Effect of Aloe vera on the expression of nerve factors, p75 and TrkA receptors in the hippocampus of diabetic rats / Efecto del Aloe vera sobre la expresión de factores de crecimiento nervioso, Receptores p75 y TrkA en el hipocampo de ratas diabéticas

SUMMARY: Diabetes mellitus can lead to structural disorders in the brain. One of the most common complications of diabetes, diabetic neuropathy is associated with central nervous system disorders. Aloe vera has anti-diabetic, antioxidant, and neuroprotective effects. This study was designed to evaluate the effects of Aloe vera gel on the hippocampus changes as well as the expression of nerve growth factor and receptors TrkA and P75 in the hippocampus of streptozotocin (STZ)-induced diabetic rats. 25 male Wistar rats were randomly divided into 5 groups including: control (normal saline), diabetic (normal saline), Aloe vera gel (400 mg/kg/day; gavage), diabetic + Aloe vera gel (400 mg/kg/day; gavage) and diabetic + insulin NPH (10 IU/kg/day; subcutaneous). Experimental diabetes was induced by streptozotocin injection (60 mg/kg; intraperitoneal). All groups treated for 8 weeks. At the end of treatment course, the rat brains were removed for measuring the expression of nerve growth factor, p75 and TrkA receptors were evaluated in the hippocampus. Diabetes induction after 8 weeks caused NGF and P75 expression levels in the diabetic group than other groups significantly increased (p<0.05). The TrkA receptor expression in the diabetic group compared with the control had a significant reduction (p<0.05). On the other hand in the diabetic group receiving Aloe vera gel expression of NGF and P75 expression levels compared to the diabetic group was significantly reduced (p<0.05) and the TrkA receptor expression compared with the diabetic group had a significant increase (p<0.05). The results showed that oral administration of Aloe vera gel in diabetic rats ameliorates diabetes-induced hyperglycemia. On the other hand, Aloe vera gel cause modulation of the expression of NGF neurotrophic factor via increased expression of TrkA receptor-specific and non-specific receptor down-regulation of P75 in the hippocampus of STZ-induced diabetic rats.

RESUMEN: La diabetes mellitus puede provocar trastornos estructurales en el cerebro. Es una de las complicaciones más comunes de la diabetes y la neuropatía diabética y está relacionada con trastornos del sistema nervioso central. El Aloe vera tiene efectos antidiabéticos, antioxidantes y neuroprotectores. Este estudio fue diseñado para evaluar los efectos del gel de Aloe vera en los cambios del hipocampo, así como la expresión del factor de crecimiento nervioso y los receptores TrkA y P75 en el hipocampo de ratas diabéticas inducidas por estreptozotocina (STZ). Se dividieron al azar 25 ratas Wistar macho en 5 grupos de: control (solución salina normal), diabéticos (solución salina normal), gel de Aloe vera (400 mg / kg / día; sonda), diabéticos + gel de Aloe vera (400 mg / kg / día; sonda) y diabéticos + insulina NPH (10 UI / kg / día; subcutánea). La diabetes experimental fue inducida por inyección de estreptozotocina (60 mg / kg; intraperitoneal). Todos los grupos fueron tratados durante 8 semanas. Al final del tratamiento, se extrajeron los cerebros de las ratas para medir la expresión del factor de crecimiento nervioso y se evaluaron los receptores p75 y TrkA en el hipocampo. La inducción de diabetes después de 8 semanas provocó que los niveles de expresión de NGF y P75 en el grupo de diabéticos aumentaran significativamente en comparación con otros grupos (p <0,05). La expresión del receptor TrkA en el grupo diabético comparado con el control tuvo una reducción significativa (p <0,05). Por otro lado, el grupo de ratas diabéticas que recibieron la expresión en gel de Aloe vera de NGF y los niveles de expresión de P75 en comparación con el grupo de ratas diabéticas se redujo significativamente (p <0,05) y la expresión del receptor de TrkA en comparación con el grupo de ratas diabéticas tuvo un aumento significativo (p <0,05). Los resultados mostraron que la administración oral de gel de Aloe vera en ratas diabéticas mejora la hiperglucemia inducida por la diabetes. Por otro lado, el gel de Aloe vera causa modulación de la expresión del factor neurotrófico NGF a través del aumento de la expresión de receptor TrkA específico y no específico y regulación negativa del receptor de P75 en el hipocampo de ratas diabéticas inducidas por STZ.

