lncRNA-056298 Regulates GAP43 and Promotes Cardiac Intrinsic Autonomic Nerve Remodelling in a Canine Model of Atrial Fibrillation Induction after Ganglionated Plexus Ablation.

BACKGROUND: Cardiac intrinsic autonomic nerve remodelling has been reported to play an important role in the recurrence of atrial fibrillation after radiofrequency ablation, which significantly affects the long-term efficacy of this procedure. lncRNAs have been shown to interact in the pathological processes underlying heart diseases. However, the roles and mechanisms of lncRNAs in cardiac intrinsic autonomic nerve remodelling during atrial fibrillation reduction after ganglionated plexus ablation remain unknown. OBJECTIVES: The aim of this study was to investigate the mechanism by which lncRNA- 056298 modulates GAP43 to affect cardiac intrinsic autonomic nerve remodelling and facilitate the induction of atrial fibrillation after ganglionated plexus ablation. METHODS: A canine model of right atrial ganglionated plexus ablation was established. The atrial electrophysiological characteristics and neural markers were detected before and after 6 months of ganglionated plexus ablation. High-throughput sequencing was used to screen differentially expressed lncRNAs in target atrial tissues, and lncRNA- 056298 was selected to further explore its effects and mechanisms on cardiac intrinsic autonomic nerve remodelling. RESULTS: The induction rate of atrial fibrillation increased in dogs after ganglionated plexus ablation. Overexpression of lncRNA-056298 by lentivirus can shorten the atrial effective refractory period and increase the induction of atrial fibrillation. lncRNA- 056298 promoted cardiac intrinsic autonomic nerve remodelling via endogenous competition with cfa-miR-185 to induce transcription of its target gene GAP43, thereby affecting the induction of atrial fibrillation. CONCLUSIONS: lncRNA-056298 regulates GAP43 by sponging miR-185, which affects cardiac intrinsic autonomic nerve remodelling and mediates atrial fibrillation induction after ganglionated plexus ablation.

Calcium-Associated Proteins in Neuroregeneration.

The dysregulation of intracellular calcium levels is a critical factor in neurodegeneration, leading to the aberrant activation of calcium-dependent processes and, ultimately, cell death. Ca2+ signals vary in magnitude, duration, and the type of neuron affected. A moderate Ca2+ concentration can initiate certain cellular repair pathways and promote neuroregeneration. While the peripheral nervous system exhibits an intrinsic regenerative capability, the central nervous system has limited self-repair potential. There is evidence that significant variations exist in evoked calcium responses and axonal regeneration among neurons, and individual differences in regenerative capacity are apparent even within the same type of neurons. Furthermore, some studies have shown that neuronal activity could serve as a potent regulator of this process. The spatio-temporal patterns of calcium dynamics are intricately controlled by a variety of proteins, including channels, ion pumps, enzymes, and various calcium-binding proteins, each of which can exert either positive or negative effects on neural repair, depending on the cellular context. In this concise review, we focus on several calcium-associated proteins such as CaM kinase II, GAP-43, oncomodulin, caldendrin, calneuron, and NCS-1 in order to elaborate on their roles in the intrinsic mechanisms governing neuronal regeneration following traumatic damage processes.

Associations of Growth-Associated Protein 43 with Cerebral Microbleeds: A Longitudinal Study.

