GAP43 Located on Corticostriatal Terminals Restrains Novelty-Induced Hyperactivity in Mice.

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 (Gap43fl/fl ;Nex1Cre mice, hereafter Glu-GAP43-/- mice) or forebrain GABAergic neurons (Gap43fl/fl ;Dlx5/6Cre 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 Gi-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.

The complex relationship between late-onset caloric restriction and synaptic plasticity in aged Wistar rats.

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.

Investigating the association between the GAP-43 concentration with diffusion tensor imaging indices in Alzheimer's dementia continuum.

BACKGROUND: Synaptic degeneration, axonal injury, and white matter disintegration are among the pathological events in Alzheimer's disease (AD), for which growth-associated protein 43 (GAP-43) and diffusion tensor imaging (DTI) could be an indicator. In this study, the cerebrospinal fluid (CSF) GAP-43 clinical trajectories and their association with progression and AD hallmarks with white matter microstructural changes were evaluated. METHODS: A total number of 133 participants were enrolled in GAP-43 and DTI values were compared between groups, both cross-sectionally and longitudinally with two and four-year follow-ups. Subsequently, the correlation between GAP-43 levels in the CSF and DTI values was investigated using Spearman's correlation. RESULTS: The CSF level of GAP-43 is negatively correlated with the mean diffusivity measures in Fornix (Cres)/Stria terminals in early and late MCI (rs=-0.478 p = 0.021 and rs=-0.425 p = 0.038). Additionally, the CSF level of GAP-43 is negatively correlated with fractional anisotropy in the cingulum in late MCI (rs=-0.437 p = 0.033). Moreover, the axial diffusivity in superior corona radiate (rs=-0.562 p = 0.005 and rs=-0.484 p = 0.036) and radial diffusivity in superior fronto-occipital fasciculus was negatively correlated with GAP-43 level in the early and mid-MCI participants (rs=-0.520 p = 0.011 and rs=-0.498 p = 0.030). CONCLUSIONS: Presynaptic marker GAP-43 in combination with DTI can be used as a novel biomarker to identify microstructural synaptic degeneration in the early MCI. In addition, it can be used as a biomarker for tracking the progression of AD and monitoring treatment efficacy.

Deletion of Slc1a4 Suppresses Single Mauthner Cell Axon Regeneration In Vivo through Growth-Associated Protein 43.

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.

Evaluation of the concentration of growth associated protein-43 and glial cell-derived neurotrophic factor in degenerated intervertebral discs of the lumbosacral region of the spine.

Important neurotrophic factors that are potentially involved in degenerative intervertebral disc (IVD) disease of the spine's lumbosacral (L/S) region include glial cell-derived neurotrophic factor (GDNF) and growth associated protein 43 (GAP-43). The aim of this study was to determine and compare the concentrations of GAP-43 and GDNF in degenerated and healthy IVDs and to quantify and compare the GAP-43-positive and GDNF-positive nerve fibers. The study group consisted of 113 Caucasian patients with symptomatic lumbosacral discopathy (confirmed by a specialist surgeon), an indication for surgical treatment. The control group included 81 people who underwent postmortem examination. GAP-43 and GDNF concentrations were significantly higher in IVD samples from the study group compared with the control group, and the highest concentrations were observed in the degenerated IVDs that were graded 4 on the Pfirrmann scale. In the case of GAP-43, it was found that as the degree of IVD degeneration increased, the number of GAP-43-positive nerve fibers decreased. In the case of GDNF, the greatest number of fibers per mm2 of surface area was found in the IVD samples graded 3 on the Pfirrmann scale, and the number was found to be lower in samples graded 4 and 5. Hence, GAP-43 and GDNF are promising targets for analgesic treatment of degenerative IVD disease of the lumbosacral region of the spine.

Growth associated protein 43 deficiency promotes podocyte injury by activating the calmodulin/calcineurin pathway under hyperglycemia.