Small molecule modulation of the p75 neurotrophin receptor suppresses age- and genotype-associated neurodegeneration in HIV gp120 transgenic mice.

The persistence of HIV in the central nervous system leads to cognitive deficits in up to 50% of people living with HIV even with systemic suppression by antiretroviral treatment. The interaction of chronic inflammation with age-associated degeneration places these individuals at increased risk of accelerated aging and other neurodegenerative diseases and no treatments are available that effectively halt these processes. The adverse effects of aging and inflammation may be mediated, in part, by an increase in the expression of the p75 neurotrophin receptor (p75NTR) which shifts the balance of neurotrophin signaling toward less protective pathways. To determine if modulation of p75NTR could modify the disease process, we treated HIV gp120 transgenic mice with a small molecule ligand designed to engage p75NTR and downregulate degenerative signaling. Daily treatment with 50 mg/kg LM11A-31 for 4 months suppressed age- and genotype-dependent activation of microglia, increased microtubule associated protein-2 (MAP-2), reduced dendritic varicosities and slowed the loss of parvalbumin immunoreactive neurons in the hippocampus. An age related accumulation of microtubule associated protein Tau was identified in the hippocampus in extracellular clusters that co-expressed p75NTR suggesting a link between Tau and p75NTR. Although the significance of the relationship between p75NTR and Tau is unclear, a decrease in Tau-1 immunoreactivity as gp120 mice entered old age (>16 months) suggests that the Tau may transition to more pathological modifications; a process blocked by LM11A-31. Overall, the effects of LM11A-31 are consistent with strong neuroprotective and anti-inflammatory actions that have significant therapeutic potential.

Differentiation of NSC-34 cells is characterized by expression of NGF receptor p75, glutaminase and NCAM L1, activation of mitochondria, and sensitivity to fatty acid intervention.

Motor neuronal damage due to diseases, traumatic insults or de-afferentation of the spinal cord is often incurable because of poor intrinsic regenerative capacity. Hence, medical basic research has to provide a better understanding of development-/regeneration-related cellular processes as only way to develop new and successful therapeutic strategies. Here, we investigated the neuronal differentiation of the NSC-34 hybrid cell line, which is an accepted model for spinal cord motor neurons. Their differentiation was stimulated by switching from normal to differentiation medium and by supplementation with palmitic and oleic acid. To characterize neuro-differentiation of NSC-34 cells, expression of nicotinic acetylcholine receptor alpha 4, NGF p75 receptor, IGF I alpha receptor, glutaminase, NCAM L1, ADAM10 and myelin basic protein as well as activation of mitochondria were analyzed. Both switch from normal to differentiation medium and fatty acid application stimulated NSC-34 differentiation. Differentiation was characterized by diminishing expression of the nicotinic acetylcholine receptor alpha 4 and enhancing expression of the NGF receptor p75, of glutaminase, of NCAM L1 and it's partially transformation from the cell surface into the cell. Fatty acid intervention stabilized the expression of the nicotinic acetylcholine receptor alpha 4, diminished the expression of the NGF receptor p75, consolidated the expression profile of NCAM L1, and intensified the expression of the relevant for NCAM L1 cleavage ADAM10. However, NCAM L1 cleavage itself was unaffected by fatty acid intervention, as was the differentiation-relevant activation of mitochondria and their transformation into neuronal filopodia.

Targeting p75 neurotrophin receptors ameliorates spinal cord injury-induced detrusor sphincter dyssynergia in mice.

AIMS: To determine the role of p75 neurotrophin receptor (p75NTR ) and the therapeutic effect of the selective small molecule p75NTR modulator, LM11A-31, in spinal cord injury (SCI) induced lower urinary tract dysfunction (LTUD) using a mouse model. METHODS: Adult female T8 -T9 transected mice were gavaged daily with LM11A-31 (100 mg/kg) for up to 6 weeks, starting 1 day before, or 7 days following injury. Mice were evaluated in vivo using urine spot analysis, cystometrograms (CMGs), and external urethral sphincter (EUS) electromyograms (EMGs); and in vitro using histology, immunohistochemistry, and Western blot. RESULTS: Our studies confirm highest expression of p75NTRs in the detrusor layer of the mouse bladder and lamina II region of the dorsal horn of the lumbar-sacral (L6 -S1 ) spinal cord which significantly decreased following SCI. LM11A-31 prevented or ameliorated the detrusor sphincter dyssynergia (DSD) and detrusor overactivity (DO) in SCI mice, significantly improving bladder compliance. Furthermore, LM11A-31 treatment blocked the SCI-related urothelial damage and bladder wall remodeling. CONCLUSION: Drugs targeting p75NTRs can moderate DSD and DO in SCI mice, may identify pathophysiological mechanisms, and have therapeutic potential in SCI patients.