Background: Cerebral microbleeds (CMB) play an important role in neurodegenerative pathology. Objective: The present study aims to test whether cerebrospinal fluid (CSF) growth-associated protein 43 (GAP-43) level is linked to CMBs in elderly people. Methods: A total of 750 participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI) who had measurements of GAP-43 and CMBs were included in the study. According to the presence and extent of CMBs, participants were stratified into different groups. Regression analyses were used to assess cross-sectional and longitudinal associations between GAP-43 and CMBs. Results: Participants with CMB were slightly older and had higher concentrations of CSF GAP43. In multivariable adjusted analyses for age, gender, APOEɛ4 status, and cognitive diagnoses, higher CSF GAP-43 concentrations were modestly associated with CMB presence (OR = 1.169, 95% CI = 1.001-1.365) and number (ß= 0.020, SE = 0.009, p = 0.027). Similarly, higher CSF GAP43 concentrations were accrual of CMB lesions, associated with higher CMB progression (OR = 1.231, 95% CI = 1.044-1.448) and number (ß= 0.017, SE = 0.005, p = 0.001) in the follow up scan. In stratified analyses, slightly stronger associations were noted in male participants, those 65 years and older, carriers of APOEɛ4 alleles, and with more advanced cognitive disorders. Conclusions: CSF GAP-43 was cross-sectionally associated with the presence and extent of CMBs. GAP-43 might be used as a biomarker to track the dynamic changes of CMBs in elderly persons.

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

OBJECTIVES: In this study, we employed an in vitro culturing technique to investigate the impact of p53 on the modulation of growth-associated protein-43 (GAP-43) within the primary cortical neurons of rat specimens. METHODS: (1) Within the first 24 hours after birth, the bilateral cortex was extracted from newborn Wistar rats and primary cortical neurons were cultured and identified. (2) The changes in the mRNA and protein expressions of GAP-43 induced by p53 in rat primary cortical neurons cultured in vitro were identified utilizing real-time polymerase chain reaction and western blot techniques. RESULTS: (1) Lentiviral transfection of p53 within primary cortical neurons of rats elicited elevated levels of both mRNA and protein expressions of GAP-43, consequently culminating in a noteworthy augmentation of p53 expression. (2) The introduction of a p53 inhibitor in rat primary cortical neurons resulted in a reduction in both mRNA and protein expressions of GAP-43. CONCLUSION: Within primary rat cortical neurons, p53 has the potential to prompt an augmentation in both the transcriptional and protein expression levels of the GAP-43 protein.

Vof16-miR-185-5p-GAP43 network improves the outcomes following spinal cord injury via enhancing self-repair and promoting axonal growth.

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

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.

Restoring Synaptic Function: How Intranasal Delivery of 3D-Cultured hUSSC Exosomes Improve Learning and Memory Deficits in Alzheimer's Disease.

Memory problems are often the first signs of cognitive impairment related to Alzheimer's disease (AD), and stem cells and stem cell-derived exosomes (EXOs) have been studied for their therapeutic potential to improve the disease signs. While many studies have shown the anti-inflammatory and immunomodulatory effects of stem cells and exosomes on improving memory in different AD models, there is still insufficient data to determine how they modulate neural plasticity to enhance spatial memory and learning ability. Therefore, we conducted a study to investigate the effects of exosomes derived from 3D-cultured human Unrestricted Somatic Stem Cells (hUSSCs) on spatial memory and neuroplasticity markers in a sporadic rat model of AD. Using male Wistar rats induced by intracerebral ventricle injection of streptozotocin, we demonstrated that intranasal administration of hUSSC-derived exosomes could decrease Aß accumulation and improve learning and memory in the Morris water maze test. We also observed an increase in the expression of pre-synaptic and post-synaptic molecules involved in neuronal plasticity, including NMDAR1, integrin ß1, synaptophysin, pPKCα, and GAP-43, in the hippocampus. Our findings suggest that intranasal administration of exosomes can ameliorate spatial learning and memory deficits in rats, at least in part, by increasing the expression of neuroplasticity proteins. These results may encourage researchers to further investigate the molecular pathways involved in memory improvement after stem cell and exosome therapy, with the goal of increasing the efficacy and safety of exosome-based treatments for AD.

Analysis of medaka GAP43 gene promoter activity in transgenic lines.

