Doses Of Nerve Growth Factor Research Articles

SİNİR BÜYÜME FAKTÖRÜ İLE FARKLILAŞTIRILMIŞ PC12 HÜCRELERİNİN MORFOMETRİK VE FLORESANS ANALİZİ

Objective
 PC12 is a rat pheochromocytoma cell line. These
 cells characteristically undergo differentiation when
 cultured with nerve growth factor (NGF). Depending
 on the dose of NGF, the length of neurite extensions
 changes. Thanks to this differentiation property,
 the cells are used in neuroscience and in modeling
 pathophysiological diseases such as Alzheimer's,
 Parkinson's, and Amyotrophic Lateral Sclerosis.
 However, literature studies showing the effect of NGF
 on neurite extensions formed in PC12 cells are very
 limited. This study aimed to investigate the effect of
 NGF on neurite extensions and cell viability depending
 on dose and incubation time.
 Materials and Methods
 In this study, PC12 cells were incubated with 50 ng/ml
 and 100 ng/ml NGF for 3, 6 and 7 days. The lengths
 of neurite outgrowths and dead cell ratios were
 calculated in incubated cells.
 Results
 The results showed that the length of neurite
 extensions and dead cell ratio increased depending
 on NGF doses and incubation time. When NGF
 incubation times were compared, no difference was
 found between 50 ng/ml NGF 6 days and 100 ng/ml
 NGF 3 days groups.
 Conclusion
 When the dead cell ratios and sizes of neurite
 extensions in the experimental groups are evaluated,
 it is thought that 100 ng/ml NGF and 3 days incubation
 time parameters are ideal for PC12 cell differentiation.

Nerve growth factor protects against cadmium-induced hypertension in mice via vascular remodeling

Purpose: To investigate the effect of nerve growth factor (NGF) on cadmium ion (Cd2+)-induced hypertension in a mouse model, and the mechanism of action involved.
 Methods: Hypertension was induced in mice by administration of cadmium chloride (CdCl2) at a dose of 100 mg/L in deionised water. Then, NGF was administered daily for 45 days via the intragastric route. Immunohistochemical technique employing Vectastain ABC kit was used for determination of the levels of matrix metalloproteinases in mice aorta.
 Results: Treatment with NGF significantly and dose-dependently alleviated Cd-induced increase in blood pressure in mice (p < 0.05). The Cd-induced elevation in mean arterial pressure (MAP) in mice was also reduced on treatment with NGF at doses of 5 and 10 mg/kg. At the two doses of NGF, vascular responsiveness was enhanced in Cd-administered mice. Exposure to NGF dose-dependently reversed Cd-mediated suppression of eNOS expression and elevation in iNOS level. Moreover, NGF at doses of 5 and 10 mg/kg, reversed Cd-mediated enhancement in nitrate/nitrite levels in urine samples, and reversed Cd-induced elevation in vascular smooth muscle cell (VSMC) count and collagen content in mice arterial wall. The NGF treatment also significantly (p < 0.05) reduced Cd-induced increases in levels of MMP’s.
 Conclusion: The present study demonstrates that NGF increased vascularity and arterial stiffness, and protected against Cd-induced hypertension in mice. Moreover, NGF treatment elevated eNOS expression, suppressed iNOS level and inhibited MMP expression in mice exposed to Cd. Thus, NGF has anti-hypertensive potential and may be beneficial in the treatment of hypertension.

Trewioidesine A, an unsaturated fatty acid from rhizomes of Alchornea trewioides, shows synergy with NGF in inducing differentiation of pheochromocytoma PC12 cells

A new unsaturated fatty acid trewioidesine A (1), together with seven known compounds (2 − 8) were isolated from the rhizomes of Alchornea trewioides (Benth.) Muell. Arg. Their structures were established on the basis of extensive spectroscopic data interpretation (1 D and 2 D NMR, and HRESIMS). The absolute configuration of 1 was determined by electronic circular dichroism (ECD) calculations, confirming as trewioidesine A. The functionality of isolated compounds was tested in cultured PC12 cells, a cell line from rat pheochromocytoma. Trewioidesine A was the one showing robust activity in inducing neuronal differentiation: the induction was synergized when co-applied with nerve growth factor (NGF). In addition, a neurofilament 200 (NF200) promoter-luciferase (pNF200-Luc) reporter was used to evaluate the differentiating ability in the transfected PC12 cells for the isolated compounds. Trewioidesine A exhibited a strong NF200 promoter activation, and application of trewioidesine A with low dose of NGF significantly induced the promoter activity over 50%.