Nerve growth factor/p75 neurotrophin receptor-mediated sensitization of rat sensory neurons depends on membrane cholesterol.

Nerve growth factor (NGF) is an important mediator in the initiation of the inflammatory response and NGF via activation of the p75 neurotrophin receptor (p75(NTR)) and downstream sphingomyelin signaling leads to significant enhancement of the excitability of small-diameter sensory neurons. Because of the interaction between sphingomyelin and cholesterol in creating membrane liquid-ordered domains known as membrane or lipid rafts, we examined whether neuronal NGF-induced sensitization via p75(NTR) was dependent on the integrity of membrane rafts. Here, we demonstrate that the capacity of NGF to enhance the excitability of sensory neurons may result from the interaction of p75(NTR) with its downstream signaling partner(s) in membrane rafts. Two agents known to disrupt membrane rafts, edelfosine and methyl-ß-cyclodextrin (MßCD), block the increase in excitability produced by NGF. In contrast, treatment with MßCD containing saturated amounts of cholesterol does not alter the capacity of NGF to augment excitability. In addition, adding back MßCD with cholesterol restored the NGF-induced sensitization in previously cholesterol-depleted neurons, suggesting that cholesterol and the structural integrity of rafts are key to promoting NGF-mediated sensitization. Using established protocols to isolate detergent-resistant membranes, both p75(NTR) and the neuronal membrane raft marker, flotillin, localize to raft fractions. These results suggest that downstream signaling partners interacting with p75(NTR) in sensory neurons are associated with membrane raft signaling platforms.

Oral administration of a small molecule targeted to block proNGF binding to p75 promotes myelin sparing and functional recovery after spinal cord injury.

The lack of effective therapies for spinal cord injury points to the need for identifying novel targets for therapeutic intervention. Here we report that a small molecule, LM11A-31, developed to block proNGF-p75 interaction and p75-mediated cell death crosses the blood-brain barrier efficiently when delivered orally. Administered starting 4 h postinjury, LM11A-31 promotes functional recovery without causing any toxicity or increased pain in a mouse model of spinal contusion injury. In both weight-bearing open-field tests and nonweight-bearing swim tests, LM11A-31 was effective in improving motor function and coordination. Such functional improvement correlated with a >50% increase in the number of surviving oligodendrocytes and myelinated axons. We also demonstrate that LM11A-31 indeed inhibits proNGF-p75 interaction in vivo, thereby curtailing the JNK3-mediated apoptotic cascade. These results thus highlight p75 as a novel therapeutic target for an orally delivered treatment for spinal cord injury.

Design of HIV-1 integrase inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75: a scaffold hopping approach using salicylate and catechol groups.

HIV-1 integrase (IN) is a validated therapeutic target for antiviral drug design. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands the discovery of novel inhibitors that are structurally as well mechanistically different. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC(50)=5 µM) with more than 40-fold selectivity for the strand transfer over the 3'-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC(50) value of 8 µM. Using molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. Furthermore, the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site on IN. This work provides a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.

p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells.

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75(NTR)), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75(NTR) and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75(NTR) and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75(NTR)/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75(NTR) or TrkA. Interestingly, immunoreactivity to anti-p75(NTR) antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75(NTR), when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75(NTR) is turned on.

Effect of betamethasone on the degree of macrophage recruitment and nerve growth factor receptor p75 immunoreaction during recovery of the sciatic nerve after injury: an experimental study in rats.

