Effects Of Neurotrophins Research Articles

The Retrograde Delivery of Adenovirus Vector Carrying the Gene for Brain-Derived Neurotrophic Factor Protects Neurons and Oligodendrocytes From Apoptosis in the Chronically Compressed Spinal Cord of twy/twy Mice

The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of β-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS.: Four weeks after injection, the retrograde delivery of the LacZ marker gene was identified in cervical spinal neurons and some glial cells, including oligodendrocytes in the white matter of the spinal cord, in both the twy/twy mouse and the heterozygous Institute of Cancer Research mouse (+/twy). In the compressed spinal cord of twy/twy mouse, AdV-BDNF gene transfection resulted in a significant decrease in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells present in the spinal cord and a downregulation in the caspase apoptotic pathway compared with AdV-LacZ (control) gene transfection. There was a marked and significant increase in the areas of the spinal cord of AdV-BDNF-injected mice that were NF- and NG2-immunopositive compared with AdV-LacZ-injected mice, indicating the increased presence of neurons and oligodendrocytes in response to BDNF transfection. Our results demonstrate that targeted retrograde BDNF gene delivery suppresses apoptosis in neurons and oligodendrocytes in the chronically compressed spinal cord of twy/twy mouse. Further work is required to establish whether this method of gene delivery may provide neuroprotective effects in other situations of compressive spinal cord injury.

Neurotrophin serum concentrations and polymorphisms of neurotrophins and their receptors in children with asthma

In the recent years numerous studies have analysed the effects of neurotrophins on allergic inflammation in airway diseases reporting increased neurotrophin levels locally in the airways as well as in serum of asthmatic patients. We aimed to investigate if levels of neurotrophins in serum of asthmatic children are influenced by the genotype of functional variants within genes encoding analysed neurotrophins and their specific receptors. In the study we included 98 children diagnosed with asthma. Genotyping of 9 polymorphisms located in neurotrophins genes and their receptors genes was done with use of TaqMan SNP genotyping assays or PCR-RFLP. The serum levels of four neurotrophins (BDNF, NGF, NTF3, NTF4) were analysed during exacerbation of asthma symptoms with use of DuoSet ELISA Development Kit (R&D). The two patients with the genetic variant A/A of NTRK1 (rs6334) showed significantly higher NGF serum concentrations (113.4 and 218.1 pg/mL) as compared to the mean NGF serum concentrations in the total group of patients (34.8 pg/mL). We also observed a significant epistatic interactions between variants of NGF rs6330 and NTRK1 rs6334 that influenced NGF serum level (P = 0.0004). Analysis of four neurotrophins serum levels in relation to different genotypes of analysed neurotrophins genes showed no significant differences among analysed asthmatic children. Our results suggest that, among analysed neurotrophins, NGF serum levels may be influenced by the genotype of NTRK1 gene individually as well as in the interaction with NGF functional genetic variant suggesting their involvement in allergic inflammation in asthma. Serum levels of the other neurotrophins do not seem to be affected by the variants in the analysed genes.

Neurotrophins: The therapeutic potential and concept of minipeptides

Neurotrophins belong to a family of polypeptides that exert control over many aspects of the survival, development, and functioning of structures within the central and peripheral nervous system. Neurotrophins, the nerve growth factor (NGF), the brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), as well as extracellular tyrosine kinase receptors (TrkA, TrkB, and TrkC) are specific targets for therapeutic intervention during different diseases. All these neurotrophins also bind to the p75 receptor, which has many functions depending on the type of cell where it is present. The diversity of neurotrophin effects is determined by ligand-receptor binding and the type of signaling responses that are specific for this interaction. NGF and other neurotrophins are involved in many pathological conditions. It has been shown that an insufficient level of neurotrophins in certain brain structures may be not only an initial cause of Alzheimer’s disease but also may be typical for cerebrovascular dysfunction, brain trauma, cognitive impairments, etc. The therapeutic potential of neurotrophins has been shown in many studies in the last decade but the effectiveness of neurotrophic therapy is limited by the poor diffusion of molecules across the blood-brain barrier and toxic adverse effects. The solution to this problem may be the creation of minipeptides and peptidomimetics that affect the activity of tyrosine-kinase receptors. Some of these structures are combinations of cyclic pentapeptides that facilitate interaction with Trk receptors and exert neuroprotective activity. In this review, we discuss the clinical and experimental data on the results of an alternative strategy that uses these peptidomimetics. These compounds comprise a new group of perspective agents in the therapy of neurodegenerative disorders.