Hypothalamic involvement and the role of progesterone in the NGF-induced LH surge pathway.

To elucidate the mechanism by which nerve growth factor (NGF) influences the LH secretory pathway in camelids, a series of experiments were done to determine the involvement of the hypothalamus (Experiment 1), the role of GnRH neurons (Experiment 2), and the effect of progesterone (Experiment 3) on the NGF-induced LH surge and ovulation in llamas. In Experiment 1, the declining phase of the NGF-induced LH surge was used to determine if the decline is a result of pituitary depletion or hypothalamic unresponsiveness. Female llamas were treated with NGF and, 7 h later, assigned to three groups and given a second dose of NGF (n = 5), a dose of GnRH (n = 5), or saline (n = 6). The LH response was attenuated after the second dose of NGF vs GnRH. In Experiment 2, Fos expression (marker of neuronal activation) in GnRH neurons was examined in the hypothalamus of llamas after NGF or saline treatment (n = 3 per group). Despite an LH surge in the NGF group but not in the saline group, no differences were detected between groups in Fos/GnRH co-expression. In Experiment 3, llamas in low-, medium-, and high-plasma progesterone groups (n = 4 per group) were treated with NGF. The NGF-induced LH surge did not differ among treatment groups. Results from the present study show that the induction of a preovulatory LH surge by NGF may be controlled by a novel pathway involving GnRH neuro-terminals downstream of the hypothalamus and is independent of progesterone influence.

Luteolin stimulates the NGF-induced neurite outgrowth in cultured PC12 cells through binding with NGF and potentiating its receptor signaling.

Luteolin, a flavonoid in fruits and vegetables, has neurotrophic functions without a well-characterized mechanism. Here, we hypothesize a direct interaction of luteolin with nerve growth factor (NGF); as such, the functionality of the NGF could be potentiated. The direct binding of luteolin with NGF was validated by ultra-filtration, Biacore, and docking analyses. In cultured PC12 cells, application of luteolin in combination with a low dose of NGF potentiated the NGF-induced differentiation of neurons by an increase of the differentiated cell number to 25.4 ± 4.8% (p < 0.01), as well as the increased expression of neurofilaments by 119 ± 32.1% (p < 0.05), 191 ± 12.6% (p < 0.01), and 110 ± 23.4% (p < 0.05) for NF68, NF160 and NF200, respectively. The co-treatment induced the phosphorylations of tropomyosin receptor kinase A (TrkA), extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (Akt), phospholipase C-γ1 (PLCγ1), and cAMP response element-binding protein (CREB) by 2 to 3 fold: these induced phosphorylations were mimicking that of a high dose of NGF. Moreover, the application of the TrkA inhibitor, K252a, blocked the luteolin-mediated induction of neurofilament expression and neurite outgrowth in cultured PC12 cells, suggesting the target specificity. The result supports the development of luteolin as a therapeutic, or preventive, agent for NGF insufficiency-associated neurodegenerative diseases.

123 Nerve growth factor-induced ovulation in llamas: Evidence of hypothalamic refractoriness to nerve growth factor during the declining phase of the luteinising hormone surge

