NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons.

Cancer patients undergoing treatment with antineoplastic drugs often experience chemotherapy-induced neuropathic pain (CINP), and the therapeutic options for managing CINP are limited. Here, we show that systemic paclitaxel administration upregulates the expression of neurotrophin-3 (Nt3) mRNA and NT3 protein in the neurons of dorsal root ganglia (DRG), but not in the spinal cord. Blocking NT3 upregulation attenuates paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities and spontaneous pain without altering acute pain and locomotor activity in male and female mice. Conversely, mimicking this increase produces enhanced responses to mechanical, heat, and cold stimuli and spontaneous pain in naive male and female mice. Mechanistically, NT3 triggers tropomyosin receptor kinase C (TrkC) activation and participates in the paclitaxel-induced increases of C-C chemokine ligand 2 (Ccl2) mRNA and CCL2 protein in the DRG. Given that CCL2 is an endogenous initiator of CINP and that Nt3 mRNA co-expresses with TrkC and Ccl2 mRNAs in DRG neurons, NT3 likely contributes to CINP through TrkC-mediated activation of the Ccl2 gene in DRG neurons. NT3 may be thus a potential target for CINP treatment.

Neurotrophin-3 promotes peripheral nerve regeneration by maintaining a repair state of Schwann cells after chronic denervation via the TrkC/ERK/c-Jun pathway.

BACKGROUND: Maintaining the repair phenotype of denervated Schwann cells in the injured distal nerve is crucial for promoting peripheral nerve regeneration. However, when chronically denervated, the capacity of Schwann cells to support repair and regeneration deteriorates, leading to peripheral nerve regeneration and poor functional recovery. Herein, we investigated whether neurotrophin-3 (NT-3) could sustain the reparative phenotype of Schwann cells and promote peripheral nerve regeneration after chronic denervation and aimed to uncover its potential molecular mechanisms. METHODS: Western blot was employed to investigate the relationship between the expression of c-Jun and the reparative phenotype of Schwann cells. The inducible expression of c-Jun by NT-3 was examined both in vitro and in vivo with western blot and immunofluorescence staining. A chronic denervation model was established to study the role of NT-3 in peripheral nerve regeneration. The number of regenerated distal axons, myelination of regenerated axons, reinnervation of neuromuscular junctions, and muscle fiber diameters of target muscles were used to evaluate peripheral nerve regeneration by immunofluorescence staining, transmission electron microscopy (TEM), and hematoxylin and eosin (H&E) staining. Adeno-associated virus (AAV) 2/9 carrying shRNA, small molecule inhibitors, and siRNA were employed to investigate whether NT-3 could signal through the TrkC/ERK pathway to maintain c-Jun expression and promote peripheral nerve regeneration after chronic denervation. RESULTS: After peripheral nerve injury, c-Jun expression progressively increased until week 5 and then began to decrease in the distal nerve following denervation. NT-3 upregulated the expression of c-Jun in denervated Schwann cells, both in vitro and in vivo. NT-3 promoted peripheral nerve regeneration after chronic denervation, mainly by upregulating or maintaining a high level of c-Jun rather than NT-3 itself. The TrkC receptor was consistently presented on denervated Schwann cells and served as NT-3 receptors following chronic denervation. NT-3 mainly upregulated c-Jun through the TrkC/ERK pathway. CONCLUSION: NT-3 promotes peripheral nerve regeneration by maintaining the repair phenotype of Schwann cells after chronic denervation via the TrkC/ERK/c-Jun pathway. It provides a potential target for the clinical treatment of peripheral nerve injury after chronic denervation.

Electroacupuncture facilitates the integration of a grafted TrkC-modified mesenchymal stem cell-derived neural network into transected spinal cord in rats via increasing neurotrophin-3.

