Expression Of Growth-associated Protein Research Articles

Heterologous expressing melittin in a probiotic yeast to evaluate its function for promoting NSC-34 regeneration

Melittin is a bioactive peptide and the predominant component in bee venom (BV), studied for its many medical properties, such as antibacterial, anti-inflammatory, anti-arthritis, nerve damage reduction, and muscle cell regeneration. Melittin is primarily obtained through natural extraction and chemical synthesis; however, both methods have limitations and cannot be used for mass production. This study established a heterologous melittin expression system in the probiotic yeast Kluyveromyces marxianus. This yeast was selected for its advantages in stress tolerance and high secreted protein yields, simplifying purification. A > 95% high-purity melittin (MET) and its precursor promelittin (ProMET) were successfully produced and purified at 1.68 μg/mL and 3.33 μg/mL concentrations and verified through HPLC and mass spectrum. The functional test of the NSC-34 cell regeneration revealed that MET achieved the best activity compared to ProMET and the natural-extracted BV groups. Growth-related gene expressions were evaluated, including microtubule-associated protein 2 (MAP2), microtubule-associated protein Tau (MAPT), growth-associated protein 43 (GAP-43), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholine esterase (AChE). The results indicated that treating MET increased MAP2, GAP-43, and VAChT expressions, in which cholinergic signaling is related to neurological functions. A heterologously expressed melittin in a probiotic yeast and its potential for promoting NSC-34 regeneration described here facilitate commercial and therapeutic use.Key points• MET and its precursor ProMET were successfully hetero-expressed in K. marxianus• > 95% high-purity MET and ProMET were purified at 1.68 μg/mL and 3.33 μg/mL• MET has no cytotoxicity toward NSC-34 and significantly promotes NSC-34 growth

Urinary dysfunction after spinal cord injury: Comparing outcomes after thoracic spinal transection and contusion in the rat

Spinal cord injury (SCI) above the lumbosacral spinal cord induces loss of voluntary control over micturition. Spinal cord transection (SCT) was the gold standard method to reproduce SCI in rodents, but its translational value is arguable and other experimental SCI methods need to be better investigated, including spinal cord contusion (SCC). At present, it is not fully investigated if urinary impairments arising after transection and contusion are comparable. To explore this, we studied bladder-reflex activity and lower urinary tract (LUT) and spinal cord innervation after SCT and different severities of SCC. Severe-contusion animals presented a longer spinal shock period and the tendency for higher residual volumes, followed by SCT and mild-contusion animals. Urodynamics showed that SCT animals presented higher basal and peak bladder pressures. Immunostaining against growth-associated protein-43 (GAP43) and calcitonin gene-related peptide (CGRP) at the lumbosacral spinal cord demonstrated that afferent sprouting is dependent on the injury model, reflecting the severity of the lesion, with a higher expression in SCT animals. In LUT organs, the expression of GAP43, CGRP cholinergic (vesicular acetylcholine transporter (VAChT)) and noradrenergic (tyrosine hydroxylase (TH)) markers was reduced after SCI in the LUT and lumbosacral cord, but only the lumbosacral expression of VAChT was dependent on the injury model. Overall, our findings demonstrate that changes in LUT innervation and function after contusion and transection are similar but result from distinct neuroplastic processes at the lumbosacral spinal cord. This may impact the development of new therapeutic options for urinary impairment arising after spinal cord insult.

Nrg1 intracellular signaling regulates the development of interhemispheric callosal axons in mice.

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.