Nerve growth factor (NGF) in semen is responsible for triggering ovulation after copulation in camelids. Interaction of NGF with its cognate receptors results in a preovulatory luteinising hormone (LH) surge that leads to ovulation, but the pharmacokinetics of NGF and the mechanism by which it mediates LH release are unknown. In an effort to elucidate the site and mechanism of action involved, the objective of this study was to determine whether the decline in the LH surge occurs as a result of pituitary depletion (i.e. diminished response to gonadotrophin-releasing hormone (GnRH)) or as an obtunded response at the level of the hypothalamus (diminished GnRH). Adult nonpregnant, nonlactating llamas (n=18) were synchronized by the administration of an ovulatory dose of a GnRH analogue (100μg IM; Fertiline, Vetoquinol). At 10 to 14 days later, the ovaries were examined to confirm the presence of a dominant follicle measuring ≥ 7mm, and a jugular catheter was put in place. The following day, a pretreatment blood sample was taken, and llamas were treated intravenously with 1mg of purified NGF from llama seminal plasma. Blood samples were taken every 30min for 7h from the time of NGF treatment. After the last blood sample was taken, llamas were treated with NGF (n=6; 1mg IV), GnRH (n=5; 100μg IV), or saline (Sal; IV; n=6), and blood samples were taken every 30min for another 7h. The ovaries were examined 48h after initial NGF treatment, via transrectal ultrasonography, to detect ovulation. Plasma was harvested and stored for analysis of LH concentration by radioimmunoassay. Data were compared using ANOVA for repeated measures, and single-point data were analysed using paired t-tests. As expected, most llamas ovulated in response to the initial NGF treatment (5 out of 5 in NGF-GnRH; 5 of 6 in NGF-NGF; 5 of 6 in NGF-Sal). Compared with pretreatment values, all llamas showed a 4-fold increase in plasma LH concentrations within 2h of the initial NGF treatment (P&amp;lt;0.05). Plasma LH concentrations peaked at 3h after initial NGF treatment and began to decline 4.5 to 5h after treatment (P&amp;lt;0.05). Plasma LH concentrations continued to decline following the second dose of NGF or Sal, whereas a transient elevation of LH was detected in llamas treated with GnRH (P&amp;lt;0.05). The LH concentration returned to basal levels (pretreatment) 8, 12, and 13h after NGF treatment in llamas treated with NGF-Sal, NGF-GnRH, and NGF-NGF, respectively. We conclude that the lack of LH response to the second dose of NGF is not because of pituitary depletion but rather due to diminished GnRH. The latter may be attributed to either a downregulation of NGF receptors within the hypothalamus or to temporary depletion of GnRH at the nerve terminals within the median eminence. Research was supported by the Natural Sciences and Engineering Research Council of Canada.

In vitro effect of nerve growth factor on the main traits of rabbit sperm

BackgroundThe nerve growth factor (NGF), a member of the neurotrophins family, plays an important role not only in the nervous but also in other non-nervous systems such as the reproductive system. The aim of the paper is to study the in vitro effect of NGF on rabbit sperm functions.MethodsTen adult rabbit bucks were collected five times, and pooled semen samples have been analysed. NGF was quantified in seminal plasma, and the distribution of NGF receptors (TrKA and p75NTR) in sperm was established. Moreover, the dose-effect of NGF on motility rate and track speed was evaluated. Successively, the effect of the neutralisation of NGF receptors was assessed to verify the specific role of each receptor. Untreated sperm were used as control.ResultsOur study identified several interesting results: i) We detected NGF in seminal plasma and TrKA and p75NTR in sperm surface. In particular, TrKA is localised in the head and p75NTR in the midpiece and tail of rabbit sperm. ii) Once the optimal dose of NGF (100 ng/mL) was established, its addition affected both kinetics and other physiological traits (capacitation, apoptosis and necrosis) of rabbit sperm. (iii) The neutralisation of TrKA and p75NTR receptors affected sperm traits differently. In particular, sperm speed, apoptosis and capacitation seemed mainly modulated via p75NTR receptor, whereas motile, live cells, necrosis and acrosome reaction were modulated via TrKA.ConclusionFor the first time, we showed the presence of p75NTR in rabbit sperm. NGF affects kinetic and other physiological traits of rabbit sperm. Most of these changes are modulated by the receptors involved (TrKA or p75NTR). Considering that some seminal disorders in human have been correlated with a lower NGF concentration and no studies have been done on the possible involvement of NGF receptors, these findings also provide new insights on human fertility.

