Sham group), the cerebral ischemia model group (MCAO group), the astaxanthin intervention group (gavage with 30 mg/kg astaxanthin for 28 days, twice a day; Ast group), and astaxanthin + H89 group (Ast + H89 group). At 3, 7, 14, and 28 d after the operation, the Rotarod test and the balance beam footstep error test were performed. The brain tissues were taken for immunofluorescence to observe the expression of the growth-associated protein 43 (GAP43) in the cortex around the infarction. The GAP43 protein and mRNA levels in the cortex around the infarction were detected via Western blotting, and the Reverse Transcription-Polymerase Chain Reaction (RT-PCR), the levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in the bilateral cerebral cortex were detected via enzyme-linked immunosorbent assay (ELISA), and the PKAc and phosphorylated-cAMP-response element-binding protein (p-CREB) levels in the bilateral cerebral cortex were detected via Western blotting. Biotin dextran amine (BDA) was injected at 14 d after the operation, and the brain was taken at 28 d. The BDA-labeled neurons or axons were observed in the bilateral cortex via immunohistochemistry and immunofluorescence, and the colocalization of BDA and GAP43 in the cortex around the infarction was observed using double immunofluorescence staining., Results: Compared with those in the MCAO group, the mean residence time in the Rotarod test was significantly increased, and the times of the footstep error on the balance beam were significantly reduced in the Ast group. In the Ast group, the expression of GAP43 in the cortex around the infarction, the GAP43 protein, and the mRNA levels were all significantly elevated. Immunofluorescence showed that in the Ast group, the number of the labeled neurons and axons in the bilateral cortex was slightly larger than that in the other groups, and the number of labeled axonal fibers in the ischemic cortex was significantly increased. The colocalization area of BDA and GAP43 was observed, and it was found that the positive area in the Ast group was significantly larger than that in the MCAO group. The cAMP level was higher in the Ast group and Ast + H89 group at 7, 14, and 28 d after operation, while the PKA level was lower in the Ast + H89 group at 7 and 14 d after operation and higher in the Ast group at 7, 14, and 28 d after operation. The results of the Western blotting manifested that the PKAc and p-CREB levels were upregulated in the Ast group at 7, 14, and 28 d after the operation, and downregulated in the Ast + H89 group at 7, 14, and 28 d after the operation., Conclusions: Astaxanthin activates the cAMP/PKA/CREB signaling pathway by increasing the cAMP concentration in brain tissues, ultimately promoting the axonal regeneration in the cerebral cortex and improving the motor function.

Exercise intensity is known to affect neuroplasticity. Although corticosterone and lactate levels have been linked to neuroplasticity, the effect of different endurance exercise intensity-dependent production of these biochemicals on the behaviour of hippocampal growth cone markers remains incompletely explored. Here, we investigated the effects of three different endurance treadmill training episodes for six weeks on GAP-43 and CAP-1 expression in the hippocampus of adult male Wistar rats. Our findings showed that mild exercise intensity (MEI) with a lactate production slightly higher than the lactate threshold (LT) is the optimal form of physical activity for elevating GAP-43 without changing CAP-1 expression. It was further observed that high exercise intensity (HEI) with the highest level of corticosterone and lactate production, reduced GAP-43 expression, yet increased CAP-1 expression in the hippocampus. Like HEI, we further identified similar expression patterns for these markers in low exercise intensity (LEI) with blood lactate production below LT and corticosterone level similar to MEI. The findings suggested that in high-intensity exercise, the negative pattern of hippocampal neuroplasticity depends on both corticosterone and lactate levels, whereas in low-intensity exercise, the most important factor determining this negative pattern is the lactate level. Generally, MEI with a lactate production of slightly higher than LT is the most optimal intensity for improving hippocampal neuroplasticity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts., Competing Interests: The authors have declared that no competing interests exist.