AIMS: This study was aimed to investigate whether electroacupuncture (EA) would increase the secretion of neurotrophin-3 (NT-3) from injured spinal cord tissue, and, if so, whether the increased NT-3 would promote the survival, differentiation, and migration of grafted tyrosine kinase C (TrkC)-modified mesenchymal stem cell (MSC)-derived neural network cells. We next sought to determine if the latter would integrate with the host spinal cord neural circuit to improve the neurological function of injured spinal cord. METHODS: After NT-3-modified Schwann cells (SCs) and TrkC-modified MSCs were co-cultured in a gelatin sponge scaffold for 14 days, the MSCs differentiated into neuron-like cells that formed a MSC-derived neural network (MN) implant. On this basis, we combined the MN implantation with EA in a rat model of spinal cord injury (SCI) and performed immunohistochemical staining, neural tracing, electrophysiology, and behavioral testing after 8 weeks. RESULTS: Electroacupuncture application enhanced the production of endogenous NT-3 in damaged spinal cord tissues. The increase in local NT-3 production promoted the survival, migration, and maintenance of the grafted MN, which expressed NT-3 high-affinity TrkC. The combination of MN implantation and EA application improved cortical motor-evoked potential relay and facilitated the locomotor performance of the paralyzed hindlimb compared with those of controls. These results suggest that the MN was better integrated into the host spinal cord neural network after EA treatment compared with control treatment. CONCLUSIONS: Electroacupuncture as an adjuvant therapy for TrkC-modified MSC-derived MN, acted by increasing the local production of NT-3, which accelerated neural network reconstruction and restoration of spinal cord function following SCI.

Growth Factors in the Carotid Body-An Update.

The carotid body may undergo plasticity changes during development/ageing and in response to environmental (hypoxia and hyperoxia), metabolic, and inflammatory stimuli. The different cell types of the carotid body express a wide series of growth factors and corresponding receptors, which play a role in the modulation of carotid body function and plasticity. In particular, type I cells express nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, insulin-like-growth factor-I and -II, basic fibroblast growth factor, epidermal growth factor, transforming growth factor-α and -ß, interleukin-1ß and -6, tumor necrosis factor-α, vascular endothelial growth factor, and endothelin-1. Many specific growth factor receptors have been identified in type I cells, indicating autocrine/paracrine effects. Type II cells may also produce growth factors and express corresponding receptors. Future research will have to consider growth factors in further experimental models of cardiovascular, metabolic, and inflammatory diseases and in human (normal and pathologic) samples. From a methodological point of view, microarray and/or proteomic approaches would permit contemporary analyses of large groups of growth factors. The eventual identification of physical interactions between receptors of different growth factors and/or neuromodulators could also add insights regarding functional interactions between different trophic mechanisms.

Diterpene Ginkgolides Exert an Antidepressant Effect Through the NT3-TrkA and Ras-MAPK Pathways.

BACKGROUND: Depression is a highly prevalent mental illness that severely impacts the quality of life of affected individuals. Our recent studies demonstrated that diterpene ginkgolides (DG) have antidepressant effects in mice. However, the underlying molecular mechanisms remained much unclear. METHODS: In this study, we assessed the antidepressant effects of chronic DG therapy in rats by evaluating depression-related behaviors, we also examined potential side effects using biochemical indicators. Furthermore, we performed an in-depth molecular network analysis of gene-protein-metabolite interactions on the basis of metabolomics. RESULTS: Chronic DG treatment significantly ameliorated the depressive-like behavioral phenotype. Furthermore, the neurotrophin signaling-related NT3-TrkA and Ras-MAPK pathways may play an important role in the antidepressant effect of DG in the hippocampus. CONCLUSION: These findings provide novel insight into the mechanisms underlying the antidepressant action of DG, and should help advance the development of new therapeutic strategies for depression.

Neurotrophin gene therapy to promote survival of spiral ganglion neurons after deafness.