Dexamethasone induces developmental axon damage in the offspring hippocampus by activating miR-210-3p/miR-362-5p to target the aberrant expression of Sonic Hedgehog

Given the extensive application of dexamethasone in both clinical settings and the livestock industry, human exposure to this drug can occur through various sources and pathways. Prior research has indicated that prenatal exposure to dexamethasone (PDE) heightens the risk of cognitive and emotional disorders in offspring. Axonal development impairment is a frequent pathological underpinning for neuronal dysfunction in these disorders, yet it remains unclear if it plays a role in the neural damage induced by PDE in the offspring. Through RNA-seq and bioinformatics analysis, we found that various signaling pathways related to nervous system development, including axonal development, were altered in the hippocampus of PDE offspring. Among them, the Sonic Hedgehog (SHH) signaling pathway was the most significantly altered and crucial for axonal development. By using miRNA-seq and targeting miRNAs and glucocorticoid receptor (GR) expression, we identified miR-210-3p and miR-362-5p, which can target and suppress SHH expression. Their abnormal high expression was associated with GR activation in PDE fetal rats. Further testing of PDE offspring rats and infant peripheral blood samples exposed to dexamethasone in utero showed that SHH expression was significantly decreased in peripheral blood mononuclear cells (PBMCs) and was positively correlated with SHH expression in the hippocampus and the expression of the axonal development marker growth-associated protein-43. In summary, PDE-induced hippocampal GR-miR-210-3p/miR-362-5p-SHH signaling axis changes lead to axonal developmental damage. SHH expression in PBMCs may reflect axonal developmental damage in PDE offspring and could serve as a warning marker for fetal axonal developmental damage.

P53-induced GAP-43 Upregulation in Primary Cortical Neurons of Rats.

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. (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. (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. 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.

A highly bioavailable curcumin formulation ameliorates inflammation cytokines and neurotrophic factors in mice with traumatic brain injury.

A novel curcumin formulation increases relative absorption by 46 times (CurcuWIN®) of the total curcuminoids over the unformulated standard curcumin form. However, the exact mechanisms by which curcumin demonstrates its neuroprotective effects are not fully understood. This study aimed to investigate the impact of a novel formulation of curcumin on the expression of brain-derived neurotrophic factor (BDNF), glial fibrillary acidic protein (GFAP), a main component of the glial scar and growth-associated protein-43 (GAP-43), a signaling molecule in traumatic brain injury (TBI). Mice (adult, male, C57BL/6j) were randomly divided into three groups as follows: TBI group (TBI-induced mice); TBI + CUR group (TBI mice were injected i.p. curcumin just after TBI); TBI+ CurcuWIN® group (TBI mice were injected i.p. CurcuWIN® just after TBI). Brain injury was induced using a cold injury model. Injured brain tissue was stained with Cresyl violet to evaluate infarct volume and brain swelling, analyzed, and measured using ImageJ by Bethesda (MD, USA). Western blot analysis was performed to determine the protein levels related to injury. While standard curcumin significantly reduced brain injury, CurcuWIN® showed an even greater reduction associated with reductions in glial activation, NF-κB, and the inflammatory cytokines IL-1β and IL-6. Additionally, both standard curcumin and CurcuWIN® led to increased BDNF, GAP-43, ICAM-1, and Nrf2 expression. Notably, CurcuWIN® enhanced their expression more than standard curcumin. This data suggests that highly bioavailable curcumin formulation has a beneficial effect on the traumatic brain in mice.

Transplantation of autologous bone marrow–derived mononuclear cells into cerebrospinal fluid in a canine model of spinal cord injury