The fragile X mental retardation 1 knockout (Fmr1 KO) mouse replicates behavioral deficits associated with autism, fragile X syndrome, and schizophrenia. Less is known whether protein expression changes are consistent with findings in subjects with schizophrenia. In the current study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics to determine the protein expression of four subcellular fractions in the forebrains of Fmr1 KO mice vs. C57BL/6 J mice and the effect of a negative allosteric modulator of mGluR5-2-Methyl-6-(phenylethynyl)pyridine (MPEP)-on protein expression. Strain- and treatment-specific differential expression of proteins was observed, many of which have previously been observed in the brains of subjects with schizophrenia. Western blotting verified the direction and magnitude of change for several proteins in different subcellular fractions as follows: neurofilament light protein (NEFL) and 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) in the total homogenate; heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC) and heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) in the nuclear fraction; excitatory amino acid transporter 2 (EAAT2) and ras-related protein rab 3a (RAB3A) in the synaptic fraction; and ras-related protein rab 35 (RAB35) and neuromodulin (GAP43) in the rough endoplasmic reticulum fraction. Individuals with FXS do not display symptoms of schizophrenia. However, the biomarkers that have been identified suggest that the Fmr1 KO model could potentially be useful in the study of schizophrenia., (© 2018 Wiley Periodicals, Inc.)

The present study explored a key miRNA that plays a vital role in sciatic nerve conditioning injury promoting repair of injured dorsal column, and validated its function. Microarray analysis revealed miR-17-5p expression decreased sharply at 3, 7 and 14 days in the sciatic nerve conditioning injury group compared with the simple dorsal column lesion group. After miR-17-5p inhibition in DRG neurons, GAP-43 expression was upregulated and neurite growth was increased. STAT3 together with p-STAT3 showed opposite trends with miR-17-5p. MiR-17-5p inhibition extended neurite and upregulated STAT3, p-STAT3 and GAP-43. To further determine a substitution therapy for sciatic nerve conditioning injury, beta-phenethyl isothiocyanate (PEITC), which downregulates miR-17-5p, was assessed. The results showed that treatment with 10 µM PEITC resulted in longest neurite length. Further experiments demonstrated PEITC induced neurite growth by inhibiting miR-17-5p and further upregulating STAT3, p-STAT3 and GAP-43. The somatosensory evoked potential test confirmed similar treatment effects for PEITC, Ad-miRNA-17-5p inhibitor, and sciatic nerve conditioning injury on the dorsal column lesion. In conclusion, the miR-17-5p/STAT3/GAP-43 axis is an indispensable component of sciatic nerve conditioning injury promoting repair of injured dorsal column. PEITC could promote repair of injured dorsal column via the miR-17-5p/STAT3/GAP-43 axis, and could mimic the treatment effect of sciatic nerve conditioning injury.

Background: Brain metastasis is an extremely serious sequela with a dismal prognosis in non-small cell lung cancer (NSCLC). The present study aimed to identify novel biomarkers and potential therapeutic targets for brain metastases of NSCLC., Methods: We performed high-throughput Luminex assays to profile the transcriptional levels of 36 genes in 70 operable NSCLC patients, among whom 37 developed brain metastases as the first relapse within 3 years after surgery. The Cox proportional hazards regression model was used to evaluate the association between genes and brain metastases. Wound healing assay and transwell assay was carried out to estimate the function of target gene in vitro. And left ventricular injection on nude mice was used to evaluate the effect of target gene in vivo., Results: Growth-associated protein 43 (GAP43) was found to be related to brain metastasis. Multivariate Cox regression analysis showed that NSCLC patients with elevated GAP43 had a 3.29-fold increase in the risk for brain metastasis compared with those with low levels (95% confidence interval: 1.55-7.00; P = 0.002). Kaplan-Meier survival curves revealed that GAP43 was also associated with overall survival. Analysis of a cohort of 1926 NSCLC patients showed similar results: patients with high levels of GAP43 had worse progression-free and overall survival rates. Furthermore, in vitro experiments showed that GAP43 facilitated cell migration. Animal studies demonstrated that GAP43-silenced NSCLC cells were less likely to metastasize to the brain and bone than control cells. Immunofluorescence and F-actin/G-actin in vivo assays indicated that GAP43 knockdown triggered depolymerization of the F-actin cytoskeleton. Rho GTPase activation assays showed that Rac1 was deactivated after GAP43 was silenced., Conclusions: Our findings suggest that GAP43 is an independent predictor of NSCLC brain metastasis and that it may facilitate metastasis by regulating the Rac1/F-actin pathway.