Podocyte injury is a crucial factor in the pathogenesis of diabetic kidney disease (DKD), and finding potential therapeutic interventions that can mitigate podocyte injury holds significant clinical relevance. This study was to elucidate the role of growth associated protein-43(Gap43) in podocyte injury of high glucose (HG). We confirmed the expression of Gap43 in human glomerulus and found that Gap43 expression was downregulated in podocytes of patients with DKD and HG-treated podocytes in vitro. Gap43 knockdown in podocytes promoted podocyte apoptosis, increased migration ability and decreased nephrin expression, while overexpression of Gap43 markedly suppressed HG-induced injury. Moreover, the increased expression and activity of calcineurin (CaN) were also abrogated by overexpression Gap43 in HG. Pretreatment with a typical CaN inhibitor FK506 in Gap43 knockdown podocytes restored the injury. Mechanistically, co-immunoprecipitation experiments suggested that Gap43 could bind to calmodulin (CaM). Pull-down assay further demonstrated that Gap43 and CaM directly interacts with each other via amino acids 30-52 of Gap43 and amino acids 133-197 of CaM. In addition, we also identified Pax5 as potential transcription inhibitor factor mediating Gap43 expression. In conclusion, the study indicated that the Gap43/CaM-CaN pathway may be exploited as a promising therapeutic target for protecting against podocyte injury in high glucose.

Smad4-dependent morphogenic signals control the maturation and axonal targeting of basal vomeronasal sensory neurons to the accessory olfactory bulb.

The vomeronasal organ (VNO) contains two main types of vomeronasal sensory neurons (VSNs) that express distinct vomeronasal receptor (VR) genes and localize to specific regions of the neuroepithelium. Morphogenic signals are crucial in defining neuronal identity and network formation; however, if and what signals control maturation and homeostasis of VSNs is largely unexplored. Here, we found transforming growth factor ß (TGFß) and bone morphogenetic protein (BMP) signal transduction in postnatal mice, with BMP signaling being restricted to basal VSNs and at the marginal zones of the VNO: the site of neurogenesis. Using different Smad4 conditional knockout mouse models, we disrupted canonical TGFß/BMP signaling in either maturing basal VSNs (bVSNs) or all mature VSNs. Smad4 loss of function in immature bVSNs compromises dendritic knob formation, pheromone induced activation, correct glomeruli formation in the accessory olfactory bulb (AOB) and survival. However, Smad4 loss of function in all mature VSNs only compromises correct glomeruli formation in the posterior AOB. Our results indicate that Smad4-mediated signaling drives the functional maturation and connectivity of basal VSNs.

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.

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.

Role of miR-219a-5p in regulating NMDAR in nonylphenol-induced synaptic plasticity damage.

Nonylphenol (NP) is a typical environmental endocrine disruptor with estrogenic effects. It serves as an emulsifier and as the main ingredient of detergents and has become an increasingly common pollutant in both fresh and salt water, vegetables, and fruits. This study aimed to clarify whether NP exposure could lead to cognitive dysfunction and synaptic plasticity impairment, and also explore the mechanism of microRNA (miR)- 219a-5p regulation of N-methyl-D-aspartate receptor (NMDAR) in NP-induced synaptic plasticity impairment in vivo and in vitro. In vivo, 30 male Sprague-Dawley rats were randomly divided into 2 groups: blank control group (pure corn oil) and NP-exposed group [NP 80 mg/(kg·d)], with 15 rats in each group. In vitro, the extracted hippocampal neurons were divided into 6 groups: blank control group, mimics NC group, miR-219 mimics group, NP group (70 µmol/L NP), NP + mimics NC group, and NP + miR-219 mimics group. In vivo, the content of NP in hippocampal tissues after 90 days of NP exposure was significantly higher in the NP-stained group than in the blank control group. NP exposure could lead to a decrease in the ability to learn and memory, ability to remember, and space spatial memory ability in rats. The dendrites in the NP-stained group were disordered, with few dendritic spines and significantly decreased dendritic spine density. The postsynaptic densities were loosely arranged, the thickness and length of the postsynaptic densities shortened, and the length and width of the synaptic gap increased. Glutamine (Glu) and γ-aminobutyric acid (GABA) contents in hippocampal tissues decreased in the NP-stained group. The expression of miR-219a-5p mRNA decreased in the NP-stained group after 3 months of NP exposure. The expression of NMDAR1, NMDAR2A, NMDAR2B, nerve growth-associated protein (GAP-43), and Ca/calmodulin-dependent kinase II (CaMKII) mRNA/proteins decreased in the NP-stained group. In vitro, NMDAR protein expression decreased, while GAP-43 and CaMKII protein expression increased in the miR-219 mimics group compared with the control group. The expression levels of NMDAR and GAP-43 and CaMKII proteins were higher in the NP + miR-219 mimics group compared with the NP group. The levels of neurotransmitters Glu and GABA decreased in the NP and NP + mimics NC groups compared with the blank group. Shortened synaptic active band length, decreased thickness of postsynaptic densities, and shortened length of postsynaptic densities were observed in the NP, NP + mimics NC, and NP + miR-219 mimics groups compared with the blank control group. In vivo, NP exposure reduced learning memory capacity and neurotransmitter content in rats and caused a decrease in dendritic spine density and synaptic number density and a decrease in miR-219a-5p expression. In vitro, high expression of miR-219a-5p inhibited the expression of NMDAR, thus reducing the effect of NP on synaptic plasticity impairment in hippocampal neurons. Our study provided a scientific basis for the prevention of cognitive impairment owing to NP exposure and the development of targeted drug treatment strategies.