IntroductionSpinal cord injury (SCI) is associated with severe dysfunction of nervous tissue, and repair via the transplantation of bone marrow–derived mononuclear cells (BM-MNCs) into cerebrospinal fluid yields promising results. It is essential to understand the underlying mechanisms; therefore, this study aimed to evaluate the regenerative potential of autologous BM-MNC transplantation in a canine model of acute SCI. MethodsSix dogs were included in this study, and SCI was induced using an epidural balloon catheter between L2 and L3, particularly in the area of the anterior longitudinal ligament. BM-MNC transplantation was performed, and T2-weighted magnetic resonance imaging (MRI) was conducted at specific time points (i.e., immediately after inducing SCI and at 1, 2, and 4 weeks after inducing SCI); moreover, the expression of growth-associated protein 43 (GAP-43) was evaluated. ResultsMRI revealed that the signal intensity reduced over time in both BM-MNC–treated and control groups. However, the BM-MNC–treated group exhibited a significantly faster reduction than the control group during the early stages of SCI induction (BM-MNC–treated group: 4.82 ± 0.135 cm [day 0], 1.71 ± 0.134 cm [1 week], 1.37 ± 0.036 cm [2 weeks], 1.21 cm [4 weeks]; control group: 4.96 ± 0.211 cm [day 0], 2.49 ± 0.570 cm [1 week], 1.56 ± 0.045 cm [2 weeks], 1.32 cm [4 weeks]). During the early stages of treatment, GAP-43 was significantly expressed at the proximal end of the injured spinal cord in the BM-MSC–treated group, whereas it was scarcely expressed in the control group. ConclusionsIn SCI, transplanted BM-MNCs can activate the expression of GAP-43, which is involved in axonal elongation (an important process in spinal cord regeneration). Thus, cell therapy with BM-MNCs can provide favorable outcomes in terms of better regenerative capabilities compared with other therapies.

Effect and mechanism of Zuogui Pills on neural function recovery in ischemic stroke mice based on OPN/IGF-1/mTOR

To explore the effect and mechanism of Zuogui Pills in promoting neural tissue recovery and functional recovery in mice with ischemic stroke. Male C57BL/6J mice were randomly divided into a sham group, a model group, and low-, medium, and high-dose Zuogui Pills groups(3.5, 7, and 14 g·kg~(-1)), with 15 mice in each group. The ischemic stroke model was established using photochemical embolization. Stiker remove and irregular ladder walking behavioral tests were conducted before modeling and on days 7, 14, 21, and 28 after medication. Triphenyl tetrazolium chloride(TTC) staining was performed on day 3 after modeling, and T2-weighted imaging(T2WI) and diffusion-weighted imaging(DWI) were performed on day 28 after medication to evaluate the extent of brain injury. Hematoxylin-eosin(HE) staining was performed to observe the histology of the cerebral cortex. Axonal marker proteins myelin basic protein(MBP), growth-associated protein 43(GAP43), mammalian target of rapamycin(mTOR), and its downstream phosphorylated s6 ribosomal protein(p-S6), as well as mechanism-related proteins osteopontin(OPN) and insulin-like growth factor 1(IGF-1), were detected using immunofluorescence and Western blot. Zuogui Pills had a certain restorative effect on the neural function impairment caused by ischemic stroke in mice. TTC staining showed white infarct foci in the sensory-motor cortex area, and T2WI imaging revealed cystic necrosis in the sensory-motor cortex area. The Zuogui Pills groups showed less brain tissue damage, fewer scars, and more capillaries. The number of neuronal axons in those groups was higher than that in the model group, and neuronal activity was stronger. The expression of GAP43, OPN, IGF-1, and mTOR proteins in the Zuogui Pills groups was higher than that in the model group. In summary, Zuogui Pills can promote the recovery of neural function and axonal growth in mice with ischemic stroke, and its mechanism may be related to the activation of the OPN/IGF-1/mTOR signaling pathway.

Alternating current stimulation promotes neurite outgrowth and plasticity in neurons through activation of the PI3K/AKT signaling pathway.