mRNA translation in axons enables neurons to introduce new proteins at sites distant from their cell body. mRNA-protein interactions drive this post-transcriptional regulation, yet knowledge of RNA binding proteins (RBP) in axons is limited. Here we used proteomics to identify RBPs interacting with the axonal localizing motifs of Nrn1 , Hmgb1 , Actb , and Gap43 mRNAs, revealing many novel RBPs in axons. Interestingly, no RBP is shared between all four RNA motifs, suggesting graded and overlapping specificities of RBP-mRNA pairings. A systematic assessment of axonal mRNAs interacting with hnRNP H1, hnRNP F, and hnRNP K, proteins that bound with high specificity to Nrn1 and Hmgb1 , revealed that axonal mRNAs segregate into axon growth-associated RNA regulons based on hnRNP interactions. Axotomy increases axonal transport of hnRNPs H1, F, and K, depletion of these hnRNPs decreases axon growth and reduces axonal mRNA levels and axonal protein synthesis. Thus, subcellular hnRNP-interacting RNA regulons support neuronal growth and regeneration., (© 2018 Lee et al.)

Background: Adipose derived stem cells can be stimulated to produce a growth factor rich secretome which enhances axon regeneration. In this study we investigated the importance of exosomes, extracellular vesicles released by many different cell types, including stem cells and endogenous nervous system Schwann cells (SCs), on neurite outgrowth., Methods: Adipose derived stem cells were differentiated towards a Schwann cell-like phenotype (dADSCs) by in vitro stimulation with a mix of factors (basic fibroblast growth factor, platelet derived growth factor-AA, neuregulin-1 and forskolin). Using a precipitation and low-speed centrifugation protocol the extracellular vesicles were isolated from the medium of the stem cells cultures and also from primary SCs. The conditioned media or concentrated vesicles were applied to neurons in vitro and computerised image analysis was used to assess neurite outgrowth. Total RNA was purified from the extracellular vesicles and investigated using qRT-PCR., Results: Application of exosomes derived from SCs significantly enhanced in vitro neurite outgrowth and this was replicated by the exosomes from dADSCs. qRT-PCR demonstrated that the exosomes contained mRNAs and miRNAs known to play a role in nerve regeneration and these molecules were up-regulated by the Schwann cell differentiation protocol. Transfer of fluorescently tagged exosomal RNA to neurons was detected and destruction of the RNA by UV-irradiation significantly reduced the dADSCs exosome effects on neurite outgrowth. In contrast, this process had no significant effect on the SCs-derived exosomes., Conclusions: In summary, this work suggests that stem cell-derived exosomes might be a useful adjunct to other novel therapeutic interventions in nerve repair.

Background: Despite the intense efforts devoted to preventing and treating cerebral ischemia, some individuals will continue to have completed infarctions. Failure of prevention or intervention does not, however, preclude therapeutic approaches to enhance recovery. Our study aims to explore the effect of multimodal rehabilitation program on the motor function recovery of rats with ischemic stroke., Methods: Rat models of ischemic stroke were established using clean-grade adult male Sprague-Dawley rats. Motor function of rats was scored by the Bederson neurological function, balance beam test, and screen test. Nissl staining was conducted for morphological and structural changes of nerve cells in the arteriae cerebri anterior zone. Immunohistochemistry was applied to detect the expressions of growth-associated protein (GAP-43), synaptophysin (SYN) and Caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was carried out in the corpus striatum 21 days after operation; reverse transcription quantitative polymerase chain reaction and Western blot analysis were conducted for testing messager RNA (mRNA) and protein expressions of heat shock protein 70 (Hsp70) and MYC proto-oncogene (c-Myc)., Results: Rats receiving multimodal rehabilitation program had lower Bederson neurological function, balance beam, and screen test scores on the 7th, 14th and 21st days after operation; more number of neurons surviving in the arteriae cerebri anterior zone at each time point after operation, higher GAP-43 expression on the 7th and 14th days after operation, and higher SYN expression on the 14th and 21st days after operation, on the 7th, 14th and 21st days after operation, higher mRNA and protein expressions of HSP70 and C-MYC, lower Caspase-3 positive expression and TUNEL positive stained cells., Conclusions: Multimodal rehabilitation program could promote motor function recovery of rats after ischemic stroke by upregulating GAP-43 and SYN expressions at arteriae cerebri anterior zone and upregulating HSP70 and C-MYC expressions in the brain tissues., (Copyright © 2018. Published by Elsevier Inc.)