Neuronal survival of DRG neurons after neurite transection in vitro promotes by nerve growth factor and brain derived neurotrophic factor

Neurotrophic factors are growth factors that promote neuronal survival, regulate synaptic function and neurotransmitter release, and promote the plasticity and growth of axons in the peripheral and central nervous systems. This study focused on the roles of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in the survival of adult dorsal root ganglion (DRG) neurons following axotomy. To investigate this, we cultured adult mouse DRG neurons and administered NGF or BDNF to the culture medium at different doses before transection. After determining the optimal doses of NGF and BDNF, these factors were then applied in combination. Axotomy was performed using a precise laser beam and neuronal death was visualized through cell observer microscopy system, by adding propidium iodide to the culture medium. The results demonstrate that the optimal doses of NGF and BDNF for neuronal survival are 150 ng/mL and 50 ng/mL, respectively. The highest level of neuronal survival was observed in the cells treated with a combination of NGF and BDNF. In conclusion, NGF and BDNF have a positive effect, both individually and in combination, on the survival of DRG neurons following neurite transection.

Interactions of peripheral endothelin-1 and nerve growth factor as contributors to persistent cutaneous pain.

Endothelin-1 (ET-1) and Nerve Growth Factor (NGF) are proteins, released from cancer-ridden tissues, which cause spontaneous pain and hypersensitivity to noxious stimuli. Here we examined the electrophysiological and behavioral effects of these two agents for evidence of their interactions. Individual small-medium cultured DRG sensory neurons responded to both ET-1 (50 nM, n=6) and NGF (100 ng/ml, n=4), with increased numbers of action potentials and decreased slow K(+) currents; pre-exposure to ET-1 potentiated NGF´s actions, but not vice versa. Behaviorally, single intraplantar (i.pl.) injection of low doses of ET-1 (20 pmol) or NGF (100 ng), did not increase hindpaw tactile or thermal sensitivity, but their simultaneous injections sensitized the paw to both modalities. Daily i.pl. injections of low ET-1 doses in male rats caused tactile sensitization after 21 days, and enabled further tactile and thermal sensitization from low dose NGF, in ipsilateral and contralateral hindpaws. Single injections of 100 ng NGF, without changing the paw's tactile sensitivity by itself, acutely sensitized the ipsilateral paw to subsequent injections of low ET-1. The sensitization from repeated low ET-1 dosing and the cross-sensitization between NGF and ET-1 were both significantly greater in female than in male rats. These findings reveal a synergistic interaction between cutaneously administered low doses of NGF and ET-1, which could contribute to cancer-related pain.

Physiologic facet capsule stretch can induce pain & upregulate matrix metalloproteinase-3 in the dorsal root ganglia when preceded by a physiological mechanical or nonpainful chemical exposure

BackgroundNeck pain from cervical facet loading is common and induces inflammation and upregulation of nerve growth factor (NGF) that can sensitize the joint afferents. Yet, the mechanisms by which these occur and whether afferents can be pre-conditioned by certain nonpainful stimuli are unknown. This study tested the hypothesis that a nonpainful mechanical or chemical insult predisposes a facet joint to generate pain after a later exposure to typically nonpainful distraction. MethodsRats were exposed to either a nonpainful distraction or an intra-articular subthreshold dose of NGF followed by a nonpainful distraction two days later. Mechanical hyperalgesia was measured daily and C6 dorsal root ganglia (DRG) tissue was assayed for NGF and matrix metalloproteinase-3 (MMP-3) expression on day 7. FindingsThe second distraction increased joint displacement and strains compared to its first application (p = 0.0011). None of the initial exposures altered behavioral sensitivity in either of the groups being pre-conditioned or in controls; but, sensitivity was established in both groups receiving a second distraction within one day that lasted until day 7 (p < 0.024). NGF expression in the DRG was increased in both groups undergoing a pre-conditioning exposure (p < 0.0232). Similar findings were observed for MMP-3 expression, with a pre-conditioning exposure increasing levels after an otherwise nonpainful facet distraction. InterpretationThese findings suggest that nonpainful insults to the facet joint, when combined, can generate painful outcomes, possibly mediated by upregulation of MMP-3 and mature NGF.

NGF Attenuates High Glucose-Induced ER Stress, Preventing Schwann Cell Apoptosis by Activating the PI3K/Akt/GSK3β and ERK1/2 Pathways.