As a commonly used physical intervention, electrical stimulation (ES) has been demonstrated to be effective in the treatment of central nervous system disorders. Currently, researchers are studying the effects of electrical stimulation on individual neurons and neural networks, which are dependent on factors such as stimulation intensity, duration, location, and neuronal properties. However, the exact mechanism of action of electrical stimulation remains unclear. In some cases, repeated or prolonged electrical stimulation can lead to changes in the morphology or function of the neuron. In this study, immunofluorescence staining and Sholl analysis are used to assess changes in the neurite number and axon length to determine the optimal pattern and stimulation parameters of ES for neurons. Neuronal death and plasticity are detected by TUNEL staining and microelectrode array assays, respectively. mRNA sequencing and bioinformatics analysis are applied to predict the key targets of the action of ES on neurons, and the identified targets are validated by western blot analysis and qRT-PCR. The effects of alternating current stimulation (ACS) on neurons are more significant than those of direct current stimulation (DCS), and the optimal parameters are 3 μA and 20 min. ACS stimulation significantly increases the number of neurites, the length of axons and the spontaneous electrical activity of neurons, significantly elevates the expression of growth-associated protein-43 (GAP-43) without significant changes in the expression of neurotrophic factors. Furthermore, application of PI3K/AKT-specific inhibitors significantly abolishes the beneficial effects of ACS on neurons, confirming that the PI3K/AKT pathway is an important potential signaling pathway in the action of ACS.

Nicotine Decreases Nerve Regeneration and Pain Behaviors via PTEN and Downstream Inflammation-Related Pathway in Two Rat Nerve Injury Models.

Neuropathic pain is stubborn and associated with the peripheral nerve regeneration process. Nicotine has been found to reduce pain, but whether it is involved in the regulation of nerve regeneration and the underlying mechanism are unknown. In this study, we examined the mechanical allodynia thermal hyperalgesia together with the peripheral nerve regeneration after nicotine exposure in two rat neuropathic pain models. In the spinal nerve ligation model, in which anatomic nerve regeneration can be easily observed, nicotine reduced anatomic measures of regeneration as well as expression of regeneration marker growth-associated protein 43 (GAP43). In the tibial nerve crush model, nicotine treatment significantly suppressed GAP43 expression and functional reinnervation as measured by myelinated action potential and electromyography of gastrocnemius. In both models, nicotine treatment reduced macrophage density in the sensory ganglia and peripheral nerve. These effects of nicotine were reversed by the selective α7 nicotinic acetylcholine receptor (nAChR) blocker methyllycaconitine. In addition, nicotine significantly elevated expression of PTEN (the phosphatase and tensin homolog deleted on chromosome 10), a key player in both regeneration and pain. Pharmacological interference of PTEN could regulate GAP43 expression, pain-related behaviors, and macrophage infiltration in a nicotine-treated nerve crush model. Our results reveal that nicotine and its α7-nAChR regulate both peripheral nerve regeneration process and pain though PTEN and the downstream inflammation-related pathway.

Effects of Curcumin on Axon Growth and Myelin Sheath Formation in an In Vitro Model.

Although the beneficial effects of curcumin, extracted from rhizomes of the ginger family genus Curcuma, on the repair and regeneration of nerves have been evaluated in vitro, there are few studies concerning its effects on axon myelination. Here, we used pheochromocytoma cells as an in vitro model of peripheral nerves. Pheochromocytoma cells were cultured alone or cocultured with Schwann cells and treated with increasing concentrations of curcumin. Cell growth was observed, and the expression levels of growth-associated protein 43 (GAP-43), microtubule-associated protein 2 (MAP-2), myelin basic protein (MBP), myelin protein zero (MPZ), Krox-20, and octamer binding factor 6 (Oct-6) were quantified. We found a significant increase in expression of all six proteins following curcumin treatment, with a corresponding increase in the levels of MBP, MPZ, Krox-20, and Oct-6 mRNA. Upregulation was greater with increasing curcumin concentration, showing a concentration-dependent effect. The results suggested that curcumin can promote the growth of axons by upregulating the expression of GAP-43 and MAP-2, stimulate synthesis and secretion of myelin-related proteins, and facilitate formation of the myelin sheath in axons by upregulating the expression of Krox-20 and Oct-6. Therefore, curcumin could be widely applied in future strategies for the treatment of nerve injuries.