Paternal stress alters synaptic density and expression of GAP-43, GRIN1, M1 and SYP genes in the hippocampus and cortex of offspring of stress-induced male rats.

Adverse experiences during pregnancy have a negative impact on the neuronal structure and behavior of offspring, but the effects of a father's life events on the outcome of progeny are scarce. The present study is intended to investigate whether paternal stress affects the offspring brain structure, especially those regions concerned with learning and formation of memory, namely the hippocampus (HC) and prefrontal cortex (PFC), and also the expression of certain genes linked to learning and memory in the offspring. Induced stress to male rats by five stressors, one per day followed by allowing them to mate with the normal, unstressed female. Synaptophysin immunoreactivity was assessed in the tissue sections of the HC and PFC as well as expression of genes concerned with learning and memory was evaluated by RT-PCR in the progeny of stress-received males. The progeny of stressed rats had reduced antisynaptophysin immunoreactivity in the HC and PFC. The synaptic density in HC was less in the A-S (Offspring of male rats who received stress during adulthood) and PA-S (offspring of male rats who received stress during both adolescence and adulthood) than in P-S (offspring of male rats who received stress during adolescence) and C-C (offspring of control) groups. Similar results were observed even in the PFC. The results of post hoc tests proved that the HC and PFC of the progeny of stress-exposed rats exhibited considerably less synaptic density than control (P<0.05), and the levels of expression of GAP-43, GRIN1, M1, and SYP genes in HC and PFC were down-regulated. This study concludes that paternal adverse experiences can affect the offspring's synaptic plasticity and also the genes, which can regulate learning and formation of memory.

[Lianmei Qiwu Decoction relieves diabetic cardiac autonomic neuropathy by regulating AMPK/TrkA/TRPM7 signaling pathway].

This study investigated the effect of Lianmei Qiwu Decoction(LMQWD) on the improvement of cardiac autonomic nerve remodeling in the diabetic rat model induced by the high-fat diet and explored the underlying mechanism of LMQWD through the AMP-activated protein kinase(AMPK)/tropomyosin receptor kinase A(TrkA)/transient receptor potential melastatin 7(TRPM7) signaling pathway. The diabetic rats were randomly divided into a model group, an LMQWD group, an AMPK agonist group, an unloaded TRPM7 adenovirus group(TRPM7-N), an overexpressed TRPM7 adenovirus group(TRPM7), an LMQWD + unloaded TRPM7 adenovirus group(LMQWD+TRPM7-N), an LMQWD + overexpressed TRPM7 adenovirus group(LMQWD+TRPM7), and a TRPM7 channel inhibitor group(TRPM7 inhibitor). After four weeks of treatment, programmed electrical stimulation(PES) was employed to detect the arrhythmia susceptibility of rats. The myocardial cell structure and myocardial tissue fibrosis of myocardial and ganglion samples in diabetic rats were observed by hematoxylin-eosin(HE) staining and Masson staining. The immunohistochemistry, immunofluorescence, real-time quantitative polymerase chain reaction(RT-PCR), and Western blot were adopted to detect the distribution and expression of TRPM7, tyrosine hydroxylase(TH), choline acetyltransferase(ChAT), growth associated protein-43(GAP-43), nerve growth factor(NGF), p-AMPK/AMPK, and other genes and related neural markers. The results showed that LMQWD could significantly reduce the arrhythmia susceptibility and the degree of fibrosis in myocardial tissues, decrease the levels of TH, ChAT, and GAP-43 in the myocardium and ganglion, increase NGF, inhibit the expression of TRPM7, and up-regulate p-AMPK/AMPK and p-TrkA/TrkA levels. This study indicated that LMQWD could attenuate cardiac autonomic nerve remodeling in the diabetic state, and its mechanism was associated with the activation of AMPK, further phosphorylation of TrkA, and inhibition of TRPM7 expression.