Background: The formation of intrafusal muscle (IM) fibers and their contact with afferent proprioceptive axons is critical for construction, function, and maintenance of the stretch reflex. Many factors affect the formation of IM fibers. Finding new factors and mechanisms of IM fiber formation is essential for the reconstruction of stretch reflex arc after injury., Methods: We established a coculture system of organotypic dorsal root ganglion (DRG) explants and dissociated skeletal muscle (SKM) cells. The formation of IM fibers was observed in this coculture system after neuregulin-1β (NRG-1β) incubation., Results: We found that NRG-1β promoted outgrowth of neurites and migration of neurons from the organotypic DRG explants and that this correlated with an induction of growth-associated protein 43 (GAP-43) expression. NRG-1β also increased the amount of nuclear bag fibers and nuclear chain fibers by elevating the proportion of tyrosine kinase receptor C (TrkC) phenotypic DRG neurons. In addition, we found that the effects of NRG-1β could be blocked by inhibiting ERK1/2, PI3K/Akt, and JAK2/STAT3 signaling pathways., Conclusion: These data imply that NRG-1β promoted neurite outgrowth and neuronal migration from the organotypic DRG explants and that this correlated with an induction of GAP-43 expression. The modulating effects of NRG-1β on TrkC DRG neuronal phenotype may link to promote IM fiber formation. The effects produced by NRG-1β in this neuromuscular coculture system provide new data for the therapeutic potential on IM fiber formation after muscle injury.

Periodontal ligament mesenchymal stem cells (hPDLSCs), as well as all mesenchymal stem cells, show self-renewal, clonogenicity, and multi-tissue differentiation proprieties and can represent a valid support for regenerative medicine. We treated hPDLSCs with a combination of Moringin (MOR) and Cannabidiol (CBD), in order to understand if treatment could improve their survival and their in vitro differentiation capacity. Stem cells survival is fundamental to achieve a successful therapy outcome in the re-implanted tissue of patients. Through NGS transcriptome analysis, we found that combined treatment increased hPDLSCs survival, by inhibition of apoptosis as demonstrated by enhanced expression of anti-apoptotic genes and reduction of pro-apoptotic ones. Moreover, we investigated the possible involvement of PI3K/Akt/mTOR pathway, emphasizing a differential gene expression between treated and untreated cells. Furthermore, hPDLSCs were cultured for 48 h in the presence or absence of CBD and MOR and, after confirming the cellular viability through MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide) assay, we examined the presence of neuronal markers, through immunofluorescence analysis. We found an increased expression of Nestin and GAP43 (growth associated protein 43) in treated cells. In conclusion, hPDLSCs treated with Moringin and Cannabidiol showed an improved survival capacity and neuronal differentiation potential., Competing Interests: The authors declare no conflict of interest.