PURPOSE: This study was designed to explain our previous findings of beneficial effects of betamethasone given perioperatively on decreasing the incidence of neurosensory disturbance after sagittal split osteotomy and improving functional recovery after crush injury to rat sciatic nerves. We analysed the pattern of macrophage recruitment and expression of nerve growth factor p75. MATERIAL AND METHODS: The sciatic nerve was crushed in each of 42 animals by tying the nerve against a glass rod for 30s. Half the rats were given betamethasone and half were not. The effect of betamethasone was evaluated immunohistochemically in a double blind manner after 2, 7 and 17 days using antibodies against macrophage marker (ED1) and p75. RESULTS: We found an initial and significant decrease in the number of macrophages recruited after two days in the group treated with betamethasone compared with controls (p=0.001). By 7 days there were significantly more macrophages in the steroid group than in the control group (p=0.001). There was however, a tendency for the number of p75R to be higher in the in the steroid group but the difference was not significant. At 17 days, there were significantly fewer macrophages in the steroid group (p=0.008) than in the control. CONCLUSION: We conclude that the beneficial effect of a moderate perioperative dose of betamethasone on recovery of a nerve is reflected in the recruitment of macrophages but to only a small extent in expression of p75.

The actions of BDNF on dendritic spine density and morphology in organotypic slice cultures depend on the presence of serum in culture media.

We have previously shown that brain-derived neurotrophin factor (BDNF) increases dendritic spine density and the proportion of stubby spines in apical dendrites of CA1 pyramidal neurons of hippocampal slice cultures maintained in serum-free media. We show here that serum withdrawal causes an increase in the proportion of thin spines and a decrease in the fraction of stubby spines, without changing the overall density of dendritic spines. When slices are maintained in serum-containing media, BDNF also increased spine density but had the opposite effect on spine morphology: it increased the proportion of mushroom and thin spines and decreased the proportion of stubby spines. Intriguingly, slices maintained in serum media showed a lower p75NTR-to-TrkB expression level than serum-free slices, even after BDNF exposure. The differential actions of BDNF on spine morphology depending on the presence of serum in culture media, together with the difference in neurotrophin receptor expression are reminiscent of opposing functional signaling by p75NTR and Trk receptors, and reveal a complex modulation of dendritic morphology by BDNF signaling.

Postnatal lead exposure alters expression of forebrain p75 and TrkA nerve growth factor receptors.

Lead is a potent developmental neurotoxicant that affects many aspects of cognition and behavior. The hippocampus and striatum are among the areas particularly sensitive to the effects of lead and cholinergic neurons in both regions depend upon nerve growth factor (NGF) for their survival and maturation. The present study examined the extent to which postnatal lead exposure may affect the survival and expression of neuroptrophin receptors of septo-hippocampal cholinergic projection neurons in the medial septum/vertical limb of the diagonal band of Broca (MS/VDB) and cholinergic neurons of the striatum. Weanling rats were fed chow containing lead acetate for 30 days and effects on cholinergic cell number and the number of cells expressing neurotrophin receptors p75(NGFR) and trkA were assessed. A decrease in the number of cells expressing p75(NGFR) and an increase in the number of cells expressing trkA receptor was observed in the MS/VDB of lead-exposed rats, without a loss of cholinergic cell number or alteration in cell size. Lead-exposure resulted in a significant decrease in trkA-expressing cells in the striatum but no change in the number or size of cholinergic neurons. These results suggest that a brief postnatal lead exposure does not result in loss of MS/VDB or striatal cholinergic neurons but does modify the expression of neurotrophin receptors in these regions. The significance of these effects on the septo-hippocampal and striatal functioning remains to be studied.

Neuronal nitric oxide synthase is upregulated in a subset of primary sensory afferents after nerve injury which are necessary for analgesia from alpha2-adrenoceptor stimulation.