Study on in vitro differentiation of human adenoid-derived mesenchymal stem cells into olfactory sensory neurons

Objective: To investigate the feasibility of isolation and culture of human adenoid-derived mesenchymal stem cells (aMSCs) in vitro, and to observe the differentiation of aMSCs into olfactory sensory neurons. Methods: Adenoid tissues surgically removed from children with adenoid hypertrophy in the Second Xiangya Hospital of Central South University from September to November of 2020 were collected. The adenoid tissues were digested and isolated by trypsin and then cultured with adhesion method. The expressions of cell surface antigens CD45, CD73 and CD90 on aMSCs of P5 generation were tested by flow cytometry, and the ability of osteogenic and adipogenic induction were used to identify cell differentiation ability. Then, aMSCs were induced into differentiation by retinoic acid (RA), sonic hedgehog (SHH), basic fibroblast growth factor (bFGF), RA+SHH, RA+bFGF, SHH+bFGF and RA+SHH+bFGF, respectively. The morphology of differentiated cells was observed under inverted microscope. The expression of β-tubulin 3, which was the specific marker of sensory neuron, the expressions of growth associated protein-43 (GAP43) and olfactory maker protein (OMP), which were the specific markers of olfactory sensory neuron, were detected by immunofluorescence antibody assay. The expression intensities were compared by Chi-square test of four-grid table data. Results: aMSCs were successively isolated and cultured from human adenoid tissues. P0 cells generation had good adhesion and proliferation performance. P2 cells were basically purified. P5 cells expressed CD73 and CD90 with the purity of 99.3% and 99.75% respectively, without CD45 expression. P5 cells had a good ability of osteogenic differentiation and adipogenic differentiation. Neuron-like morphology and expression of β-tubulin 3 were found in differentiated cells after induced by RA, SHH, or bFGF, respectively. An induction of expression of GAP43 was found in differentiated cells of bFGF+SHH group and RA+SHH+bFGF group, without expression of OMP of each group. The intensity of GAP43 expression of RA+SHH+bFGF group was stronger than that of bFGF+SHH group (χ2=17.48, P<0.005). Conclusions: aMSCs can be cultured from human adenoid tissues, with the stably passaged and good differentiation ability. As a new population of mesenchymal stem cells, aMSCs have the neuroregenerative properties and could differentiate into immature olfactory sensory neurons under the induction of RA+SHH+bFGF in vitro.

LncRNA LOC100911717-targeting GAP43-mediated sympathetic remodeling after myocardial infarction in rats.

Sympathetic remodeling after myocardial infarction (MI) is the primary cause of ventricular arrhythmias (VAs), leading to sudden cardiac death (SCD). M1-type macrophages are closely associated with inflammation and sympathetic remodeling after MI. Long noncoding RNAs (lncRNAs) are critical for the regulation of cardiovascular disease development. Therefore, this study aimed to identify the lncRNAs involved in MI and reveal a possible regulatory mechanism. M0- and M1-type macrophages were selected for sequencing and screened for differentially expressed lncRNAs. The data revealed that lncRNA LOC100911717 was upregulated in M1-type macrophages but not in M0-type macrophages. In addition, the lncRNA LOC100911717 was upregulated in heart tissues after MI. Furthermore, an RNA pull-down assay revealed that lncRNA LOC100911717 could interact with growth-associated protein 43 (GAP43). Essentially, immunofluorescence assays and programmed electrical stimulation demonstrated that GAP43 expression was suppressed and VA incidence was reduced after lncRNA LOC100911717 knockdown in rat hearts using an adeno-associated virus. We observed a novel relationship between lncRNA LOC100911717 and GAP43. After MI, lncRNA LOC100911717 was upregulated and GAP43 expression was enhanced, thus increasing the extent of sympathetic remodeling and the frequency of VA events. Consequently, silencing lncRNA LOC100911717 could reduce sympathetic remodeling and VAs.

An Enriched Environment Promotes Motor Function through Neuroprotection after Cerebral Ischemia.