Detection of Neurotrophin‐4 (NT‐4) in Ejaculated Bull Spermatozoa and its Effect on Spermatozoa Mitochondrial Activity

Recently, we demonstrated that two members of neurotrophins, nerve growth factor and brain-derived neurotrophic factor, and two types of receptor, tyrosine kinase A (TrkA) and tyrosine kinase (TrkB), exist in ejaculated bull spermatozoa, and play a crucial role in the normal function of spermatozoa. Neurotrophin-4 (NT-4) is another neurotrophic factor that signals predominantly through the TrkB receptor tyrosine kinase, and no reports of detection of NT-4 in spermatozoa have been published. In the present study, the presence of NT-4 in mature bull spermatozoa was investigated using RT-PCR, immunofluorescence and Western blotting. The result shows that there was no RT-PCR evidence for NT-4 transcripts in bovine spermatozoa. However, the NT-4 protein was present in bovine spermatozoa, and the NT-4 immunoreactivity was localized to the equatorial segment and midpiece of bovine spermatozoa. In addition, effects of NT-4 on function of spermatozoa were studied. Significant increased mitochondria activity of mature bovine spermatozoa was observed in response to 300 or 500 ng/ml exogenous NT-4 (p < 0.05), in comparison with the control, while addition of inhibitors (40 ng/ml k252α) specific for tyrosine protein kinase significantly blocked the increase of mitochondria activity. However, NT-4 had no effects on the viability or acrosome reaction of spermatozoa (p > 0.05). Consequently, this study provided evidence that NT-4 protein was presented in the mature bull spermatozoa and can influence the mitochondrial activity of bovine spermatozoa through TrkB tyrosine kinase-dependent pathways.

Differing effects of NT-3 and GDNF on dissociated enteric ganglion cells exposed to hydrogen peroxide in vitro

Oxidative stress is widely recognized to contribute to neuronal death during various pathological conditions and ageing. In the enteric nervous system (ENS), reactive oxygen species have been implicated in the mechanism of age-associated neuronal loss. The neurotrophic factors, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF), are important in the development of enteric neurons and continue to be expressed in the gut throughout life. It has therefore been suggested that they may have a neuroprotective role in the ENS. We investigated the potential of NT-3 and GDNF to prevent the death of enteric ganglion cells in dissociated cell culture after exposure to hydrogen peroxide (H2O2). H2O2 treatment resulted in a dose-dependent death of enteric neurons and glial cells, as demonstrated by MTS assay, bis-benzimide and propidium iodide staining and immunolabelling. Cultures treated with NT-3 prior to exposure showed reduced cell death compared to untreated control or GDNF-treated cultures. GDNF treatment did not affect neuronal survival in H2O2-treated cultures. These results suggest that NT-3 is able to enhance the survival of enteric ganglion cells exposed to oxidative stress.

Serum neurotrophin-3 and neurotrophin-4 levels are associated with asthma severity in children

To the Editors: Asthma is the most common chronic disease in childhood and is characterised by chronic airway inflammation, reversible airflow obstruction and hyperresponsiveness of the airways. Among the substantial pathophysiological aspects of asthma are neuroimmune interaction mechanisms; neurotrophins are mediators of the interactions providing links between immune, structural and neuronal cells. In recent years, several studies have analysed the effects of neurotrophins on allergic inflammation and airway diseases. Elevated brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) levels have been found in bronchoalveolar lavage fluid (BALF) after allergen challenge in allergic asthmatic patients [1,2]. It has also been shown that asthmatic patients demonstrate elevated levels of neurotrophins (NGF and BDNF) in serum and locally in the airways [3,4], and that allergen provocation significantly increases neurotrophin levels in the airways [2]. Increased neurotrophin (BDNF) concentration also correlated with clinical parameters of allergic airway dysfunction, such as airflow limitation and airway hyperresponsiveness, in asthmatic patients [5], whereas its level is normalised after treatment with inhaled corticosteroids [4]. However, to our knowledge, only one study has investigated neurotrophin (BDNF) levels in a paediatric population of asthmatic patients [6]. We hypothesise that given the relationship between neuronal cells and the immune system in asthma, altered peripheral expression of neurotrophins associated with neuronal dysregulation may affect the course of asthma and disease severity in children. The present study was performed on a Polish sample of 98 …