alpha2-Adrenoceptor (AR) agonists increase in analgesic potency and efficacy after peripheral nerve injury, and their effects are blocked by neuronal nitric oxide synthase (nNOS) inhibitors and M4 muscarinic receptor antagonists only after injury. We tested whether nNOS and M4 muscarinic receptors are co-expressed in the spinal cord, and whether destruction of a subset of sensory afferents which are essential to alpha2-AR analgesia would also destroy nNOS and M4 receptor expression. Male Sprague-Dawley rats underwent left L5 and L6 spinal nerve ligation. Lumbar spinal cord was removed and immunostained for M4 muscarinic receptors and nNOS alone and for co-expression. Others received intrathecal injection of saporin linked to an antibody to the neurotrophin receptor p75(NTR), which eliminates cells expressing this receptor and the analgesic effects of alpha2-AR agonists. nNOS staining of fibers in the superficial dorsal horn was dramatically increased after spinal nerve ligation, and this was abolished by saporin linked anti-p75(NTR) treatment. In contrast, nNOS staining in dorsal horn neurons was unaltered by these manipulations. M4 receptors were present on neurons in the dorsal horn, some of which co-expressed nNOS, but their pattern of expression was not altered by these manipulations. Peripheral nerve injury increases nNOS expression in fibers in the superficial dorsal horn, some of which likely express p75(NTR), and alpha2-AR agonists may reduce injury-induced sensitization by activation of nNOS in these fibers In contrast, changes in nNOS and M4 receptor location on spinal cord neurons are not responsible for increased analgesic potency of alpha2-AR agonists after nerve injury.

Nerve growth factor eye drop administrated on the ocular surface of rodents affects the nucleus basalis and septum: biochemical and structural evidence.

It has been shown that conjunctivally applied NGF in rats can reach the retina and optic nerve. Whether topical eye NGF application reaches the central nervous system is not known. In the present study, we have addressed this question. It was found that topical eye NGF application affects brain cells. Time-course studies revealed that repeated NGF application leads to high concentration of this neurotrophins after 6 h and normal levels after 24 h. Our results also showed that topical eye application of NGF causes an enhanced expression of NGF receptors and ChAT immunoreactivity in forebrain cholinergic neurons, suggesting that ocular NGF application could have a functional role on damaged brain cells. The present findings suggest that eye NGF application can represent an alternative route to prevent degeneration of NGF-receptive neurons involved in disorders such as Alzheimer and Parkinson.

Peroxynitrite transforms nerve growth factor into an apoptotic factor for motor neurons.

Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo posttranslational oxidative or nitrative modifications that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed an exceptional ability to induce p75(NTR)-dependent motor neuron apoptosis at physiologically relevant concentrations. Whereas native NGF requires an external source of nitric oxide (NO) to induce motor neuron death, peroxynitrite-treated NGF induced motor neuron apoptosis in the absence of exogenous NO. Nevertheless, NO potentiated the apoptotic activity of peroxynitrite-modified NGF. Blocking antibodies to p75(NTR) or downregulation of p75(NTR) expression by antisense treatment prevented motor neuron apoptosis induced by peroxynitrite-treated NGF. We investigated what oxidative modifications were responsible for inducing a toxic gain of function and found that peroxynitrite induced tyrosine nitration in a dose-dependent manner. Moreover, peroxynitrite triggered the formation of stable high-molecular-weight oligomers of NGF. Preventing tyrosine nitration by urate abolished the effect of peroxynitrite on NGF apoptotic activity. These results indicate that the oxidation of NGF by peroxynitrite enhances NGF apoptotic activity through p75(NTR) 10,000-fold. To our knowledge, this is the first known posttranslational modification that transforms a neurotrophin into an apoptotic agent.

Expression and significance of nerve growth factor receptor p75 in rats' cathartic colonic wall.

OBJECTIVE: To investigate the expression of nerve growth factor receptor p75 in a normal and cathartic colon and its significance in the formation of the cathartic colon in rats. METHODS: Sixty Sprague-Dawley rats were equally divided into normal control group, rhubarb group and phenolphthalein group. A model of the cathartic colon was constructed by gastric infusion with rhubarb or phenolphthalein in rats. The first dose of rhubarb and phenolphthalein was both 200 mg/kg/d and was increased by 200 mg/kg/d with each passing day. The last dose of rhubarb and phenolphthalein was 3200 mg/kg/d and 4200 mg/kg/d, respectively. The transit function of colon was measured by the Chinese ink expulsion test; the p75 in colon wall was determined by the immunohistochemical method. RESULTS: The transit speed was much slower in the cathartic colon group than that in the control group. The imprinted Chinese ink length and the ratio of imprinted length/total colon length in the rhubarb-induced cathartic colon was significantly shorter than that of the control group (77.38 +/- 8.42 vs 94.25 +/- 7.07 cm, P < 0.01). Those in the phenolphthalein-induced group (83.38 +/- 9.75 cm) were also significantly shorter than those of the control group but to a lesser degree (P < 0.05). p75 was abundantly expressed in the submucosal nerve plexus and weakly expressed in the myenteric plexus. The expression of p75 was much higher in the rhubarb-induced group. The expression was strongly positive in the submucosal nerve plexus, significantly higher than that in the controls (P < 0.01). In the myenteric plexus, p75 was also highly expressed (P < 0.05). In the case of the phenolphthalein-induced group, the expression of p75 was positive in the submucosal nerve plexus but was positive in the myenteric plexus of three rats only. The remaining rats were negative or weakly positive. This was not significantly different from that of the control group. CONCLUSIONS: The abnormal expression of p75 in cathartic colon probably has some effect on the degeneration or apoptosis of neuronal cells in the enteric nerve plexus, with a subsequent pathological change of the enteric nervous system, and thus leads to abnormalities in colonic dynamics. The kind of lesion is probably associated with long-term use of irritative cathartics.