We produced transgenic medaka fish lines that mimicked the expression of the GAP43 gene. Fish lines with the proximal 2-kilobase (kb) 5'-untranslated region (UTR) as the expression promoter specifically expressed enhanced green fluorescent protein (EGFP) in neural tissues, such as the brain, spinal cord, and peripheral nerves, and its expression decreased with growth, but persisted until adulthood. A functional analysis of the promoter using partially deleted UTRs revealed that functions related to neural tissue-specific promoter activity were widely distributed in the region upstream of the proximal 400-b. Furthermore, the distal half of the 2-kb UTR contributed to expression throughout the brain, while the region 400-b upstream of the proximal 600-b was strongly associated with expression in specific areas, such as the telencephalon. In addition, a region from 957 to 557b upstream of the translation initiation site was important for the long-term maintenance of promoter activity into adulthood. Among the transcription factors with recognition sequences in this region, Sp1 and CREB1 have been suggested to play important roles in the GAP43 promoter expression characteristics, such as strong expression in the telencephalon and long-term maintenance of expression.

GAP-43 closely interacts with BDNF in hippocampal neurons and is associated with Alzheimer's disease progression.

Introduction: Growth-associated protein 43 (GAP-43) is known as a neuronal plasticity protein because it is widely expressed at high levels in neuronal growth cones during axonal regeneration. GAP-43 expressed in mature adult neurons is functionally important for the neuronal communication of synapses in learning and memory. Brain-derived neurotrophic factor (BDNF) is closely related to neurodegeneration and synaptic plasticity during the aging process. However, the molecular mechanisms regulating neurodegeneration and synaptic plasticity underlying the pathogenesis and progression of Alzheimer's disease (AD) still remain incompletely understood. Methods: Remarkably, the expressions of GAP-43 and BDNF perfectly match in various neurons in the Human Brain Atlas database. Moreover, GAP-43 and BDNF are highly expressed in a healthy adults' hippocampus brain region and are inversely correlated with the amyloid beta (Aß), which is the pathological peptide of amyloid plaques found in the brains of patients with AD. Results: These data led us to investigate the impact of the direct molecular interaction between GAP-43 and BDNF in hippocampal neuron fate. In this study, we show that GAP-43 and BDNF are inversely associated with pathological molecules for AD (Tau and Aß). In addition, we define the three-dimensional protein structure for GAP-43 and BDNF, including the predictive direct binding sites via analysis using ClusPro 2.0, and demonstrate that the deprivation of GAP-43 and BDNF triggers hippocampal neuronal death and memory dysfunction, employing the GAP-43 or BDNF knock-down cellular models and 5XFAD mice. Conclusion: These results show that GAP-43 and BDNF are direct binding partners in hippocampal neurons and that their molecular signaling might be potential therapeutic targets for AD.

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.

Effect of prenatal stress and extremely low-frequency electromagnetic fields on anxiety-like behavior in female rats: With an emphasis on prefrontal cortex and hippocampus.

OBJECTIVE: Prenatal stress (PS) is a problematic situation resulting in psychological implications such as social anxiety. Ubiquitous extremely low-frequency electromagnetic fields (ELF-EMF) have been confirmed as a potential physiological stressor; however, useful neuroregenerative effect of these types of electromagnetic fields has also frequently been reported. The aim of the present study was to survey the interaction of PS and ELF-EMF on anxiety-like behavior. METHOD: A total of 24 female rats 40 days of age were distributed into four groups of 6 rats each: control, stress (their mothers were exposed to stress), EMF (their mothers underwent to ELF-EMF), and EMF/stress (their mothers concurrently underwent to stress and ELF-EMF). The rats were assayed using elevated plus-maze and open field tests. RESULTS: Expressions of the hippocampus GAP-43, BDNF, and caspase-3 (cas-3) were detected by immunohistochemistry in Cornu Ammonis 1 (CA1) and dentate gyrus (DG) of the hippocampus and prefrontal cortex (PFC). Anxiety-like behavior increased in all treatment groups. Rats in the EMF/stress group presented more serious anxiety-like behavior. In all treatment groups, upregulated expression of cas-3 was seen in PFC, DG, and CA1 and downregulated expression of BDNF and GAP-43 was seen in PFC and DG and the CA1. Histomorphological study showed vast neurodegenerative changes in the hippocampus and PFC. CONCLUSION: The results showed ,female rats that underwent PS or/and EMF exhibited critical anxiety-like behavior and this process may be attributed to neurodegeneration in PFC and DG of the hippocampus and possibly decreased synaptic plasticity so-called areas.