Hearing impairment is a major health and economic concern worldwide. Currently, the cochlear implant (CI) is the standard of care for remediation of severe to profound hearing loss, and in general, contemporary CIs are highly successful. But there is great variability in outcomes among individuals, especially in children, with many CI users deriving much less or even marginal benefit. Much of this variability is related to differences in auditory nerve survival, and there has been substantial interest in recent years in exploring potential therapies to improve survival of the cochlear spiral ganglion neurons (SGN) after deafness. Preclinical studies using osmotic pumps and other approaches in deafened animal models to deliver neurotrophic factors (NTs) directly to the cochlea have shown promising results, especially with Brain-Derived Neurotrophic Factor (BDNF). More recent studies have focused on the use of NT gene therapy to force expression of NTs by target cells within the cochlea. This could provide the means for a one-time treatment to promote long-term NT expression and improve neural survival after deafness. This review summarizes the evidence for the efficacy of exogenous NTs in preventing SGN degeneration after hearing loss and reviews the animal research to date suggesting that NT gene therapy can elicit long-term NT expression in the cochlea, resulting in significantly improved SGN and radial nerve fiber survival after deafness. In addition, we discuss NT gene therapy in other non-auditory applications and consider some of the remaining issues with regard to selecting optimal vectors, timing of treatment, and place/method of delivery, etc. that must be resolved prior to considering clinical application.

NT3P75-2 gene-modified bone mesenchymal stem cells improve neurological function recovery in mouse TBI model.

BACKGROUND: The attainment of extensive neurological function recovery remains the key challenge for the treatment of traumatic brain injury (TBI). Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) has been shown to improve neurological function recovery after TBI. However, the survival of BMSCs after transplantation in early-stage TBI is limited, and much is unknown about the mechanisms mediating this neurological function recovery. Secretion of neurotrophic factors, including neurotrophin 3 (NT3), is one of the critical factors mediating BMSC neurological function recovery. Gene mutation of NT3 (NT3P75-2) has been shown to enhance the biological function of NT3 via the reduction of the activation of the P75 signal pathway. Thus, we investigated whether NT3P75-2 gene-modified BMSCs could enhance the survival of BMSCs and further improve neurological function recovery after TBI. METHODS: The ability of NT3P75-2 induction to improve cell growth rate of NSC-34 and PC12 cells in vitro was first determined. BMSCs were then infected with three different lentiviruses (green fluorescent protein (GFP), GFP-NT3, or GFP-NT3P75-2), which stably express GFP, GFP-NT3, or GFP-NT3P75-2. At 24 h post-TBI induction in mice, GFP-labeled BMSCs were locally transplanted into the lesion site. Immunofluorescence and histopathology were performed at 1, 3, and/or 7 days after transplantation to evaluate the survival of BMSCs as well as the lesion volume. A modified neurological severity scoring system and the rotarod test were chosen to evaluate the functional recovery of the mice. Cell growth rate, glial activation, and signaling pathway analyses were performed to determine the potential mechanisms of NT3P75-2 in functional recovery after TBI. RESULTS: Overall, NT3P75-2 improved cell growth rate of NSC-34 and PC12 cells in vitro. In addition, NT3P75-2 significantly improved the survival of transplanted BMSCs and neurological function recovery after TBI. Overexpression of NT3P75-2 led to a significant reduction in the activation of glial cells, brain water content, and brain lesion volume after TBI. This was associated with a reduced activation of the p75 neurotrophin receptor (P75NTR) and the c-Jun N-terminal kinase (JNK) signal pathway due to the low affinity of NT3P75-2 for the receptor. CONCLUSIONS: Taken together, our results demonstrate that administration of NT3P75-2 gene-modified BMSCs dramatically improves neurological function recovery after TBI by increasing the survival of BMSCs and ameliorating the inflammatory environment, providing a new promising treatment strategy for TBI.

Protection from noise-induced cochlear synaptopathy by virally mediated overexpression of NT3.

Noise exposures causing only transient threshold shifts can destroy auditory-nerve synapses without damaging hair cells. Here, we asked whether virally mediated neurotrophin3 (NT3) overexpression can repair this damage. CBA/CaJ mice at 6 wks were injected unilaterally with adeno-associated virus (AAV) containing either NT3 or GFP genes, via the posterior semicircular canal, 3 wks prior to, or 5 hrs after, noise exposure. Controls included exposed animals receiving vehicle only, and unexposed animals receiving virus. Thresholds were measured 2 wks post-exposure, just before cochleas were harvested for histological analysis. In separate virus-injected animals, unexposed cochleas were extracted for qRT-PCR. The GFP reporter showed that inner hair cells (IHCs) were transfected throughout the cochlea, and outer hair cells mainly in the apex. qRT-PCR showed 4- to 10-fold overexpression of NT3 from 1-21 days post-injection, and 1.7-fold overexpression at 40 days. AAV-NT3 delivered prior to noise exposure produced a dose-dependent reduction of synaptopathy, with nearly complete rescue at some cochlear locations. In unexposed ears, NT3 overexpression did not affect thresholds, however GFP overexpression caused IHC loss. In exposed ears, NT3 overexpression increased permanent threshold shifts. Thus, although NT3 overexpression can minimize noise-induced synaptic damage, the forced overexpression may be harmful to hair cells themselves during cochlear overstimulation.