Mice lacking growth-associated protein 43 develop cardiac remodeling and hypertrophy.

Growth-associated protein 43 (GAP43) is found in skeletal muscle, localized near the calcium release units. In interaction with calmodulin (CaM), it indirectly modulates the activity of dihydropyridine and ryanodine Ca2+ channels. GAP43-CaM interaction plays a key role in intracellular Ca2+ homeostasis and, consequently, in skeletal muscle activity. The control of intracellular Ca2+ signaling is also an important functional requisite in cardiac physiology. The aim of this study is to define the impact of GAP43 on cardiac tissue at macroscopic and cellular levels, using GAP43 knockout (GAP43-/-) newborn C57/BL6 mice. Hearts from newborn GAP43-/- mice were heavier than hearts from wild-type (WT) ones. In these GAP43-/- hearts, histological section analyses revealed a thicker ventricular wall and interventricular septum with a reduced ventricular chamber area. In addition, increased collagen deposits between fibers and increased expression levels of myosin were observed in hearts from GAP43-/- mice. Cardiac tropism and rhythm are controlled by multiple intrinsic and extrinsic factors, including cellular events such those linked to intracellular Ca2+ dynamics, in which GAP43 plays a role. Our data revealed that, in the absence of GAP43, there were cardiac morphological alterations and signs of hypertrophy, suggesting that GAP43 could play a role in the functional processes of the whole cardiac muscle. This paves the way for further studies investigating GAP43 involvement in signaling dynamics at the cellular level.

Galectin-3 is elevated in CSF and is associated with Aß deposits and tau aggregates in brain tissue in Alzheimer's disease.

Galectin-3 (Gal-3) is a beta-galactosidase binding protein involved in microglial activation in the central nervous system (CNS). We previously demonstrated the crucial deleterious role of Gal-3 in microglial activation in Alzheimer's disease (AD). Under AD conditions, Gal-3 is primarily expressed by microglial cells clustered around Aß plaques in both human and mouse brain, and knocking out Gal-3 reduces AD pathology in AD-model mice. To further unravel the importance of Gal-3-associated inflammation in AD, we aimed to investigate the Gal-3 inflammatory response in the AD continuum. First, we measured Gal-3 levels in neocortical and hippocampal tissue from early-onset AD patients, including genetic and sporadic cases. We found that Gal-3 levels were significantly higher in both cortex and hippocampus in AD subjects. Immunohistochemistry revealed that Gal-3+ microglial cells were associated with amyloid plaques of a larger size and more irregular shape and with neurons containing tau-inclusions. We then analyzed the levels of Gal-3 in cerebrospinal fluid (CSF) from AD patients (n = 119) compared to control individuals (n = 36). CSF Gal-3 levels were elevated in AD patients compared to controls and more strongly correlated with tau (p-Tau181 and t-tau) and synaptic markers (GAP-43 and neurogranin) than with amyloid-ß. Lastly, principal component analysis (PCA) of AD biomarkers revealed that CSF Gal-3 clustered and associated with other CSF neuroinflammatory markers, including sTREM-2, GFAP, and YKL-40. This neuroinflammatory component was more highly expressed in the CSF from amyloid-ß positive (A+), CSF p-Tau181 positive (T+), and biomarker neurodegeneration positive/negative (N+/-) (A + T + N+/-) groups compared to the A + T-N- group. Overall, Gal-3 stands out as a key pathological biomarker of AD pathology that is measurable in CSF and, therefore, a potential target for disease-modifying therapies involving the neuroinflammatory response.