Stroke seriously affects human health. Many studies have shown that enriched environment (EE) can promote functional recovery after stroke, but the intrinsic mechanisms remain unclear. In order to study the internal mechanisms of EE involved in functional recovery after ischemic stroke and which mechanism plays a leading role in the recovery of limb function after cerebral infarction, key proteins potentially involved in neuronal protection and synaptic remodeling in the ischemic penumbra have been investigated. In this study, adult C57BL/6 mice after permanent middle cerebral artery occlusion (pMCAO) were assigned to the EE and standard housing (SH) groups 3 days after operation. The EE house was spacious that contained a large variety of small toys; the SH was a normal sized cage. Sham-operated mice without artery occlusion were housed under standard conditions and were fed a normal diet. On days 3, 7, 14, and 21, postoperative motor functional recovery was tested using the modified neurological severity score (mNSS) and the Rotarod test. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), growth-associated protein-43 (GAP-43), and synaptophysin (SYN) was examined by western blotting and immunofluorescence staining. The motor functional recovery (based on the mNSS and Rotarod test 3, 7, 14, and 21 days post operation) of mice in the EE group improved significantly compared to the SH group. The expression of GAP-43 and SYN and the ratio of Bcl-2/Bax were all upregulated in the EE group compared to the SH group. In addition, we also explored the relationship between neuronal protection and synaptic remodeling in the EE-mediated recovery of limb function after cerebral infarction by correlation analysis. Correlation analysis showed that compared with the increase of Bcl-2/Bax ratio, the increased expression of GAP-43 and SYN was more closely related to the recovery of limb function in ischemic mice. These data support the hypothesis that EE can promote the process of improvement of limb dysfunction induced by ischemic stroke, and this behavior restoration may, via promoting neuroprotection in the ischemic penumbra, be dependent on the regulation of the expression of GAP-43, SYN, Bcl-2, and Bax. A limitation of the study was that we only observed several representative key indicators of synaptic remodeling and neuronal apoptosis, without an in-depth study of the potential mechanisms involved.

Effect of External Magnetic Field on the Efficacy of ADSC Transplantation in Rats with Spinal Cord Injury.

To assess the effect of intravenously injected superparamagnetic iron oxide nanoparticle (SPION)-labeled adipose-derived stem cells (ADSCs) under an external magnetic field on the efficacy of ADSC transplantation in rats with spinal cord injury (SCI). ADSCs were isolated from rats, labeled with SPIONs, and divided into magnetic and non-magnetic groups. A rat model of SCI was established, and SCI rats were randomly divided into magnetic, non-magnetic, and control groups, with ten rats in each group. Rats in the magnetic and non-magnetic groups were injected with SPION-labeled ADSCs via the tail vein. A 300-mT neodymium iron boron magnet was placed externally at the SCI site of the rats in the magnetic group. One and two weeks after successful modeling, SCI rats were scored for the degree of SCI followed by histopathology of the spinal cord, number of ADSCs at the SCI site, and growth-associated protein-43 (GAP-43) expression were determined in the spinal cord tissues. One and two weeks after modeling, the Basso-beattie bresnahan (BBB) scores were the highest in the magnetic group, followed by the non-magnetic group, and the lowest in the control group. HE staining showed that the histopathological manifestations of the spinal cord in the magnetic group were somewhat improved compared to those in the non-magnetic and control groups. Two weeks after modeling, Prussian blue staining revealed that the number of ADSCs was significantly higher in the spinal cord tissue of the magnetic group than in that of the non-magnetic group. One and two weeks after modeling, western blotting revealed that the magnetic group exhibited the highest GAP-43 expression. An external magnetic field applied at the SCI site in rats exerted a directional effect on SPION-labeled ADSCs, directing their migration and improving the efficacy of stem cell-targeted therapies for SCI.

Peripheral nerve regeneration through nerve conduits evokes differential expression of growth-associated protein-43 in the spinal cord.