Hirschsprung disease (HSCR) is a severe multifactorial genetic disorder. Microarray studies indicated GAL, GAP43 and NRSN1 might contribute to the altered risk in HSCR. Thus, we focused on genetic variations in GAL, GAP43 and NRSN1, and the gene-gene interactions involved in HSCR susceptibility. We recruited a strategy combining case-control study and MassArray system with interaction network analysis. For GAL, GAP43 and NRSN1, a total of 18 polymorphisms were assessed in 104 subjects with sporadic HSCR and 151 controls of Han Chinese origin. We found statistically significant differences between HSCR and control groups at 5 genetic variants. For each gene, the haplotypes combining all polymorphisms were the most significant. Based on SNPsyn, MDR and GeneMANIA analyses, we observed significant gene-gene interactions among GAL, GAP43, NRSN1 and our previous identified RELN, GABRG2 and PTCH1. Our study for the first time indicates that genetic variants within GAL, GAP43 and NRSN1 and related gene-gene interaction networks might be involved in the altered susceptibility to HSCR in the Han Chinese population, which might shed more light on HSCR pathogenesis., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Neuromas are pathologic nerve distensions caused by a nerve's response to trauma, resulting in a dysfunctional to non-functional nerve. Depending on the severance of the affected nerve, the resulting neuroma can be differentiated into continuous and stump neuroma. While neuroma formation has been investigated in animal models with enormous regenerative capacity, the search for differences in human response to nerve trauma on a molecular level ultimately seeks to identify reasons for functionally successful versus unsuccessful regeneration after peripheral nerve trauma in man., Methods: In the present study, the regenerative potential of axons and the capability of Schwann cells (SC) to remyelinate regenerating axons was quantitatively and segmentally analyzed and compared within human neuroma in-continuity and discontinuity., Results: For the stump neuroma and the neuroma in-continuity, there was a significant reduction of the total number of axons (86% stump neuroma and 91% neuroma in-continuity) from the proximal to the distal part of the neuroma, while the amount of fibrotic tissue increased, respectively. Labeling the myelin sheath of regenerating axons revealed a remyelination of regenerating axons by SCs in both neuroma types. The segmented analysis showed no distinct alterations in the number and spatial distribution of regenerating, mature, and myelinated axons between continuous and discontinuous neuroma., Conclusions: The quantitative and segmented analysis showed no distinct alterations in the number and spatial distribution of regenerating, mature, and myelinated axons between continuous and discontinuous neuroma, while the extensive expression of Gap43 in up to 55% of the human neuroma axons underlines their regenerative capacity independent of whether the neuroma is in continuity or discontinuity. Remyelination of Gap43-positive axons suggests that the capability of SCs to remyelinate regenerating axons is preserved in neuroma tissue.

Valproic acid (VPA), a medication primarily used to treat epilepsy and bipolar disorder, has been applied to the repair of central and peripheral nervous system injury. The present study investigated the effect of VPA on functional recovery, survival of facial motor neurons (FMNs), and expression of proteins in rats after facial nerve trunk transection by functional measurement, Nissl staining, TUNEL, immunofluorescence, and Western blot. Following facial nerve injury, all rats in group VPA showed a better functional recovery, which was significant at the given time, compared with group NS. The Nissl staining results demonstrated that the number of FMNs survival in group VPA was higher than that in group normal saline (NS). TUNEL staining showed that axonal injury of facial nerve could lead to neuronal apoptosis of FMNs. But treatment of VPA significantly reduced cell apoptosis by decreasing the expression of Bax protein and increased neuronal survival by upregulating the level of brain-derived neurotrophic factor (BDNF) and growth associated protein-43 (GAP-43) expression in injured FMNs compared with group NS. Overall, our findings suggest that VPA may advance functional recovery, reduce lesion-induced apoptosis, and promote neuron survival after facial nerve transection in rats. This study provides an experimental evidence for better understanding the mechanism of injury and repair of peripheral facial paralysis.