Exonized Alu repeats in the 3'UTR of a CYP20A1_Alu-LT transcript act as a miRNA sponge.

OBJECTIVE: Alu repeats have gained huge importance in the creation and modification of regulatory networks. We previously reported a unique isoform of human CYP20A1 i.e. CYP20A1_Alu-LT with 23 Alu repeats exonized in its 9 kb long 3'UTR with 4742 potential binding sites for 994 miRNAs. The role of this transcript was hypothesized as a potential miRNA sponge in primary neurons as its expression correlated with that of 380 genes having shared miRNA sites and enriched in neuro-coagulopathy. This study provides experimental evidence for the miRNA sponge activity of CYP20A1_Alu-LT in neuronal cell lines. RESULTS: We studied the Alu-rich fragment of the CYP20A1_Alu-LT extended 3'UTR with > 10 binding sites for miR-619-5p and miR-3677-3p. Enrichment of the Alu-rich fragment with Ago2 confirmed miRNA association of this transcript. Cloning the fragment downstream of a reporter gene led to a 90% decrease in luciferase activity. Overexpression and knockdown studies revealed a positive correlation between the expression of CYP20A1_Alu-LT and miR-619-5p / miR-3677-3p target genes. GAP43, one of the key modulators of nerve regeneration, was significantly altered by the expression of CYP20A1_Alu-LT. This study, for the first time, provides evidence for a unique regulatory function of exonized Alu repeats as miRNA sponges.

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.

Synergistic role of resveratrol and exercise training in management of diabetic neuropathy and myopathy via SIRT1/NGF/GAP43 linkage.

AIMS: Oxidative stress also plays an important role in the pathogenesis of diabetic neuropathy (DN). Both resveratrol (RES) and exercise (EX) have potent anti-oxidative benefits. Low levels of nerve growth factor (NGF) and SIRT1 (a member of sirtuin family) have been reported in patients with DN. The current study has been designed to investigate the role of serum NGF and SIRT1 on DN-induced hyperalgesia and motor incoordination and to evaluate the possible protective role of RES and/or EX. MAIN METHODS: A total of 40 male adult albino rats divided into five groups; control, DN, DN + RES, DN + EX, and DN + RES and EX. DN was confirmed by sensorimotor disturbance and diminished nerve conduction velocity (NCV). NGF and SIRT1 levels were measured by western blot. Calcitonin gene-related peptide (CGRP) was measured by PCR. Myofibrillar degeneration and inflammation scores were revealed via H&E microscopic analysis of the gastrocnemius muscle. Immunohistochemical evaluation of caspase3 and TNF-α was performed in the lumber segment of spinal cord and gastrocnemius muscle sections. Ultrastructural evaluation of sciatic nerve axonal degeneration has also been assessed. KEY FINDINGS: DN group showed decreased SIRT1 level, decreased NGF level and correlated with CGRP level and Na+/K+ ATPase. Treatment with RES and/or EX resulted in improvement of sensorimotor disturbance. DN characterized by reduced SOD level, whereas RES and/or EX could limit oxidative damage by up-regulation Bcl2, Akt and GAP-43 and down-regulation of caspase3 and TNF-α. In conclusion, increased level of SIRT1and NGF by incorporation of RES (natural supplementation) and EX (life style modification) could improve the neuroinflammatory state in DN.

Sex differences in maternal odor preferences and brain levels of GAP-43 and sonic hedgehog proteins in infant SHR and Wistar Kyoto rats.

Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder that presents sex differences in the severity and presentation of symptoms, whose neurobiological basis is still unknown. Both Growth-associated Protein 43 (GAP-43) and Sonic hedgehog (Shh) are considered essential proteins for the appropriate brain development, but their participation in ADHD neurobiology have not been investigated yet. In this study, we hypothesized that alterations in these proteins could be related to behavioral traits to ADHD phenotype. Thus, both sexes of infant Spontaneously hypertensive rats (SHR, used as ADHD animal model) were evaluated for developmental milestones, locomotor activity, olfactory and recognition memory. Both GAP-43 and Shh were assessed in the olfactory bulb, frontal cortex and hippocampus in early and late infancy. During early infancy, SHR reached three developmental milestones later, and females showed olfactory memory impairment accompanied by increased levels of Shh in the olfactory bulb. In later infancy, hyperlocomotion, impaired recognition memory, and decreased Shh in the hippocampus were observed in SHR from both sexes. While in early infancy GAP-43 was not altered, it was decreased in the frontal cortex and hippocampus of female SHR in late infancy. Therefore, both Shh and GAP-43 are involved in the sex-dependent behavioral alterations showed by infant SHR. Despite the disorder's complexity and heterogeneity, our findings reveal important developmental parameters during SHR development and also emphasizes the relevance of studying sex differences in the ADHD context.

Expressions of NeuroD and GAP43 as diagnostic markers for olfactory neuroblastoma.

OBJECTIVE: Olfactory neuroblastoma (ONB) is often difficult to pathologically distinguish from other small round cell tumors (SRCTs) arising in the nasal cavities. Although there are several diagnostic markers used for differential diagnosis of ONB, these molecules are also expressed in various neuronal derived tumors. Here, we examined the expression of NeuroD, GAP43, and olfactory marker protein (OMP) in ONB and non-ONB SRCT to determine their utility in the differential diagnosis of ONB. METHODS: Twenty-six patients diagnosed with and treated for ONB at Kobe University Hospital between 1997 and 2017 with formalin-fixed, paraffin-embedded biopsy or surgical resection specimens were included. The expressions of NeuroD, GAP43, and OMP were immunohistochemically examined in these 26 ONB specimens and specimens from 13 SRCTs arising in the nasal cavities for reference. RESULTS: Among the 26 ONB samples, focal, patchy, and marked staining for NeuroD was observed in 4, 3, and 9 samples, respectively. Focal, patchy, and marked GAP43 staining was observed in 5, 3, and 11 samples, respectively. Consequently, marked positive staining for either NeuroD or GAP43 was observed in 54% (14/26) of ONBs. Among the 13 SRCTs, marked staining for NeuroD was observed in two small cell carcinomas, one undifferentiated carcinoma, and one neuroendocrine carcinoma, whereas marked positive staining for GAP43 was observed only in one undifferentiated carcinoma. No specimen in this study exhibited OMP staining. CONCLUSIONS: Our results suggest possible roles of GAP43 immunostaining in the differential diagnosis of ONB.

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.

Bestrophin-1 Participates in Neuropathic Pain Induced by Spinal Nerve Transection but not Spinal Nerve Ligation.

Previous studies have reported that L5/L6 spinal nerve ligation (SNL), but not L5 spinal nerve transection (SNT), enhances anoctamin-1 in injured and uninjured dorsal root ganglia (DRG) of rats suggesting some differences in function of the type of nerve injury. The role of bestrophin-1 in these conditions is unknown. The aim of this study was to investigate the role of bestrophin-1 in rats subjected to L5 SNT and L5/L6 SNL. SNT up-regulated bestrophin-1 protein expression in injured L5 and uninjured L4 DRG at day 7, whereas it enhanced GAP43 mainly in injured, but also in uninjured DRG. In contrast, SNL enhanced GAP43 at day 1 and 7, while bestrophin-1 expression increased only at day 1 after nerve injury. Accordingly, intrathecal injection of the bestrophin-1 blocker CaCCinh-A01 (1-10 µg) reverted SNT- or SNL-induced tactile allodynia in a concentration-dependent manner. Intrathecal injection of CaCCinh-A01 (10 µg) prevented SNT-induced upregulation of bestrophin-1 and GAP43 at day 7. In contrast, CaCCinh-A01 did not affect SNL-induced up-regulation of GAP43 nor bestrophin-1. Bestrophin-1 was mainly expressed in small- and medium-size neurons in naïve rats, while SNT increased bestrophin-1 immunoreactivity in CGRP+, but not in IB4+ neuronal cells in DRG. Intrathecal injection of bestrophin-1 plasmid (pCMVBest) induced tactile allodynia and increased bestrophin-1 expression in DRG and spinal cord in naïve rats. CaCCinh-A01 reversed bestrophin-1 overexpression-induced tactile allodynia and restored bestrophin-1 expression. Our data suggest that bestrophin-1 plays a relevant role in neuropathic pain induced by SNT, but not by SNL. PERSPECTIVE: SNT, but not SNL, up-regulates bestrophin-1 and GAP43 protein expression in injured L5 and uninjured L4 DRG. SNT increases bestrophin-1 immunoreactivity in CGRP+ neurons in DRG. Bestrophin-1 overexpression induces allodynia. CaCCinh-A01 reduces allodynia and restores bestrophin-1 expression. Our data suggest bestrophin-1 is differentially regulated depending on the neuropathic pain model.