Interaction between neurotrophin 4 and gonadotrophin in bovine oviducts

The expression and localization of neurotrophin 4 (NT4) and its receptor, tyrosine kinase B (TRKB), in the bovine oviduct, and their interaction with gonadotrophins in bovine oviduct epithelial cells (BOECs), were examined. Transcripts for NT4 and TRKB were detected by reverse transcription polymerase chain reaction (RT-PCR) in bovine oviducts in the follicular and luteal phases, and their proteins were immunolocalized in BOECs. Based on real time PCR, NT4 mRNA did not differ significantly between the two phases of the cycle, although TRKB mRNA expression was higher (P < 0.05) in the luteal phase than that in follicular phase. The BOECs were treated with various concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in vitro; for NT4, mRNA and protein were higher (P < 0.05) than those in the control (based on real time PCR and enzyme-linked immunosorbent assay (ELISA) assays). The effects of NT4 and the TRKB inhibitor (K252a) on the expression of LH receptor (LHR) and FSH receptor (FSHR) in the oviduct epithelial cells were also studied using a monolayer culture model. Expression levels of LHR and FSHR mRNA in BOECs treated with various concentrations of NT4 were higher (P < 0.05) than those in the control. However, these expressions were blocked by treatment with K252α. We concluded that neurotrophin 4 may have a role in regulating the function of bovine oviducts by interacting with gonadotrophins.

Combined delivery of Nogo-A antibody, neurotrophin-3 and the NMDA-NR2d subunit establishes a functional ‘detour’ in the hemisected spinal cord

To encourage re-establishment of functional innervation of ipsilateral lumbar motoneurons by descending fibers after an intervening lateral thoracic (T10) hemisection (Hx), we treated adult rats with the following agents: (i) anti-Nogo-A antibodies to neutralize the growth-inhibitor Nogo-A; (ii) neurotrophin-3 (NT-3) via engineered fibroblasts to promote neuron survival and plasticity; and (iii) the NMDA-receptor 2d (NR2d) subunit via an HSV-1 amplicon vector to elevate NMDA receptor function by reversing the Mg2+ block, thereby enhancing synaptic plasticity and promoting the effects of NT-3. Synaptic responses evoked by stimulation of the ventrolateral funiculus ipsilateral and rostral to the Hx were recorded intracellularly from ipsilateral lumbar motoneurons. In uninjured adult rats short-latency (1.7-ms) monosynaptic responses were observed. After Hx these monosynaptic responses were abolished. In the Nogo-Ab + NT-3 + NR2d group, long-latency (approximately 10 ms), probably polysynaptic, responses were recorded and these were not abolished by re-transection of the spinal cord through the Hx area. This suggests that these novel responses resulted from new connections established around the Hx. Anterograde anatomical tracing from the cervical grey matter ipsilateral to the Hx revealed increased numbers of axons re-crossing the midline below the lesion in the Nogo-Ab + NT-3 + NR2d group. The combined treatment resulted in slightly better motor function in the absence of adverse effects (e.g. pain). Together, these results suggest that the combination treatment with Nogo-Ab + NT-3 + NR2d can produce a functional ‘detour’ around the lesion in a laterally hemisected spinal cord. This novel combination treatment may help to improve function of the damaged spinal cord.

