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.

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.

Hypoxic preconditioned MSCs-derived small extracellular vesicles for photoreceptor protection in retinal degeneration.

Photoreceptor apoptosis is an important pathogenesis of retinal degeneration and a primary cause of vision loss with limited treatment methods. Mesenchymal stem/stromal cells-derived small extracellular vesicles (MSC-sEVs) have shown therapeutic value in various ocular disorders. Recent studies have revealed that hypoxic preconditioning can improve the effectiveness of MSC-sEVs in tissue regeneration. However, whether hypoxic preconditioned MSC-sEVs (Hyp-sEVs) exert superior effects on photoreceptor protection relative to normoxic conditioned MSC-sEVs (Nor-sEVs) remains unclear. Here, we reported that Hyp-sEVs further improved retinal structure, recovered retinal function, and suppressed photoreceptor apoptosis in N-methyl-N-nitrosourea (MNU)-induced mouse model compared with Nor-sEVs. Hyp-sEVs also exhibited enhanced anti-apoptotic roles in MNU-provoked 661 W cell injury in vitro. We then analyzed the protein profiles of Nor-sEVs and Hyp-sEVs by LC-MS/MS and found that growth-associated protein 43 (GAP43) was enriched in Hyp-sEVs. The knockdown of GAP43 abolished the retinal therapeutic effects of Hyp-sEVs. Mechanistically, hypoxic stimulation-induced hypoxia-inducible factor-1α (HIF-1α) activation was responsible for preventing tripartite motif-containing protein 25 (TRIM25)-mediated GAP43 ubiquitination and degradation, leading to the upregulation of GAP43 in Hyp-sEVs. Together, our findings uncover the efficacy and mechanism of Hyp-sEVs-based photoreceptor protection and highlight the potential of Hyp-sEVs as optimized therapeutics for retinal degeneration.

GAP-43 targeted indocyanine green-loaded near-infrared fluorescent probe for real-time mapping of perineural invasion lesions in pancreatic cancer in vivo.

OBJECTIVE: Perineural invasion (PNI) is associated with local recurrence, distant metastasis, and a poor prognosis in pancreatic cancer. However, rare attempt was made to identified the PNI intraoperative. To facilitate precise R0 excision of the tumor, we planned to develop a fluorescent probe for intraoperative imaging of the PNI using GAP-43 as the target and indocyanine green (ICG) as the carrier. METHODS: The probe was created by binding peptide antibody and ICG. Its targeting was tested in vitro and in vivo using a co-culture model of PC12 and tumor cells to create an in vitro neural invasion model and a mouse sciatic nerve invasion model. The small animal imaging system and surgical navigation system confirmed the probe's potential clinical applicability. The sciatic nerve damage model was created to confirm the probe's targeting. RESULTS: We used the pancreatic cancer samples and the public database to confirm that GAP-43 was preferentially overexpressed in pancreatic cancer, particularly in PNI. PC12 cells showed high GAP-43RA-PEG-ICG probe-specific absorption after being co-cultured with tumor cells in vitro. In the sciatic nerve invasion experiment, animals in probe group displayed a significantly stronger fluorescence signal at the PNI compared to ICG-NP and the contralateral normal nerves groups. Although only 60 % of mice appeared to have R0 resections by the naked eye, small animal imaging systems and surgical fluorescence navigation systems could remove the tumor with R0 precision. The injury model used in the probe imaging experimental trials demonstrated that the probe was specifically targeted to the injured nerve, regardless of whether the injury was infiltrated by a tumor or physical. CONCLUSION: We developed the GAP-43Ra-ICG-PEG, an active-targeting near-infrared fluorescent (NIRF) probe, that specifically binds to GAP-43-positive neural cells in an in vitro model of PNI. The probe efficiently visualized PNI lesions in pancreatic cancer in preclinical models, opening up new possibilities for NIRF-guided pancreatic surgery, particularly for PNI patients.

Presynaptic membrane protein dysfunction occurs prior to neurodegeneration and predicts faster cognitive decline.