Diabetic peripheral neuropathy (DPN) is one of the most common and troublesome complications of diabetes mellitus. It has been demonstrated that nerve growth factor (NGF) exerts a pivotal role in the regulation of neuronal growth and the promotion of DPN recovery. However, the exact molecular mechanisms are not well understood. Recent studies have indicated that as a novel therapeutic target, endoplasmic reticulum (ER) stress participates in the onset and progression of DPN. In the present study, it has been demonstrated that NGF prevents the sciatic nerve from degeneration and demyelination in DPN rats. Thus, RSC 96 cells, which retain the characteristic features of Schwann cells (SCs), were cultured in medium containing 30mM glucose (high glucose, HG) to mimic SCs in DPN mice. The 50-ng/ml dose of NGF was identified to be the optimal concentration for treating an excessive ER stress level under HG conditions for 24h. We found that NGF treatment significantly inhibits HG-induced ER stress and subsequently suppresses ER-related apoptosis. Further, NGF administration also activates the upstream signaling pathway of ER stress, PI3K/Akt/GSK3β signaling and ERK1/2 signaling. Co-treatment with the PI3K inhibitor LY294002 or ERK1/2 inhibitor U0126 significantly reverses the protective role of NGF on HG-induced excessive ER stress and subsequent apoptosis. These observations suggest that the neuroprotective role of NGF in DPN is mediated by the inhibition of excessive ER stress via the activation of the PI3K/Akt/GSK3β and ERK1/2 signaling pathways.

P2X1 Receptor-Mediated Ca2+ Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

Negletein as a neuroprotectant enhances the action of nerve growth factor and induces neurite outgrowth in PC12 cells.

Negletein has been shown to have therapeutic potential for inflammation-associated diseases, but its effect on neurite outgrowth is still unknown. The present study showed that negletein alone did not trigger PC12 cells to differentiate and extend neurites. When compared with the cells in the untreated control, a significant (P<0.05) induction and a higher neurite outgrowth activity was observed when the cells were cotreated with negletein (10µM) and a low dose of nerve growth factor (NGF; 5ng/mL). The neurite outgrowth process was blocked by the tyrosine kinase receptor (Trk) inhibitor, K252a, suggesting that the neuritogenic effect was NGF-dependent. Negletein (10µM) together with NGF (5ng/mL) enhanced the phosphorylation of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and cAMP response element-binding protein (CREB). The growth associated protein-43 (GAP-43) and the NGF level were also upregulated by negletein (10µM) and a low dose of NGF (5ng/mL). Negletein at nanomolar concentration also was found to be sufficient to mediate the survival of serum-deprived PC12 cells up to 72h. Taken together, negletein might be useful as an efficient bioactive compound to protect neurons from cell death and promote neuritogenesis. © 2016 BioFactors, 42(6):591-599, 2016.

In Vitro Differential Effect of Nerve Growth Factor on Functional Parameters of Murrah Buffalo Spermatozoa in Low and High Fertile Groups

Aim of the present study was to examine the influence of in vitro supplementation of Nerve growth factor (NGF) on functional parameters of Murrah buffalo (Bubalusbubalis) spermatozoa from fresh semen, like, motility, plasmalemma integrity, acrosomal integrity, ATP concentration. Fresh semen samples (n=6) were washed in Tris buffer and divided into two equal parts (control and NGF groups). Only in the NGF group, NGF was added to a final concentration of 50 and 100 ng/ml. The samples were incubated at 37o C for different time intervals in TCM 199 medium supplemented with BSA and the effects were observed at 0, 30, 60 and 120 min of incubation. The experiment was performed in low (LF) and high fertile(HF) groups based on previous three years conception rate and taking cutoff value , below and above this value were categorized as LF and HF group. The mean concentration of the buffalo seminal plasma (n=12) NGF was 67.7±3.25ng/ml and 65.5± 2.76 in LF and HF groups respectively. The concentration of NGF in blood plasma was 83.5±7.82 and 68.6±3.82 in HF and LF groups respectively. The concentration of blood plasma NGF being higher (P<0.05) in HF group. With either dose of NGF in vitro significant effect on the total motility (P<0.05), progressive forward motility (P<0.05) was observed. It could be maintained in HF group till 120 min but in LF group it was restricted to 60 min when compared with their respective control. The functional membrane integrity did not differ significantly between groups (control and NGF treated) in both LF and HF groups with either concentration of NGF. The plasma lemma integrity was significantly less (P<0.05) at 120 min of incubation when compared with the initial value at 0 min of incubation. The percentage of acrosomal intact spermatozoa decreased continuously over a period of time in both the groups. As compared to 0 min of incubation, the significant (P<0.05) loss of acrosome was observed at 60 and 120 min of incubation in LF and HF control groups and NGF supplementation could maintain acrosome integrity in HF group for 60 min where as in LF group it could be maintained significantly only till 30 min of incubation when compared with the initial values of respective groups. Viability of spermatozoa was not significantly different when compared between groups with their respective control, However when compared at different time intervals, the viability of sperms in HF and LF supplemented groups was significantly different from initial values only at 120 min of incubation whereas in respective control groups the loss in viability was significant from 60 min itself. With respect to ATP concentration of spermatozoa in different groups it was observed that 100ng dose could increase the concentration of ATP in HF group significantly (P<0.05) at 60 min of incubation only with NGF in vitro when compared with its respective control. In conclusion, all the parameters decreased significantly at 120 min of incubation when compared with their respective initial values for all the groups. In HF group supplementation of NGF@50ng/ml could maintain functional parameters of spermatozoa for a greater duration of time when compared with LF or control group.