Differential Clinical Significance of Neurotrophin-3 Expression according to MYCN Amplification and TrkC Expression in Neuroblastoma.

BACKGROUND: Neurotrophin-3 (NT-3), a member of the NT family, has only been considered an ancillary compound that provides anti-apoptotic benefits by inactivating tropomyosin receptor kinase C (TrkC)-induced apoptotic signals. However, little is known about the clinical relevance of NT-3 expression itself in neuroblastoma. The purpose of this study was to assess NT-3 expression in patients with neuroblastoma and its relevance to clinicopathologic findings and treatment outcomes. METHODS: In this study, expression of NT-3 and TrkC was analyzed using immunohistochemistry in 240 patients with newly diagnosed neuroblastoma. RESULTS: The results of the study revealed that NT-3 expression was associated with older age at diagnosis, localized tumors, and more differentiated tumors but was not associated with early treatment response (degree of residual tumor volume after three cycles of chemotherapy) and progression-free survival (PFS). However, when analysis was confined to patients with MYCN amplified tumors, NT-3 expression was associated with better early treatment response with borderline significance (P = 0.092) and higher PFS (86.9% vs. 58.2%; P = 0.044). In multivariate analysis in patients with MYCN amplified tumors, NT-3 was independent prognostic factor (hazard ratio, 0.246; 95% confidence interval, 0.061-0.997; P = 0.050). In another subgroup analysis, the early treatment response was better if NT-3 was expressed in patients without TrkC expression (P = 0.053) while it was poorer in patients with TrkC expression (P = 0.023). CONCLUSION: This study suggests that NT-3 expression in neuroblastoma has its own clinical significance independent of TrkC expression, and its prognostic significance differs depending on the status of MYCN amplification and/or TrkC expression.

Dorsal Amygdala Neurotrophin-3 Decreases Anxious Temperament in Primates.

BACKGROUND: An early-life anxious temperament (AT) is a risk factor for the development of anxiety, depression, and comorbid substance abuse. We validated a nonhuman primate model of early-life AT and identified the dorsal amygdala as a core component of AT's neural circuit. Here, we combine RNA sequencing, viral-vector gene manipulation, functional brain imaging, and behavioral phenotyping to uncover AT's molecular substrates. METHODS: In response to potential threat, AT and brain metabolism were assessed in 46 young rhesus monkeys. We identified AT-related transcripts using RNA-sequencing data from dorsal amygdala tissue (including central nucleus of the amygdala [Ce] and dorsal regions of the basal nucleus). Based on the results, we overexpressed the neurotrophin-3 gene, NTF3, in the dorsal amygdala using intraoperative magnetic resonance imaging-guided surgery (n = 5 per group). RESULTS: This discovery-based approach identified AT-related alterations in the expression of well-established and novel genes, including an inverse association between NTRK3 expression and AT. NTRK3 is an interesting target because it is a relatively unexplored neurotrophic factor that modulates intracellular neuroplasticity pathways. Overexpression of the transcript for NTRK3's endogenous ligand, NTF3, in the dorsal amygdala resulted in reduced AT and altered function in AT's neural circuit. CONCLUSIONS: Together, these data implicate neurotrophin-3/NTRK3 signaling in the dorsal amygdala in mediating primate anxiety. More generally, this approach provides an important step toward understanding the molecular underpinnings of early-life AT and will be useful in guiding the development of treatments to prevent the development of stress-related psychopathology.