INTRODUCTION: Although presynaptic loss measured by cerebrospinal fluid (CSF) growth-associated protein-43 (GAP-43) is significantly involved in Alzheimer's disease (AD), the sequential association between CSF GAP-43 and AD-typical neurodegeneration is poorly understood. METHODS: We compared baseline CSF GAP-43 levels (n = 730) and longitudinal CSF GAP-43 changes (n = 327) in various biological stages of AD, and investigated their relationships with cross-sectional and longitudinal measures of residual hippocampal volume, 18 F-fluorodeoxyglucose PET, regional gray matter volume and cortical thickness, and cognition. RESULTS: Elevated CSF GAP43 levels were significantly associated with faster rates of hippocampal atrophy, AD-signature hypometabolism and cortical thinning, and middle temporal gray matter atrophy-related and AD-signature hypometabolism-related cognitive decline. In contrast, baseline levels of all these neurodegeneration biomarkers did not predict longitudinal CSF GAP-43 increases. DISCUSSION: These findings suggest that presynaptic loss may occur prior to neurodegeneration, highlighting the importance of lowing tau aggregation and tau-related synaptic dysfunction in elderly adults and AD patients.

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.

Cacna2d2 inhibits axonal regeneration following surgical decompression in a rat model of cervical spondylotic myelopathy.

BACKGROUND: Cervical spondylotic myelopathy (CSM) is a clinically symptomatic condition due to spinal cord compression, leading to spinal cord dysfunction. Surgical decompression is the main treatment of CSM, but the mechanisms of axonal regeneration after surgical decompression are still fragmentary. METHODS: In a rat model of CSM, the cacna2d2 (α2δ2) expression levels in anterior horn of spinal cord were observed following compression and decompression by western blot and immunofluorescence. The expression levels of 5 hydroxytryptamine (5HT) and GAP43 were also analyzed by immunofluorescence. Furthermore, gabapentin intervention was performed for 4 weeks after decompression to analyze the changes of behaviors and anterior horn of spinal cords. RESULTS: Following decompression, the expression levels of α2δ2 in the anterior horn of spinal cord were decreased, but the expression levels of 5HT andGAP43 were increased. Compared with the vehicle treated rats, gabapentin treatment for 4 weeks ameliorated the behaviors of rats and improved the damaged anterior horn of spinal cord. Besides, inhibition of α2δ2 through gabapentin intervention enhanced the axonal regeneration in the anterior horn of damaged spinal cord. CONCLUSIONS: Inhibition of α2δ2 could enhance axonal recovery in anterior horn of damaged spinal cord induced by CSM after surgical decompression, providing a potential method for promoting axon regeneration following surgery.

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.

Urokinase-type plasminogen activator (uPA) regulates the expression and function of growth-associated protein 43 (GAP-43) in the synapse.

Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N-methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.

Pre- and early postnatal enriched environmental experiences prevent neonatal hypoxia-ischemia late neurodegeneration via metabolic and neuroplastic mechanisms.

Prenatal and early postnatal periods are important for brain development and neural function. Neonatal insults such as hypoxia-ischemia (HI) causes prolonged neural and metabolic dysregulation, affecting central nervous system maturation. There is evidence that brain hypometabolism could increase the risk of adult-onset neurodegenerative diseases. However, the impact of non-pharmacologic strategies to attenuate HI-induced brain glucose dysfunction is still underexplored. This study investigated the long-term effects of early environmental enrichment in metabolic, cell, and functional responses after neonatal HI. Thereby, male Wistar rats were divided according to surgical procedure, sham, and HI (performed at postnatal day 3), and the allocation to standard (SC) or enriched condition (EC) during gestation and lactation periods. In-vivo cerebral metabolism was assessed by means of [18 F]-FDG micro-positron emission tomography, and cognitive, biochemical, and histological analyses were performed in adulthood. Our findings reveal that HI causes a reduction in glucose metabolism and glucose transporter levels as well as hyposynchronicity in metabolic brain networks. However, EC during prenatal or early postnatal period attenuated these metabolic disturbances. A positive correlation was observed between [18 F]-FDG values and volume ratios in adulthood, indicating that preserved tissue by EC is metabolically active. EC promotes better cognitive scores, as well as down-regulation of amyloid precursor protein in the parietal cortex and hippocampus of HI animals. Furthermore, growth-associated protein 43 was up-regulated in the cortex of EC animals. Altogether, results presented support that EC during gestation and lactation period can reduce HI-induced impairments that may contribute to functional decline and progressive late neurodegeneration.

Transcriptome profiling unveils GAP43 regulates ABC transporters and EIF2 signaling in colorectal cancer cells.

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.

Bone marrow-derived mesenchymal stem cells improve post-ischemia neurological function in rats via the PI3K/AKT/GSK-3ß/CRMP-2 pathway.