Effect of neurotrophin-4 on endoplasmic reticulum stress-related neuronal apoptosis in diabetic and high glucose exposed rat retinas

The purpose of this study was to investigate the effect of NT-4 on the endoplasmic reticulum (ER) stress-related apoptosis of retinal neurons of isolated retinas. The retinas were isolated from normal and diabetic rats, and the normal retinas were exposed to high glucose (HG). Our results showed that the number of TUNEL-positive, and PERK- and CHOP-positive cells was significantly higher in diabetic and HG exposed retinas than in normal retinas. In diabetic and HG exposed retinas supplemented with NT-4, the number of TUNEL-positive, and PERK- and CHOP-positive cells was significantly lower than in retinas without NT-4. The neuroprotective effect of NT-4 on retinas cultured under diabetic stress was correlated with the suppression in the expression of PERK and CHOP, ER stress-related factors.

Growth-inhibitory effect of neurotrophin-3-secreting adipose tissue-derived mesenchymal stem cells on the D283-MED human medulloblastoma cell line

Medulloblastoma (MBL), the most common malignant pediatric brain tumor, is incurable in about one-third of patients and can lead to long-term disabilities despite current multimodal treatments. The purpose of this study was to demonstrate in vitro biological effects of neurotrophins-3 (NT-3) on MBL cells and to evaluate the growth-inhibitory effect of neurotrophin-3 (NT-3)-secreting stem cells on tumor cells. We confirmed by western blotting that D283-MED cells express tyrosine kinase C, a specific receptor for NT-3. Analyzing the biological effects of NT-3 on MBL cells, we evaluated autophagy, apoptosis, senescence, and differentiation of tumor cells with NT-3. The NT-3 induced a concentration-dependent increase in apoptosis in the tumor cell line (P < 0.001). The high concentrations of NT-3 increased the expression of class III β-tubulin (P < 0.001) and decreased the expression of Nestin (P < 0.05). NT-3-secreting stem cells were produced by nucleofecting pIRES2.EGFP-NT3 into human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and their tropic property toward MBL cells was confirmed by migration assay. Double-layered co-culture experiments with the NT-3-secreting hAT-MSCs and D283-MED MBL cells were performed, and NT-3-induced cell death was studied by 3-(4,5-dimethylathiazol-2-yl)-2,5-dephenyl-tetrazolium bromide (MTT) assay. Consequently, the high concentrations of NT-3-secreting hAT-MSCs significantly (P < 0.05) increased the death of D283-MED cells in vitro. The present study demonstrated that both apoptotic cell death and neuronal differentiation of tumor cells were the mechanisms of growth-inhibitory effect of NT-3-secreting hAT-MSCs on MBL cell line.

Abstract 3450: Growth-inhibitory effect of neurotrophin-3-secreting adipose tissue-derived mesenchymal stem cells on the D283-MED human medulloblastoma cell line

Abstract Medulloblastoma (MBL), the most common malignant pediatric brain tumor, is incurable in about one-third of patients and can lead to long-term disabilities despite current multimodal treatments. We evaluate the in vitro, growth-inhibitory effect of neurotrophin-3 (NT-3)-secreting stem cells on an MBL cell line and propose that this cell therapy may have therapeutic effects for MBL. NT-3-secreting stem cells were produced by nucleofecting pIRES2.EGFP-NT3 into human adipose tissue-derived mesenchymal stem cells (hAT-MSCs). Double-layered co-culture experiments with the NT-3-secreting hAT-MSCs and D283-MED MBL cells were performed, and NT-3-induced apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) test. The growth-inhibitory effects of these stem cells on human MBL in vitro were confirmed by 3-(4, 5-dimethylathiazol-2-yl)-2, 5-dephenyl-tetrazolium bromide (MTT) assay. We confirmed by western blotting that D283-MED cells express tyrosine kinase C, a specific receptor for NT-3, and we showed by fluorescence microscopy and RT-PCR that transfected hAT-MSCs express NT-3. The double-layered co-culture of D283-MED with NT-3-secreting hAT-MSCs induced a concentration-dependent increase in apoptosis in the tumor cell line. Consequently, the high concentrations of NT-3-secreting hAT-MSCs significantly (P &amp;lt; 0.001) increased the death of D283-MED cells in vitro. The present studies demonstrate the potential of NT-3-secreting hAT-MSCs as an effective therapeutic cell therapy for human MBL in vitro, but in vivo experiments and clinical studies will be required in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3450. doi:10.1158/1538-7445.AM2011-3450