Intermittent vs Continuous Administration of Nerve Growth Factor to Injured Medial Septal Cholinergic Neurons in Rat Basal Forebrain

Many medial septal neurons of the basal forebrain are dependent on nerve growth factor (NGF) from the hippocampus for survival and maintenance of a cholinergic phenotype. When deprived of their source of NGF by axotomy, medial septal neuronal cell bodies atrophy and lose their cholinergic markers. This is similar to what is observed in the basal forebrain during Alzheimer’s disease (AD). In the present study, medial septal neurons were axotomized in female rats by way of a fimbria/fornix lesion. For fourteen days following axotomy, varying NGF doses (1 – 250 μg/ml) were administered to the lateral cerebral ventricle with either mini-osmotic infusion or daily injection. The responsiveness of medial septal neurons was evaluated with choline acetyltransferase immunohistochemistry. Within the mini-osmotic pumps, NGF activity diminished greatly during the first five days of implantation, but increased dramatically in the CSF after five days of infusion. The responsiveness of medial septal neurons to NGF was dose dependent and the ED50 for NGF injection was determined to be 14.08 μg/ml compared to 27.60 μg/ml for NGF infusion. Intermittent injections at varying intervals were evaluated with 30 μg/ml NGF over a fourteen-day time period (2, 3, 6, or 12 injections). No differences occurred in the number of choline acetyltransferase neurons from rats that received weekly injections to those that received daily injections of NGF. NGF administration has been suggested as a therapy for AD. The results of these studies continue to highlight the need for NGF stability within the delivery system and AD patient CSF, the choice of delivery system, frequency of administration, and the NGF dose for maintaining basal forebrain cholinergic neurons during AD.

Intranasal “painless” Human Nerve Growth Factors Slows Amyloid Neurodegeneration and Prevents Memory Deficits in App X PS1 Mice

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that “painless” hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of “painless” hNGF variants as a new generation of therapeutics for neurodegenerative diseases.

Encapsulated Cell Biodelivery of Transposon-Mediated High-Dose NGF to the Göttingen Mini Pig Basal Forebrain

Nerve Growth Factor (NGF) has therapeutic effects on the cholinergic neurodegeneration in Alzheimer's disease (AD). We have previously described an implantable Encapsulated Cell Biodelivery™ device, NsG0202, capable of local delivery of NGF to the human cholinergic basal forebrain. Results from a small Phase 1b clinical study showed that the NGF dose could advantageously be increased. We have therefore developed a second generation clinical device named NsG0202.1, containing an RPE cell line (NGC0211) generated with transposon expression technology for high- dose NGF production. Furthermore, to promote cell attachment and long-term viability of NGC0211, a polyethylene terephthalate (PET) yarn scaffolding was used. The safety was tested in Gottingen minipigs during a six months period with NsG0202.1 implants placed in the basal forebrain. The devices were well tolerated and the NGC0211 viability and NGF secretion remained after 6 months in vivo. The NGF induced relevant biological responses in the surrounding cholinergic target neurons.

Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer

Nerve growth factor (NGF) is a potential therapeutic agent for Alzheimer's disease (AD) as it has positive effects on the basal forebrain cholinergic neurons whose degeneration correlates with the cognitive decline in AD. We have previously described an encapsulated cell biodelivery device, NsG0202, capable of local delivery of NGF by a genetically modified human cell line, NGC-0295. The NsG0202 devices have shown promising safety and therapeutic results in a small phase 1b clinical study. However, results also show that the NGF dose could advantageously be increased. We have used the sleeping beauty transposon expression technology to establish a new clinical grade cell line, NGC0211, with at least 10 times higher NGF production than that of NGC-0295. To test whether encapsulation of this cell line provides a relevant dose escalation step in delivering NGF for treatment of the cognitive decline in AD patients, we have validated the bioactivity of devices with NGC0211 and NGC-0295 cells in normal rat striatum as well as in the quinolinic acid striatal lesion model. These preclinical animal studies show that implantation of devices with NGC0211 cells lead to significantly higher NGF output, which in both cases correlate with highly improved potency.

Influence of nerve growth factor on proliferation, differentiation and apoptosis of neural stem cells

Objective To evaluate the influence of different doses of nerve growth factor (NGF) on the proliferation, differentiation and apoptosis of embryonic rat neural stem cells. Methods The neural stem cells were isolated from subventricular zone of rat embryonic brains and cultured in serum-free medium DMEM/F12 with B27. NGF with different doses of 10, 20, 50 μg/L were added into the different experimental groups. The proliferation ability of the cells was measured by methyl thiazol tetrazolium (MTT) assay at 10th day. All cells were collected in plates at 7th day. Streptvidin-peroxidase immunocytochemistry was used to detect the expression of neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) , and the number of NSE( + ) and GFAP( + ) cells was counted. The apoptosis rat at 5th day after culture was assessed. Results There was no significant difference in the the number of neurospheres between 10 μg/L NGF group (0. 150 ±0. 025) and positive control group (0. 158 ±0. 017), but there was significant difference between 10 μg/L NGF group and blank control group (0. 128 ±0. 015). In 10, 20,50 μg/L N GF groups, the rate of NSE ( + ) cells was ( 15. 21 ± 1.27) %, (20. 64 ± 2. 61 )%, (24. 20 ± 3. 10) %, and that of GFAP( + ) cells was (27.98 ± 2. 35 ) %, ( 28.94 ± 2. 84 ) %, ( 30. 79 ± 3.23 ) % respectively. The rate of cells differentiation was increased with the dose of NGF added[( FNsE (+ ) = 3.47,FGFAp( + ) = 3. 47 ,PGFAP( + ) ＜ 0. 05)]. Flow cytometry revealed that in 20, 50 μg/L NGF groups the apoptosis rate was significantly increased as compared with other groups. The proliferation index (PI) in 10 μg/L NGF group was higher than other groups. Conclusion NGF at a rational dose can promote the proliferation and differentiation of human NSC. Low dose of NGF mainly facilitates the proliferation of NSCs, and high dose of NGF shows obvious influence on proliferation of NSC. Key words: Neural growth factor; Neural stem cells; Proliferation; Differentiation

Dose and duration of nerve growth factor (NGF) administration determine the extent of behavioral recovery following peripheral nerve injury in the rat

Nerve growth factor (NGF) has been previously shown to support neuron survival and direct neurite outgrowth in vitro, and to enhance axonal regeneration in vivo. However, a systematic analysis of NGF dose and dose duration on behavioral recovery following peripheral nerve injury in rodents has not been previously investigated. Here, we show that NGF promotes a bell shaped dose–response, with an optimal threshold effect occurring at 800 pg/μl. High dose NGF inhibited regeneration. However, this effect could be reversed through functional blockade of p75 receptors, thus implicating these receptors as mediators of the inhibitory response. Longer term evaluation showed that animals administered NGF at 80 ng/day for 3 weeks had greater sensorimotor recovery compared to all other treatment groups. These animals made significantly fewer errors during skilled locomotion, and displayed both increased vertical and fore-aft ground reaction forces during flat surface locomotion. Furthermore, terminal electrophysiological and myological assessments (EMG, wet gastrocnemius muscle weights) corroborated the behavioral data. Overall, these data support the hypothesis that both appropriate dose and duration of NGF are important determinants of behavioral recovery following nerve injury in the rat.