BACKGROUND: Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) is a potential therapy for cerebral ischemia. However, the underlying protective mechanism remains undetermined. Here, we tested the hypothesis that transplantation of BMSCs via intravenous injection can alleviate neurological functional deficits through activating PI3K/AKT signaling pathway after cerebral ischemia in rats. METHODS: A cerebral ischemic rat model was established by the 2 h middle cerebral artery occlusion (MCAO). Twenty-four hours later, BMSCs (1 × 106 in 1 ml PBS) from SD rats were injected into the tail vein. Neurological function was evaluated by modified neurological severity score (mNSS) and modified adhesive removal test before and on d1, d3, d7, d10 and d14 after MCAO. Protein expressions of AKT, GSK-3ß, CRMP-2 and GAP-43 were detected by Western-bolt. NF-200 was detected by immunofluorescence. RESULTS: BMSCs transplantation did not only significantly improve the mNSS score and the adhesive-removal somatosensory test after MCAO, but also increase the density of NF-200 and the expression of p-AKT, pGSK-3ß and GAP-43, while decrease the expression of pCRMP-2. Meanwhile, these effects can be suppressed by LY294002, a specific inhibitor of PI3K/AKT. CONCLUSION: These data suggest that transplantation of BMSCs could promote axon growth and neurological deficit recovery after MCAO, which was associated with activation of PI3K/AKT /GSK-3ß/CRMP-2 signaling pathway.

Surface-modified engineered exosomes attenuated cerebral ischemia/reperfusion injury by targeting the delivery of quercetin towards impaired neurons.

BACKGROUND: The incidence of ischemic stroke in the context of vascular disease is high, and the expression of growth-associated protein-43 (GAP43) increases when neurons are damaged or stimulated, especially in a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). EXPERIMENTAL: DESIGN: We bioengineered neuron-targeting exosomes (Exo) conjugated to a monoclonal antibody against GAP43 (mAb GAP43) to promote the targeted delivery of quercetin (Que) to ischemic neurons with high GAP43 expression and investigated the ability of Exo to treat cerebral ischemia by scavenging reactive oxygen species (ROS). RESULTS: Our results suggested that Que loaded mAb GAP43 conjugated exosomes (Que/mAb GAP43-Exo) can specifically target damaged neurons through the interaction between Exo-delivered mAb GAP43 and GAP43 expressed in damaged neurons and improve survival of neurons by inhibiting ROS production through the activation of the Nrf2/HO-1 pathway. The brain infarct volume is smaller, and neurological recovery is more markedly improved following Que/mAb GAP43-Exo treatment than following free Que or Que-carrying exosome (Que-Exo) treatment in a rat induced by MCAO/R. CONCLUSIONS: Que/mAb GAP43-Exo may serve a promising dual targeting and therapeutic drug delivery system for alleviating cerebral ischemia/reperfusion injury.

Effects of Theranekron and alpha-lipoic acid combined treatment on GAP-43 and Krox-20 gene expressions and inflammation markers in peripheral nerve injury.

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.

Prognosis Evaluation Using 18F-Alfatide II PET in a Rat Model of Spinal Cord Injury Treated With Estrogen.

Spinal cord injury (SCI) leads to severe dysfunction below injured segment and poses a great pressure to the individual and society. In this study, we applied 18F-alfatide II positron emission tomography/computed tomography (PET/CT) to monitor angiogenesis in an SCI model after estrogen (E2) treatment, as well as to evaluate the prognosis in a noninvasive manner. The SCI model was established with male rats and the rats were randomly divided into E2-treated group (SCI + E2) and E2-untreated group (SCI). Sham group was also used as control (Sham). The angiogenesis after SCI was monitored by 18F-alfatide II PET/CT and verified by immunofluorescence of CD31 and CD61. We also evaluated the level of E2 and growth-associated protein 43 (GAP43) by enzyme-linked immunosorbent assay. Finally, Basso, Beattie, and Bresnahan (BBB) scores were determined to evaluate the exercise capacity of the rats in all 3 groups. Our results showed that the BBB score of SCI + E2 group was significantly different from that of SCI group (P < .05) and Sham group (P < .01). The uptake of 18F-alfatide II was positively correlated with the expression level of GAP43, both of which reached the peak at day 7 after injury. CD31 and CD61 immunostaining further verified increased angiogenesis in E2-treated SCI lesions. We concluded that 18F-alfatide II PET/CT can monitor the angiogenesis status after SCI in vivo and it may help clinician predict the progression of patients with SCI. This may benefit the study of vascular repair after SCI and provide a tool for evaluation of SCI treatment in clinical practices.