The Akt/mTOR cascade mediates high glucose-induced reductions in BDNF via DNMT1 in Schwann cells in diabetic peripheral neuropathy.

Brain-derived neurotropic factor (BDNF) deficiency in Schwann cells plays an important role in the pathogenesis of diabetic peripheral neuropathy (DPN). Little is known about the mechanism involved in BDNF downregulation in Schwann cells in DPN. In this study, we first confirmed downregulation of BDNF and neurotrophin 3 expression in the sciatic nerves of diabetic mice, which was accompanied by myelin sheath abnormalities. Moreover, in vitro, high glucose was revealed to cause downregulation of BDNF, but not neurotrophin 3, expression in RSC96 cells, which was accompanied by DNA hypermethylation of BDNF promoters I and II. DNMT1 was subsequently revealed to be enhanced at the mRNA and protein levels in high glucose-stimulated RSC96 cells, and inhibition of DNMT1 with 5-Aza treatment or shRNA vector transfection reversed high glucose-induced reductions in BDNF expression. Furthermore, the mTOR and upstream Akt pathways were indicated to mediate high glucose-induced DNMT1 and BDNF expression in RSC96 cells. Taken together, our results suggest that the Akt/mTOR cascade mediates high glucose-induced reductions in BDNF via DNMT1 in Schwann cells in DPN.

Neurotrophin-3 acts on the endothelial-mesenchymal transition of heterotopic ossification in rats.

Despite the fact that extensive studies have focused on heterotopic ossification (HO), its molecular mechanism remains unclear. The endothelial-mesenchymal transition (EndMT), which may be partially modulated by neuroendocrine cytokines is thought to play a major role in HO. Neurotrophin-3 (NT-3), which has neuroendocrine characteristics is believed to promote skeletal remodeling. Herein, we suggest that that NT-3 may promote HO formation through regulation of EndMT. Here, we used an in vivo model of HO and an in vitro model of EndMT induction to elucidate the effect and underlying mechanism of NT-3 on EndMT in HO. Our results showed that heterotopic bone and cartilage arose from EndMT and NT-3 promoted HO formation in vivo. Our in vitro results showed that NT-3 up-regulated mesenchymal markers (FSP-1, α-SMA and N-cadherin) and mesenchymal stem cell (MSC) markers (STRO-1, CD44 and CD90) and down-regulated endothelial markers (Tie-1, VE-cadherin and CD31). Moreover, NT-3 enhanced a chondrogenesis marker (Sox9) and osteogenesis markers (OCN and Runx2) via activation of EndMT. However, both EndMT specific inhibitor and tropomyosin-related kinase C (TrkC) specific inhibitor rescued NT-3-induced HO formation and EndMT induction in vivo and in vitro. In conclusion, our findings demonstrate that NT-3 promotes HO formation via modulation of EndMT both in vivo and in vitro, which offers a new potential target for the prevention and therapy of HO.

HIF-1α promotes bone marrow stromal cell migration to the injury site and enhances functional recovery after spinal cord injury in rats.

BACKGROUND: Spinal cord injury (SCI) is a severe health problem worldwide, and efficacious strategies to properly repair SCI have not yet been developed. Recently, gene and cell therapies as alternative treatments for SCI have been proposed to comprise safe and promising strategies. METHODS: The present study investigated the therapeutic effects and underlying mechanisms of hypoxia-inducible factor-1α carried in recombinant adenovirus (Adv-HIF-1α), as administered immediately after SCI in adult rats. RESULTS: Adv-HIF-1α-treated animals showed better functional recovery and smaller cavity volume than those in the vehicle-treated control group. Both the numbers of green fluorescent protein-labeled bone marrow stromal cells (GFP-BMSCs) and cells double-positive for GFP and a cell lineage marker (NeuN) in the injured spinal cord were larger in the Adv-HIF-1α-treated group. The expression levels of neurotrophins such as neurotrophin-3 and brain-derived neurotrophic factor were also higher in the Adv-HIF-1α-treated group. CONCLUSIONS: Adv-HIF-1α improves functional recovery in rats with SCI, and the underlying mechanism may be related to the mobilization of BMSCs to the injured area and higher expression levels of neurotrophin-3 and brain-derived neurotrophic factor.