The delivery of a nerve insult (a "conditioning lesion") prior to a subsequent test lesion increases the number of regenerating axons and accelerates the speed of regeneration from the test site. A major barrier to clinical translation is the lack of an ethically acceptable and clinically feasible method of conditioning that does not further damage the nerve. Conditioning electrical stimulation (CES), a non-injurious intervention, has previously been shown to improve neurite outgrowth in vitro. In this study, we examined whether CES upregulates regeneration-associated gene (RAG) expression and promotes nerve regeneration in vivo, similar to a traditional nerve crush conditioning lesion (CCL). Adult rats were divided into four cohorts based on conditioning treatment to the common peroneal (fibular) nerve: i) CES (1h, 20Hz); ii) CCL (10s crush); iii) sham CES (1h, 0Hz); or iv) naïve (unconditioned). Immunofluorescence and qRT-PCR revealed significant RAG upregulation in the dorsal root ganglia of both CES and CCL animals, evident at 3-14days post-conditioning. To mimic a clinical microsurgical nerve repair, all cohorts underwent a common peroneal nerve cut and coaptation one week following conditioning. Both CES and CCL animals increased the length of nerve regeneration (3.8-fold) as well as the total number of regenerating axons (2.2-fold), compared to the sham and naïve-conditioned animals (p<0.001). These data support CES as a non-injurious conditioning paradigm that is comparable to a traditional CCL and is therefore a novel means to potentially enhance peripheral nerve repair in the clinical setting., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In peripheral nerve (PN) injuries requiring surgical repair, as in PN transection, cellular and ECM remodeling at PN epineurial repair sites is hypothesized to reduce PN functional outcomes by slowing, misdirecting, or preventing axons from regrowing appropriately across the repair site. Herein this study reports on deriving and analyzing fetal porcine urinary bladder extracellular matrix (fUB-ECM) by vacuum assisted decellularization, fabricating fUBM-ECM nerve wraps, and testing fUB-ECM nerve wrap biocompatibility and bioactivity in a trigeminal, infraorbital nerve (ION) branch transection and direct end-to-end repair model in rat. FUB-ECM nerve wraps significantly improved epi- and endoneurial organization and increased both neovascularization and growth associated protein-43 (GAP-43) expression at PN repair sites, 28-days post surgery. However, the number of neurofilament positive axons, remyelination, and whisker-evoked response properties of ION axons were unaltered, indicating improved tissue remodeling per se does not predict axon regrowth, remyelination, and the return of mechanoreceptor cortical signaling. This study shows fUB-ECM nerve wraps are biocompatible, bioactive, and good experimental and potentially clinical devices for treating epineurial repairs. Moreover, this study highlights the value provided by precise, analytic models, like the ION repair model, in understanding how PN tissue remodeling relates to axonal regrowth, remyelination, and axonal response properties.

Optic nerve injury is a common disease. The present study aimed to examine the possible role of microRNA‑204 (miR‑204) in optic nerve injury through the regulation of growth‑associated protein-43 (GAP‑43). Initially, optic nerve injury models were established in Sprague‑Dawley (SD) rats, and the function of miR‑204 was either enhanced or inhibited through injection of miR‑204 mimic and inhibitor, respectively. Subsequently, the mRNA and protein levels of miR‑204, GAP‑43, toll‑like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor‑κB (NF‑κB) were examined in retinal tissues using reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The apoptosis of retinal tissue cells was also detected using a terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay. There was a significant increase in the level of miR‑204 in retinal blood vessels of the model SD rats, compared with that in the normal SD rats (P<0.05), and the expression of GAP‑43 was significantly decreased (P<0.05). The results confirmed that the expression of GAP‑43 was significantly reduced, compared with that in the normal control group when the rats were treated with miR‑204 mimic (P<0.05), which was similar to the result in the model group. By contrast, its expression of GAP‑43 was significantly increased when treated with the miR‑204 inhibitor (P<0.05). Compared with the normal control group, the expression levels of TLR4, MyD88 and NF‑κB were significantly increased in the miR‑204 mimic group and model group (P<0.05), whereas the same three factors in the miR‑204 inhibitor group were effectively inhibited, compared with those in the model group, and showed similar results to the normal control group. The apoptotic rates of retinal cells in the miR‑204 mimic group and model group were significantly increased, compared with that in the normal control group (P<0.05), whereas miR‑204 inhibitor effectively reversed the effects on apoptotic rate observed in the model group, showing similar results to those in the normal control group. Taken together, miR‑204 promoted the apoptosis of retinal cells through inhibiting GAP‑43, providing theoretical guidance for the function of GAP‑43 in retinal injury.