GAP-43 is involved in the orientation of cell division by interacting with GΑI during neurogenesis.

Purpose: Recent studies have shown that growth-associated protein-43 (GAP-43) may influence the mitotic-spindle orientation of Madin-Darby Canine Kidney (MDCK) cells through interacting with G proteins in vitro. However, whether GAP-43 interacts with the G proteins under the influence of mitotic spindle positioning related to the orientation of cell division during neurogenesis remains unclear. In order to explore the molecular mechanism in vivo, the GAP-43 transgenic mice were produced and the angles of cell division in the ventricular zone (VZ) during neurogenesis (embryonic period between 13.5 and 17.5 days) were measured in both transgenic mice and wild type mice by spindle angle analysis.Materials and methods: The interaction of GAP-43 and Gαi was detected by co-immunoprecipitation (co-IP), whereas the localization of GAP-43 was determined by immunofluorescence.Results: The results obtained using co-IP and immunofluorescence showed that GAP-43 is localized on the cell membrane and interacts with Gαi. This interaction dramatically induced a significant increase in the proportion of horizontally and intermediately dividing cells during the embryonic period of 13.5 days in the transgenic mouse brain, as observed by spindle angle analysis.Conclusions: It can be concluded that GAP-43 is involved in the orientation of cell division by interacting with Gαi, and that this may be an important mechanism for neurogenesis in the mammalian brain.

Calmodulin-Binding Proteins in Muscle: A Minireview on Nuclear Receptor Interacting Protein, Neurogranin, and Growth-Associated Protein 43.

Calmodulin (CaM) is an important Ca2+-sensing protein with numerous downstream targets that are either CaM-dependant or CaM-regulated. In muscle, CaM-dependent proteins, which are critical regulators of dynamic Ca2+ handling and contractility, include calcineurin (CaN), CaM-dependant kinase II (CaMKII), ryanodine receptor (RyR), and dihydropyridine receptor (DHPR). CaM-regulated targets include genes associated with oxidative metabolism, muscle plasticity, and repair. Despite its importance in muscle, the regulation of CaM-particularly its availability to bind to and activate downstream targets-is an emerging area of research. In this minireview, we discuss recent studies revealing the importance of small IQ motif proteins that bind to CaM to either facilitate (nuclear receptor interacting protein; NRIP) its activation of downstream targets, or sequester (neurogranin, Ng; and growth-associated protein 43, GAP43) CaM away from their downstream targets. Specifically, we discuss recent studies that have begun uncovering the physiological roles of NRIP, Ng, and GAP43 in skeletal and cardiac muscle, thereby highlighting the importance of endogenously expressed CaM-binding proteins and their regulation of CaM in muscle.

Localization and Expression of Sirtuins 1, 2, 6 and Plasticity-Related Proteins in the Recovery Period after a Photothrombotic Stroke in Mice.

Sirtuins, class III histone deacetylases, are involved in the regulation of tissue repair processes and brain functions after a stroke. The ability of some isoforms of sirtuins to circulate between the nucleus and cytoplasm may have various pathophysiological effects on the cells. In present work, we focused on the role of non-mitochondrial sirtuins SIRT1, SIRT2, and SIRT6 in the restoration of brain cells following ischemic stroke. Here, using a photothrombotic stroke (PTS) model in mice, we studied whether local stroke affects the level and intracellular localization of SIRT1, SIRT2, and SIRT6 in neurons and astrocytes of the intact cerebral cortex adjacent to the ischemic ipsilateral hemisphere and in the analogous region of the contralateral hemisphere at different time points during the recovery period after a stroke. We evaluated the co-localization of sirtuins with growth-associated protein-43 (GAP-43), the presynaptic marker synaptophysin (SYN) and acetylated α-tubulin (Ac-α-Tub), that are associated with brain plasticity and are known to be involved in brain repair after a stroke. The results show that during the recovery period, an increase in SIRT1 and SIRT2 levels occurred. The increase of SIRT1 level was associated with an increase in synaptic plasticity proteins, whereas the increase of SIRT2 level was associated with an acetylated of α-tubulin, that can reduce the mobility of neurites. SIRT6 co-localized with GAP-43, but not with SYN. Moreover, we showed that SIRT1, SIRT2, and SIRT6 are not involved in the PTS-induced apoptosis of penumbra cells. Taken together, our results suggest that sirtuins functions differ depending on cell type, intracellular localization, specificity of sirtuins isoforms to different substrates and nature of post-translational modifications of enzymes.