Remodeling of lumbar motor circuitry remote to a thoracic spinal cord injury promotes locomotor recovery.

Retrogradely-transported neurotrophin signaling plays an important role in regulating neural circuit specificity. Here we investigated whether targeted delivery of neurotrophin-3 (NT-3) to lumbar motoneurons (MNs) caudal to a thoracic (T10) contusive spinal cord injury (SCI) could modulate dendritic patterning and synapse formation of the lumbar MNs. In vitro, Adeno-associated virus serotype two overexpressing NT-3 (AAV-NT-3) induced NT-3 expression and neurite outgrowth in cultured spinal cord neurons. In vivo, targeted delivery of AAV-NT-3 into transiently demyelinated adult mouse sciatic nerves led to the retrograde transportation of NT-3 to the lumbar MNs, significantly attenuating SCI-induced lumbar MN dendritic atrophy. NT-3 enhanced sprouting and synaptic formation of descending serotonergic, dopaminergic, and propriospinal axons on lumbar MNs, parallel to improved behavioral recovery. Thus, retrogradely transported NT-3 stimulated remodeling of lumbar neural circuitry and synaptic connectivity remote to a thoracic SCI, supporting a role for retrograde transport of NT-3 as a potential therapeutic strategy for SCI.

Transplantation of mesenchymal stem cells that overexpress NT-3 produce motor improvements without axonal regeneration following complete spinal cord transections in rats.

Transplanting stem cells engineered to overexpress trophic factors can improve motor abilities and facilitate axon regeneration following spinal cord injury. This study compared several transplantation paradigms using mesenchymal stem cells (MSCs) that overexpress the multi-neurotrophin, NT-3/D15A (NT-3-MSCs), to determine if different grafting strategies can elicit improved axon regeneration and/or behavioral outcomes following a complete T9 spinal transection. At one week post-transection, NT-3-MSCs were transplanted above, and at several locations below, the lesion site. A rostral-to-caudal gradient of NT-3-MSCs was produced by incrementally increasing the number of transplanted cells at locations distal to the transection. Motor function was analyzed using the Basso, Beattie, and Bresnahan scale for 7-weeks post-injury. The corticospinal tract was traced using biotinylated dextran amines, while raphespinal fibers were visualized using immunohistochemistry. Cell viability was assessed using transplants of NT-3-MSCs that express tdTomato. Retrograde tracing using fluorogold, as well as spinal re-transections, were performed to discriminate between a supra-spinal or reflexive influence of regained motor functions. NT-3-MSC transplants improved motor outcomes and tissue continuity at the transection site, however retrograde tracing using fluorogold revealed no evidence of axon regeneration. A spinal re-transection also failed to eliminate the improvement in motor outcomes produced by the transplant. We conclude that transplantation of NT-3-MSCs can improve motor function and morphological outcomes following a complete spinal transection without promoting axonal regeneration.

[Experimental study of lentivirus-mediated Nogo extracellular peptide residues 1-40 gene and neurotrophin 3 gene co-transduction in neural stem cells].

Objective: To explore the feasibility of co-transduction and co-expression of Nogo extracellular peptide residues 1-40 (NEP1-40) gene and neurotrophin 3 (NT-3) gene into neural stem cells (NSCs). Methods: NSCs were derived from the cortex tissue of Sprague Dawley rat embryo. The experiment included 5 groups: no-load lentiviral vector transducted NSCs (group A), NEP1-40 transducted NSCs (group B), NT-3 transducted NSCs (group C), NEP1-40 and NT-3 corporately transducted NSCs (group D), and blank control (group E). Target genes were transducted into NSCs by lentiviral vectors of different multiplicity of infection (MOI; 5, 10, 15) for different time (24, 48, 72 hours). Fluorescent microscope was used to observe the expression of fluorescence protein and acquire the optimum MOI and optimum collection time. Real-time fluorescence quantitative PCR and Western blot tests were utilized to evaluate the gene expressions of NEP1-40 and NT-3 in NSCs and protein expressions of NEP1-40 and NT-3 in NSCs and in culture medium. Results: The optimum MOI for both target gene was 10 and the optimum collection time was 48 hours. The real-time fluorescence quantitative PCR and Western blot results showed that the mRNA and protein relative expressions of NEP1-40 in groups B and D were significantly higher than those in groups A and C ( P<0.05), but no significant difference was found between groups B and D, and between groups A and C ( P>0.05). The mRNA and protein relative expressions of NT-3 in groups C and D were significantly higher than those in groups A and B ( P<0.05), but no significant difference was found between groups A and B, and between groups C and D ( P>0.05). Conclusion: NEP1-40 and NT-3 gene can be successfully co-transducted into NSCs by the mediation of lentiviral vector. The expressions of the two target genes are stable and have no auxo-action or antagonism between each other.