Role of neurotrophins on postnatal neurogenesis in the thalamus: prenatal exposure to ethanol

A second wave of neuronal generation occurs in the ventrobasal nucleus of the rat thalamus (VB) during the first three postnatal weeks. The present study tested the hypotheses (1) that postnatal neurogenesis in the VB is neurotrophin-regulated and (2) that ethanol-induced changes in this proliferation are mediated by neurotrophins. The first studies examined the effects of neurotrophins on the numbers of cycling cells in ex vivo preparations of the VB from 3-day-old rats. The proportion of cycling (Ki-67-positive) VB cells was higher in cultured thalamic slices treated with neurotrophins than in controls. Interestingly, this increase occurred with nerve growth factor (NGF) alone or with a combination of NGF and brain-derived neurotrophic factor (BDNF), but not with BDNF alone. Based on these data, the VBs from young offspring of pregnant rats fed an ethanol-containing or an isocaloric non-alcoholic liquid diet were examined between postnatal day (P) 1 and P31. Studies used enzyme-linked immunosorbent assays and immunoblots to explore the effects of ethanol on the expression of neurotrophins, their receptors, and representative signaling proteins. Ethanol altered the expression of neurotrophins and receptors throughout the first postnatal month. Expression of NGF increased, but there was no change in the expression of BDNF. The high affinity receptors (TrkA and TrkB) were unchanged but ethanol decreased expression of the low affinity receptor, p75. One downstream signaling protein, extracellular signal-regulated kinase (ERK), decreased but Akt expression was unchanged. Thus, postnatal cell proliferation in the VB of young rat pups is neurotrophin-responsive and is affected by ethanol.

The Recent Understanding of the Neurotrophin′s Role in Skeletal Muscle Adaptation

This paper summarizes the various effects of neurotrophins in skeletal muscle and how these proteins act as potential regulators of the maintenance, function, and regeneration of skeletal muscle fibers. Increasing evidence suggests that this family of neurotrophic factors influence not only the survival and function of innervating motoneurons but also the development and differentiation of myoblasts and muscle fibers. Muscle contractions (e.g., exercise) produce BDNF mRNA and protein in skeletal muscle, and the BDNF seems to play a role in enhancing glucose metabolism and may act for myokine to improve various brain disorders (e.g., Alzheimer's disease and major depression). In adults with neuromuscular disorders, variations in neurotrophin expression are found, and the role of neurotrophins under such conditions is beginning to be elucidated. This paper provides a basis for a better understanding of the role of these factors under such pathological conditions and for treatment of human neuromuscular disease.

The Interaction between Tropomyosin-Related Kinase B Receptors and Presynaptic Muscarinic Receptors Modulates Transmitter Release in Adult Rodent Motor Nerve Terminals

The neurotrophin brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase B (trkB) and p75(NTR) are present in the nerve terminals on the neuromuscular junctions (NMJs) of the levator auris longus muscle of the adult mouse. Exogenously added BDNF or NT-4 increased evoked ACh release after 3 h. This presynaptic effect (the size of the spontaneous potentials is not affected) is specific because it is not produced by neurotrophin-3 (NT-3) and is prevented by preincubation with trkB-IgG chimera or by pharmacological block of trkB [K-252a (C₂₇H₂₁N₃O₅)] or p75(NTR) [Pep5 (C₈₆H₁₁₁N₂₅O₁₉S₂] signaling. The effect of BDNF depends on the M₁ and M₂ muscarinic acetylcholine autoreceptors (mAChRs) because it is prevented by atropine, pirenzepine and methoctramine. We found that K-252a incubation reduces ACh release (~50%) in a short time (1 h), but the p75(NTR) signaling inhibitor Pep5 does not have this effect. The specificity of the K-252a blocking effect on trkB was confirmed with the anti-trkB antibody 47/trkB, which reduces evoked ACh release, like K-252a, whereas the nonpermeant tyrosine kinase blocker K-252b does not. Neither does incubation with the fusion protein trkB-IgG (to chelate endogenous BDNF/NT-4), anti-BDNF or anti-NT-4 change ACh release. Thus, the trkB receptor normally seems to be coupled to ACh release when there is no short-term local effect of neurotrophins at the NMJ. The normal function of the mAChR mechanism is a permissive prerequisite for the trkB pathway to couple to ACh release. Reciprocally, the normal function of trkB modulates M₁- and M₂-subtype muscarinic pathways.