JNK1-Dependent Phosphorylation of GAP-43 Serine 142 is a Novel Molecular Marker for Axonal Growth.

Mammalian axon growth has mechanistic similarities with axon regeneration. The growth cone is an important structure that is involved in both processes, and GAP-43 (growth associated protein-43 kDa) is believed to be the classical molecular marker. Previously, we used growth cone phosphoproteomics to demonstrate that S96 and T172 of GAP-43 in rodents are highly phosphorylated sites that are phosphorylated by c-jun N-terminal protein kinase (JNK). We also revealed that phosphorylated (p)S96 and pT172 antibodies recognize growing axons in the developing brain and regenerating axons in adult peripheral nerves. In rodents, S142 is another putative JNK-dependent phosphorylation site that is modified at a lower frequency than S96 and T172. Here, we characterized this site using a pS142-specific antibody. We confirmed that pS142 was detected by co-expressing mouse GAP-43 and JNK1. pS142 antibody labeled growth cones and growing axons in developing mouse neurons. pS142 was sustained until at least nine weeks after birth in mouse brains. The pS142 antibody could detect regenerating axons following sciatic nerve injury in adult mice. Comparison of amino acid sequences indicated that rodent S142 corresponds to human S151, which is predicted to be a substrate of the MAPK family, which includes JNK. Thus, we confirmed that the pS142 antibody recognized human phospho-GAP-43 using activated JNK1, and also that its immunostaining pattern in neurons differentiated from human induced pluripotent cells was similar to those observed in mice. These results indicate that the S142 residue is phosphorylated by JNK1 and that the pS142 antibody is a new candidate molecular marker for axonal growth in both rodents and human.

Dl-3-n-butylphthalide promotes neurite outgrowth of primary cortical neurons by Sonic Hedgehog signaling via upregulating Gap43.

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.

Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids.

Glioblastoma (GBM) is the most aggressive brain cancer and its relapse after surgery, chemo and radiotherapy appears to be led by GBM stem cells (GSCs). Also, tumor networking and intercellular communication play a major role in driving GBM therapy-resistance. Tunneling Nanotubes (TNTs), thin membranous open-ended channels connecting distant cells, have been observed in several types of cancer, where they emerge to drive a more malignant phenotype. Here, we investigated whether GBM cells are capable to intercommunicate by TNTs. Two GBM stem-like cells (GSLCs) were obtained from the external and infiltrative zone of one GBM from one patient. We show, for the first time, that both GSLCs, grown in classical 2D culture and in 3D-tumor organoids, formed functional TNTs which allowed mitochondria transfer. In the organoid model, recapitulative of several tumor's features, we observed the formation of a network between cells constituted of both Tumor Microtubes (TMs), previously observed in vivo, and TNTs. In addition, the two GSLCs exhibited different responses to irradiation in terms of TNT induction and mitochondria transfer, although the correlation with the disease progression and therapy-resistance needs to be further addressed. Thus, TNT-based communication is active in different GSLCs derived from the external tumoral areas associated to GBM relapse, and we propose that they participate together with TMs in tumor networking.

Learning and memory impairment induced by 1,4-butanediol is regulated by ERK1/2-CREB-BDNF signaling pathways in PC12 cells.