CSF peptides from VGF and other markers enhance prediction of MCI to AD progression using the ATN framework.

The amyloid beta, tau, neurodegenerative markers framework has been proposed to serve as a system to classify and combine biomarkers for Alzheimer's Disease (AD). Although cerebrospinal (CSF) fluid AT (amyloid beta and tau)-based biomarkers have a well-established track record to distinguish AD from control subjects and to predict conversion from mild cognitive impairment (MCI) to AD, there is not an established non-tau based neurodegenerative ("N") marker from CSF. Here, we examine the ability of several candidate peptides in the CSF to serve as "N" markers to both classify disease state and predict MCI to AD conversion. We observed that although many putative N markers involved in synaptic processing and neuroinflammation were able to, when examined in isolation, distinguish MCI converters from non-converters, a derivative from VGF, when combined with AT markers, most strongly enhanced prediction of MCI to AD conversion. Low CSF VGF levels were also predictive of MCI to dementia conversion in the setting of normal AT markers, suggesting that it may serve as a very early predictor of dementia conversion. Other markers derived from neuronal pentraxin 2, GAP-43 and a 14-3-3 protein were also able to enhance MCI to AD prediction when used as a marker of neurodegeneration, but VGF had the highest predictive capacity. Thus, we propose that low levels of VGF in CSF may serve as "N" in the amyloid beta, tau, neurodegenerative markers framework to enhance the prediction of MCI to AD conversion.

Fast Screening of Protein-Protein Interactions Using Förster Resonance Energy Transfer (FRET-) Based Fluorescence Plate Reader Assay in Live Cells.

Protein-protein interactions (PPIs) have great importance for intracellular signal transduction and sustaining the homeostasis of an organism. Thus, the identification of PPIs is necessary to better understand the downstream signaling functions of the proteins in healthy and pathological conditions. Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool for detecting PPIs in living cells. In literature, FRET analysis methods such as donor photobleaching (FLIM), acceptor photobleaching, spectral imaging, and the three-filter cube method (sensitized emission) are abundantly applied to investigate PPIs; however, they require various expensive instrumentations, and their calculation methods are very time consuming. Since confocal microscopy applications and live cell-based techniques of FRET are very costly, scientists sometimes prefer plate readers for FRET experiments. However, plate reader applications also have many disadvantages and considerations compared to confocal fluorescence microscopy, and complex calculation procedures should be performed. To overcome these problems, we propose a FRET-based high-throughput assay method with a standard monochromator-based microplate reader, which is generally available in most biochemistry laboratories, and an alternative calculation procedure. This rapid, low cost, and effective analysis method enables the scientists to prescreen PPIs in living cells as a preliminary study and quick glance at the experiment before preparing the whole experimental setup with the expensive instrumentations. Additionally, the alternative calculation procedure provides the FRET area comparison without complex bleed-through calculations in a non-conventional manner by shortening the analysis processes with this quick and uncomplicated spectral representation.