Regulation of the miR-212/132 locus by MSK1 and CREB in response to neurotrophins

Neurotrophins are growth factors that are important in neuronal development and survival as well as synapse formation and plasticity. Many of the effects of neurotrophins are mediated by changes in protein expression as a result of altered transcription or translation. To determine whether neurotrophins regulate the production of microRNAs (miRNAs), small RNA species that modulate protein translation or mRNA stability, we used deep sequencing to identify BDNF (brain-derived neurotrophic factor)-induced miRNAs in cultured primary cortical mouse neurons. This revealed that the miR-212/132 cluster contained the miRNAs most responsive to BDNF treatment. This cluster was found to produce four miRNAs: miR-132, miR-132*, miR-212 and miR-212*. Using specific inhibitors, mouse models and promoter analysis we have shown that the regulation of the transcription of the miR-212/132 miRNA cluster and the miRNAs derived from it are regulated by the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, via both MSK (mitogen and stress-activated kinase)-dependent and -independent mechanisms.

Neurotrophin 4 enhances in vitro follicular assembly in human fetal ovaries

To investigate the in vitro effect of neurotrophin 4 (NT-4) on follicular assembly in human fetal ovaries. Human ovarian tissue from fetuses at 19-20 gestational weeks was placed in organ culture for two weeks with NT-4. Control groups were cultured with a neutralizing antibody against NT-4. Infertility unit at an university-affiliated tertiary medical center. Four patients who underwent pregnancy terminations at 19-20 gestational weeks. None. Histologic findings of follicular assembly. Follicular assembly was significantly increased in the specimens cultured with NT-4 than in the uncultured specimens, the samples cultured without NT-4, and samples cultured with the neutralizing antibody. In the second week of culture, additional follicular assembly was promoted in the presence of 100 ng/mL NT-4 but not with 10 ng/mL NT-4. This is the first report showing that NT-4 seems to promote human follicular assembly in fetal ovaries, probably in a dose-dependent manner. Follicular assembly is regulated by multiple signals, and additional studies on the effects of other growth factors in combination with NT-4 are warranted.

Neurotrophin-4 (ntf4) mediates neurogenesis in mouse embryonic neural stem cells through the inhibition of the signal transducer and activator of transcription-3 (stat3) and the modulation of the activity of protein kinase B.

The effect of neurotrophin-4 (Ntf4) on mouse embryonic (day-14) neural stem cell (mE14-NSC) fate determination and the mechanisms involved were investigated. Using primary mE14-NSCs, immunocytochemistry and molecular-cell biological methods, such as Western-blotting, we characterized the effect of Ntf4 on mE14-NSC differentiation. Obtained in-vitro data revealed an interesting phenomenon of Ntf4 action resulting in enhanced mE14-NSC commitment to progenitor cells of the neuronal lineage. During this process, Ntf4 suppresses the interleukin 6 (Il6) family receptor and the Notch signalling pathways by modulating their specific receptor cleavages. The observed lineage commitment is controlled via an Ntf4-mediated modulation of protein kinase B (PKB/Akt) activity and characterized by a decreased Stat3 (signal transducer and activator of transcription-3) phosphorylation status. These findings suggest that the Ntf4-activated signalling cascade is responsible for initiating a concert among sheddases, kinases, and phosphatases to mediate neurogenesis.