AAV-mediated NT-3 overexpression protects cochleae against noise-induced synaptopathy.

The synapse between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) has been identified as a sensitive structure to noise-induced damage in the mammalian cochlea. Since this synapse provides the major information pathway from the cochlea to the auditory brain, it is important to maintain its integrity. Neurotrophin-3 (NT-3) has been known to play an important role in the development and the functional maintenance of this synapse. Application of exogenous NT-3, or overexpression of this gene in a transgenic animal model, have shown the value to protect this synapse from noise-induced damage. In the present study, NT-3 overexpression was induced by cochlear gene transfection before noise exposure via the use of an adeno-associated viral (AAV) vector. We found that such an overexpression provided a significant synaptic protection against a noise exposure that caused massive damage to the synapses, likely due to it promoting the repair of the synapse after the initial damage.

RGD/TAT-functionalized chitosan-graft-PEI-PEG gene nanovector for sustained delivery of NT-3 for potential application in neural regeneration.

In this study, we prepared a multifunctional gene delivery nanovector containing a chitosan (CS) backbone and polyethylenimine (PEI) arms with arginine-glycine-aspartate (RGD)/twin-arginine translocation (TAT) conjugated via polyethylene glycol (PEG). Branched PEI, with a molecular weight of 2000 Da, was used to achieve a balance between biocompatibility and transfection efficiency, whereas RGD/TAT peptides were conjugated for enhanced targeting ability and cellular uptake. Synthesis of the copolymers was confirmed by characterizing the chemical structure with 1H nuclear magnetic resonance and Fourier Transform Infrared Spectroscopy (FTIR). The nanovector was biocompatible with cells and showed excellent capability for DNA condensation; the resulting complexes with DNA were well-formed, and possessed small particle size and reasonable positive charge. Higher gene transfection efficiency, compared to that achieved with PEI (25 kDa), was confirmed in tumor (HeLa cells) and normal cells (293T and NIH 3T3 cells). More importantly, the cells transfected with the chitosan-graft-PEI-PEG/pCMV-EGFP-Ntf3 complex produced sustained neurotrophin-3 with a linear increase in cumulative concentration, which induced neuronal differentiation of neural stem cell and promoted neurite outgrowth. These findings suggested that our multifunctional copolymers might be ideal nanovectors for engineering cells via gene transfection, and could potentially be applied in tumor therapy and regenerative medicine. STATEMENT OF SIGNIFICANCE: We successfully prepared a multifunctional gene delivery nanovector containing branched PEI with a molecular weight of 2000 Da to balance between biocompatibility and transfection efficiency, and RGD/TAT peptides for enhanced targeting ability and cellular uptake. The well-formed CPPP/DNA complexes of small particle size and reasonable positive charges potentially enhanced gene transfection in both tumor and normal cells. More importantly, the CPPP/pCMV-EGFP-Ntf3 complex-transfected 293T cells could produce sustained NT-3 with a constant ratio, which induced neuron differentiation of NSC and promoted neurite outgrowth. Therefore, our study provided an effective strategy for producing neurotrophins by engineering cells with gene delivery, which deserved wide investigation and potential application in regenerative medicine.

Neurotrophin Gene Therapy in Deafened Ears with Cochlear Implants: Long-term Effects on Nerve Survival and Functional Measures.