1,4-butanediol (1,4-BD) is a known γ-hydroxybutyric acid (GHB) precursor which affects the nervous system after ingestion, leading to uncontrolled behavioral consequences. In the present study, we investigated whether 1,4-BD induces oxidative stress and inflammation in PC12 cells and evaluated the toxic effects of 1,4-BD associates with learning and memory. CCK-8 results revealed a dose-effect relationship between the cell viability of PC12 cells and 1,4-BD when the duration of action was 2 h or 4 h. Assay kits results showed that 1,4-BD decreased the levels of Glutathione (GSH), Glutathione peroxidase (GSH-px), Superoxide dismutase (SOD), Acetylcholine (Ach) and increased the levels of Malondialdehyde (MDA), Nitric oxide (NO) and Acetylcholinesterase (AchE). Elisa kits results indicated that 1,4-BD decreased the levels of synaptophysin I (SYN-1), Postsynaptic density protein-95 (PSD-95), Growth associated protein-43 (GAP-43) and increased the levels of Tumor necrosis factor alpha (TNF-α) and Interleukin- 6 (IL-6). RT-PCR results showed that the mRNA levels of PSD-95, SYN-1 and GAP-43 were significantly decreased. The expression of phosphorylation extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), phosphorylation cAMP response element binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) proteins were significantly decreased in PC12 cells by protein blotting. Overall, these results suggest that 1,4-BD may affect synaptic plasticity via the ERK1/2-CREB-BDNF pathway, leading to Ach release reduction and ultimately to learning and memory impairment. Furthermore, oxidative stress and inflammation induced by 1,4-BD may also result in learning and memory deficits. These findings will enrich the toxicity data of 1.4-BD associated with learning and memory impairment.

Taurine Ameliorates Oxidative Stress in Spinal Cords of Diabetic Rats via Keap1-Nrf2 Signaling.

Hyperglycemia associated with diabetes mellitus (DM) causes oxidative stress, which is involved in the onset and development of diabetic neuropathy. Taurine, a powerful antioxidant, is an effective inhibitor of oxidative stress. The present experiment was conducted to explore the effect of taurine treatment on alterations in body weight, blood glucose, oxidative stress, and Keap1-Nrf2 signaling in the spinal cords of DM rats. The DM rat model was established by STZ injection, and taurine was administered in the drinking water. Body weight and blood glucose were recorded during the experiment. The expression of Gap-43 and MBP proteins was examined by Western blot. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were examined as indicators of oxidative stress. The expression of Keap1, Nrf2, and HO-1 gene was examined by real-time PCR. The results showed that compared with the control group, the body weight was decreased, blood glucose was increased, and both Gap-43 and MBP expression were decreased in DM rats, which were all remarkably reversed by taurine treatment. Oxidative stress, as reflected by lower SOD activity and higher MDA concentration, was inhibited in taurine-treated DM rats. Supplemental taurine also downregulated the mRNA level of Keap1, while upregulating Nrf2 and HO-1 mRNA levels. These results showed that taurine inhibits oxidative stress in the spinal cords of DM rats, an effect that might involve the regulation of Keap1-Nrf2 signaling.

Pretreatment with combined low-level laser therapy and methylene blue improves learning and memory in sleep-deprived mice.

Low-level laser therapy (LLLT) and methylene blue (MB) were proved to have neuroprotective effects. In this study, we evaluated the preventive effects of LLLT and MB alone and in combination to examine their efficacy against sleep deprivation (SD)-induced cognitive impairment. Sixty Balb/c male mice were randomly divided into five groups as follows: wide platform (WP), SD, LLLT, MB, LMB (treatment with both LLLT and MB). Daily MB (0.5 mg/kg) was injected for ten consecutive days. An 810-nm, 10-Hz pulsed laser was used in LLLT every other day. We used the T-maze test, social interaction test (SIT), and shuttle box to assess learning and memory and PSD-95, GAP-43, and synaptophysin (SYN) markers to examine synaptic proteins levels in the hippocampus. Our results showed that SD decreased alternation rate in the T-maze test, sociability and social novelty in SIT, and memory index in the shuttle box. Single treatments were not able to reverse these in most of the behavioral parameters. However, behavioral tests showed a significant difference between combined therapy and the SD group. The levels of synaptic plasticity markers were also significantly reduced after SD. There was a significant difference between the MB group and SD animals in GAP-43 and SYN biomarkers. Combination treatment with LLLT and MB also increased GAP-43, PSD-95, and SYN compared to the SD group. We found that the combined use of LLLT and MB pretreatment is more effective in protecting SD-induced cognitive impairment, which may be imparted via modulation of synaptic proteins.