Growth-associated protein 43 plays a key role in neurite outgrowth through cytoskeleton remodeling. We have previously demonstrated that structural damage of peripheral nerves induces growth-associated protein 43 upregulation to promote growth cone formation. Conversely, the limited regenerative capacity of the central nervous system due to an inhibitory environment prevents major changes in neurite outgrowth and should be presumably associated with low levels of growth-associated protein 43 expression. However, central alterations due to peripheral nerve damage have never been assessed using the growth-associated protein 43 marker. In this study, we used the tubulization technique to repair 1 cm-long nerve gaps in the rat nerve injury/repair model and detected growth-associated protein 43 expression in the peripheral and central nervous systems. First, histological analysis of the regeneration process confirmed an active regeneration process of the nerve gaps through the conduit from 10 days onwards. The growth-associated protein 43 expression profile varied across regions and follow-up times, from a localized expression to an abundant and consistent expression throughout the regeneration tissue, confirming the presence of an active nerve regeneration process. Second, spinal cord changes were also histologically assessed, and no apparent changes in the structural and cellular organization were observed using routine staining methods. Surprisingly, remarkable differences and local changes appeared in growth-associated protein 43 expression at the spinal cord level, in particular at 20 days post-repair and beyond. Growth-associated protein 43 protein was first localized in the gracile fasciculus and was homogeneously distributed in the left posterior cord. These findings differed from the growth-associated protein 43 pattern observed in the healthy control, which did not express growth-associated protein 43 at these levels. Our results revealed a differential expression in growth-associated protein 43 protein not only in the regenerating nerve tissue but also in the spinal cord after peripheral nerve transection. These findings open the possibility of using this marker to monitor changes in the central nervous system after peripheral nerve injury.

Cutaneous expression of growth-associated protein 43 is not a compelling marker for human nerve regeneration in carpal tunnel syndrome.

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.

MiR-29c Inhibits TNF-α-Induced ROS Production and Apoptosis in Mouse Hippocampal HT22 Cell Line.

Recent reports have suggested that abnormal miR-29c expression in hippocampus have been implicated in the pathophysiology of some neurodegenerative and neuropsychiatric diseases. However, the underlying effect of miR-29c in regulating hippocampal neuronal function is not clear. In this study, HT22 cells were infected with lentivirus containing miR-29c or miR-29c sponge. Cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assay kit were applied to evaluate cell viability and toxicity before and after TNF-α administration. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Hoechst 33258 staining and TUNEL assay were used to evaluate cell apoptosis. The expression of key mRNA/proteins (TNFR1, Bcl-2, Bax, TRADD, FADD, caspase-3, -8 and -9) in the apoptosis pathway was detected by PCR or WB. In addition, the protein expression of microtubule-associated protein-2 (MAP-2), nerve growth-associated protein 43 (GAP-43) and synapsin-1 (SYN-1) was detected by WB. As a result, we found that miR-29c overexpression could improve cell viability, attenuate LDH release, reduce ROS production and inhibit MMP depolarization in TNF-α-treated HT22 cells. Furthermore, miR-29c overexpression was found to decrease apoptotic rate, along with decreased expression of Bax, cleaved caspase-3, cleaved caspase-9, and increased expression of Bcl-2 in TNF-α-treated HT22 cells. However, miR-29c sponge exhibited an opposite effects. In addition, in TNF-α-treated HT22 cells, miR-29c overexpression could decrease the expressions of TNFR1, TRADD, FADD and cleaved caspase-8. However, in HT22 cells transfected with miR-29c sponge, TNF-α-induced the expressions of TNFR1, TRADD, FADD and cleaved caspase-8 was significantly exacerbated. At last, TNF-α-induced the decreased expression of MAP-2, GAP-43 and SYN-1 was reversed by miR-29c but exacerbated by miR-29c sponge. Overall, our study demonstrated that miR-29c protects against TNF-α-induced HT22 cells injury through alleviating ROS production and reduce neuronal apoptosis. Therefore, miR-29c might be a potential therapeutic agent for TNF-α accumulation and toxicity-related brain diseases.