Targeted Retrograde Gene Delivery of Brain-Derived Neurotrophic Factor Suppresses Apoptosis of Neurons and Oligodendroglia After Spinal Cord Injury in Rats

Histologic and immunohistochemical studies after targeted retrograde adenovirus (AdV)-mediated brain-derived neurotrophic factor (BDNF) gene delivery via intramuscular injection in rats with injured spinal cord. To investigate the neuroprotective effect of targeted retrograde AdV-BDNF gene transfection in the traumatically injured spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Several studies investigated the neuroprotective effects of neurotrophins including BDNF on spinal cord injury, with respect to prevention of neural cell apoptosis in injured spinal cord. However, no report has described the potential effect of targeted retrograde neurotrophic factor gene delivery in injured spinal cord on prevention of neural cell apoptosis. AdV-BDNF or AdV-LacZ was used for retrograde delivery via bilateral sternomastoid muscles to the spinal accessory motoneurons immediately after spinal cord injury in rats. Localization of beta-galactosidase expression produced by LacZ gene or AdV-BDNF gene transfection was examined by immunofluorescence staining and double staining of cell markers (NeuN, RIP, GFAP, OX-42, and NG2) in the injured spinal cord. TUNEL-positive cells were counted and immunoreactivity to active caspase-3 and NG2 was examined after gene injection. Retrograde delivery of LacZ marker gene was identified in cervical spinal neurons and glial cells including oligodendrocytes in the white matter.AdV-BDNF transfection resulted in a significant decrease in the number of TUNEL-positive apoptotic cells by downregulating the caspase apoptotic pathway, with significant promotion of NG2 expression in injured spinal cord, compared with AdV-LacZ injection. Our results suggest that targeted retrograde BDNF gene delivery suppresses apoptosis of neurons and oligodendrocytes in the injured rat spinal cord.

Neuroprotection signaling pathway of nerve growth factor and brain-derived neurotrophic factor against staurosporine induced apoptosis in hippocampal H19-7 cells

Neurotrophins protect neurons against excitotoxicity; however the signaling mechanisms for this protection remain to be fully elucidated. Here we report that activation of the phosphatidyl inositol 3 kinase (PI3K)/Akt pathway is critical for protection of hippocampal cells from staurosporine (STS) induced apoptosis, characterized by nuclear condensation and activation of the caspase cascade. Both nerve growth factor (NGF) and brain-derived growth factor (BDNF) prevent STS-induced apoptotic morphology and caspase-3 activity by upregulating phosphorylation of the tropomyosin receptor kinase (Trk) receptor. Inhibition of Trk receptor by K252a altered the neuroprotective effect of both NGF and BDNF whereas inhibition of the p75 neurotrophin receptor (p75NTR) had no effect. Impairment of the PI3K/Akt pathway or overexpression of dominant negative (DN)-Akt abolished the protective effect of both neurotrophins, while active Akt prevented cell death. Moreover, knockdown of Akt by si-RNA was able to block the survival effect of both NGF and BDNF. Thus, the survival action of NGF and BDNF against STS-induced neurotoxicity was mediated by the activation of PI3K/Akt signaling through the Trk receptor.

Neurotrophic effects of brain‐derived neurotrophic factor and vascular endothelial growth factor in major pelvic ganglia of young and aged rats

To investigate the neurotrophic effect of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in cultured major pelvic ganglia (MPG) derived from young and aged rats. The dorsocaudal region of the MPG was isolated from 12 6-month-old male rats and 12 24-month-old male rats. The MPGs were treated with BDNF, VEGF, or both, at 0, 12.5, 25, 50, 100 and 150 ng/mL to determine the effective concentration for 50% activity (EC(50)) and optimum dosage for promoting neurite growth. Neurite outgrowth from treated MPGs was measured by microscopy. NADPH diaphorase and tyrosine hydroxylase (TH) staining was used to characterize neurites. Both BDNF and VEGF promoted neurite sprouting from MPG. Neurite growth was more robust in MPGs derived from young rats (6 months) than from aged rats (24 months). The EC(50) for BDNF, VEGF and combined treatment were 10.6, 11.9 and 52 ng/mL in young rats, and 11.3, 12 and 0.75 ng/mL in old rats, respectively. The optimum dosage of both factors for promoting MPG neurite growth in all groups was 25-50 ng/mL. VEGF appeared to favour NADPH diaphorase-positive neurites, whereas BDNF favoured TH-positive neurites. BDNF and VEGF promote neurite growth from cultured MPG; combined treatment produced the most robust neurite outgrowth. Neurite growth from MPGs derived from aged rats was not as robust as it was from MPGs from younger rats. Further studies on the effect of neurotrophins after cavernous nerve injury are warranted.