Because cochlear implants function by stimulating the auditory nerve, it is assumed that the condition of the nerve plays an important role in the efficacy of the prosthesis. Thus, considerable research has been devoted to methods of preserving the nerve following deafness. Neurotrophins have been identified as a potential contributor to neural health, but most of the research to date has been done in young animals and for short periods (less than 3 to 6 months) after the onset of treatment. The first objective of the current experiment was to examine the effects of a neurotrophin gene therapy delivery method on spiral ganglion neuron (SGN) preservation and function in the long term (5 to 14 months) in mature guinea pigs with cochlear implants. The second objective was to examine several potential non-invasive monitors of auditory nerve health following the neurotrophin gene therapy procedure. Eighteen mature adult male guinea pigs were deafened by cochlear perfusion of neomycin and then one ear was inoculated with an adeno-associated viral vector with an Nft3-gene insert (AAV.Ntf3) and implanted with a cochlear implant electrode array. Five control animals were deafened and inoculated with an empty AAV and implanted. Data from 43 other guinea pig ears from this and previous experiments were used for comparison: 24 animals implanted in a hearing ear, nine animals deafened and implanted with no inoculation, and ten normal-hearing non-implanted ears. After 4 to 21 months of psychophysical and electrophysiological testing, the animals were prepared for histological examination of SGN densities and inner hair cell (IHC) survival. Seventy-eight percent of the ears deafened and inoculated with AAV.Ntf3 showed better SGN survival than the 14 deafened-control ears. The degree of SGN preservation following the gene therapy procedure was variable across animals and across cochlear turns. Slopes of psychophysical multipulse integration (MPI) functions were predictive of SGN density, but only in animals with preserved IHCs. MPI was not affected by the AAV.Ntf3 treatment, but there was a minor improvement in temporal integration (TI). AAV.Ntf3 treatment had significant effects on ECAP and EABR amplitude growth func-tion (AGF) slopes; the reduction in slope in deafened ears was ameliorated by the AAV.Ntf3 treatment. Slopes of the ECAP and EABR AGFs were predictive of SGN density in a broad area near and just apical to the implant. The highest ensemble spontaneous activity (ESA) values were seen in animals with surviving IHCs, but AAV.Ntf3 treatment in deafened ears resulted in slightly higher ESA values compared to deafened untreated ears. Overall, a combination of the psychophysical and electrophysiological measures can be useful for monitoring the health of the implanted cochlea in guinea pigs. These measures should be applicable for assessing cochlear health in human subjects.

5-Aza-2'-deoxycytidine in the medial prefrontal cortex regulates alcohol-related behavior and Ntf3-TrkC expression in rats.

Recent studies have indicated that DNA methylation plays an important role in the development of alcohol abuse. 5-Aza-2'-deoxycytidine (5-Aza-dc), an inhibitor of DNA methyltransferases, was FDA approved for myelodysplastic syndrome treatment. However, it is unclear whether 5-Aza-dc is involved in alcohol abuse. In this study, using a chronic alcohol exposure model in rats, 5-Aza-dc was injected into the medial prefrontal cortex (mPFC). Alcohol-drinking behavior and the anxiety related behavior were evaluated by two-bottle choice and open field test. We found that 5-Aza-dc injection into the mPFC significantly decreased alcohol consumption and alcohol preference in alcohol-exposure rats, corresponding to the reduced blood alcohol levels. Although 5-Aza-dc potentiated the anxiety-like behavior of alcohol-exposure rats, it had no effect on the locomotor activity. Moreover, both of the mRNA and protein levels of DNA Methyltransferase 3A (DNMT3A) and DNMT3B in the mPFC were upregulated after 35 days of alcohol exposure and this upregulation could be reversed by 5-Aza-dc treatment. Additionally, 5-Aza-dc reversed the alcohol-induced downregulation of neurotrophin-3 (Ntf3), correspondingly the expression of its receptor-TrkC was reduced. These findings identified a functional role of 5-Aza-dc in alcohol-related behavioral phenotypes and one of the potential target genes, Ntf3. We also provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcohol abuse.