Intranasal administration of mitochondria alleviated cognitive impairments and mitochondrial dysfunction in the photothrombotic model of mPFC stroke in mice

ObjectivesDysfunction in mitochondrial activity may have profound role in ischemic stroke-induced neuronal death, hence maintaining the mitochondrial function seems to be valuable for neuronal viability and neurological improvement. MethodsC57BL/6J mice were allocated into sham and stroke groups. Mice in the stroke groups underwent photothrombosis-induced stroke in the medial prefrontal cortex (mPFC) and were divided into the following subgroups; RB, Mito 85, Mito 170, and Mito 340, and received their respective treatments via intra-nasal route every other day (3 days per week) for one week. A battery of behavioral tests including social interaction, passive avoidance, and the Lashley III maze was used to investigate social, contextual, and spatial memories. Moreover, changes in mitochondrial function, including reactive oxygen species (ROS) and ATP levels, and mitochondrial membrane potential, were assessed in mPFC. The expression of growth-associated protein 43 (GAP-43), post-synaptic density-95 (PSD-95), and synaptophysin (SYP) was detected by western blotting. ResultsBehavioral results revealed that mitotherapy alleviated ischemia-induced memory impairment. Also, transplantation of exogenous mitochondria lowered ROS, restored ATP generation, and improved mitochondrial membrane potential. Induction of ischemia decreased the levels of synaptic markers in mPFC while exogenous mitochondria (170 and 340µg) significantly upregulated the expression of GAP-43 and PSD-95 after ischemic stroke. ConclusionOur research highlighted the importance of mitotherapy in regulating synaptic markers expression and mitochondria function, which could represent a potential strategy for improving cognitive and memory deficits following stroke.

Study of the mechanism by which MSCs combined with LITUS treatment improve cognitive dysfunction caused by traumatic brain injury

Traumatic brain injury (TBI) substantially affects the quality of life of patients, and an effective therapy is unavailable. Previous studies have shown that mesenchymal stem cells (MSCs) and low-intensity transcranial ultrasound (LITUS) are effective treatments for neurological damage, inflammation, edema and cognitive impairment caused by TBI. However, it is unclear whether the combination of the two treatments exerts an additive effect. In this study, a rat TBI model was established using the controlled cortical impact (CCI) method. Neurological function was assessed by determining the rat modified neurological score (mNSS), and cognitive function was assessed using the Y-maze. Pathological changes in the injured tissue were observed using hematoxylin-eosin (HE) staining and immunohistochemistry (IHC), and western blot was performed to detect the expression levels of Nestin, neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), growth-associated protein-43 (GAP-43), postsynaptic density protein (PSD-95), brain-derived neurotrophic factor (BDNF), tumor necrosis factor-α (TNF-α), and aquaporin-4 (AQP-4). Real-time fluorescence quantitative polymerase chain reaction (RT–PCR) was performed to detect the expression levels of GAP-43, PSD-95, BDNF, TNF-α, and AQP-4 mRNA to investigate whether MSCs combined with LITUS exert an additive therapeutic effect of alleviating the cognitive dysfunction caused by TBI and the possible mechanisms involved. Rats exhibited cognitive dysfunction 28 days after TBI, and MSCs combined with LITUS treatment ameliorated the cognitive deficits caused by TBI via increasing Nestin, NSE, GAP-43, PSD-95, and BDNF expression and attenuating the inflammatory response and edema caused by TBI via reducing TNF-α and AQP-4 expression. According to these results, MSCs combined with LITUS is more effective than MSCs alone for the treatment of TBI, and the mechanism may be the promotion of neuronal proliferation and differentiation, and the attenuation of the inflammatory response and edema, which ameliorates the spatial learning memory impairment caused by TBI. MSCs combined with LITUS treatment represents a new approach for the clinical treatment of patients with TBI.