Nerve growth factor-induced migration of endothelial cells.

Nerve growth factor (NGF) is a well known neurotropic and neurotrophic agonist in the nervous system, which recently was shown to also induce angiogenic effects in endothelial cells (ECs). To measure NGF effects on the migration of cultured ECs, an important step in neoangiogenesis, we optimized an omnidirectional migration assay using human aortic endothelial cells (HAECs) and validated the assay with human recombinant basic fibroblast growth factor (rhbFGF) and human recombinant vascular endothelial growth factor (rhVEGF). The potencies of nerve growth factor purified from various species (viper, mouse, and recombinant human) to stimulate HAEC migration was similar to that of VEGF and basic fibroblast growth factor (bFGF) (EC50 of approximately 0.5 ng/ml). Recombinant human bFGF was significantly more efficacious than either viper NGF or rhVEGF, both of which stimulated HAEC migration by approximately 30% over basal spontaneous migration. NGF-mediated stimulation of HAEC migration was completely blocked by the NGF/TrkA receptor antagonist K252a [(8R*,9S*,11S*)-(/)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,-8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(c,d,e)trindene-1-one] (30 nM) but not by the VEGF/Flk receptor antagonist SU-5416 [3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one] (250 nM), indicating a direct effect of NGF via TrkA receptor activation on HAEC migration. Viper NGF stimulation of HAEC migration was additively increased by either rhVEGF or rhbFGF, suggesting a potentiating interaction between their tyrosine kinase receptor signaling pathways. Viper NGF represents a novel pharmacological tool to investigate possible TrkA receptor subtypes in endothelial cells. The ability of NGF to stimulate migration of HAEC cells in vitro implies that this factor may play an important role in the cardiovascular system besides its well known effects in the nervous system.

Glial-mediated neuroprotection: evidence for the protective role of the NO-cGMP pathway via neuron-glial communication in the peripheral nervous system.

The NO-cGMP pathway has emerged as a neuroprotective signaling system involved in communication between neurons and glia. We have previously shown that axotomy or nerve growth factor (NGF)-deprivation of dorsal root ganglion (DRG) neurons leads to increased production of NO and at the same time an increase in cGMP production in their satellite glia cells. Blockade of NO or its receptor, the cGMP synthesizing enzyme soluble guanylate cyclase (sGC), results in apoptosis of neurons and glia. We now show that co-culture of neonatal DRG neurons with either Schwann cells pre-treated with an NO donor or a membrane-permeant cGMP analogue; or neurons maintained in the medium from Schwann cell cultures treated in the same way, prevents neuronal apoptosis. Both NO donor and cGMP treatment of Schwann cells results in synthesis of NGF and NT3. Furthermore, if the Schwann cells are previously infected with adenoviral vectors expressing a dominant negative sGC mutant transgene, treatment of these Schwann cells with an NO donor now fails to prevent neuronal apoptosis. Schwann cells treated in this way also fail to express neither cGMP nor neurotrophins. These findings suggest NO-sGC-cGMP-mediated NGF and NT3 synthesis by Schwann cells protect neurons.

Melanopsin in the circadian timing system.

In mammals, circadian rhythms are generated by a light-entrainable oscillator located in the hypothalamic suprachiasmatic nucleus (SCN). Light signals reach the SCN via a dedicated retinal pathway, the retinohypothalamic tract (RHT). One question that continues to elude scientists is whether the circadian system has its own dedicated photoreceptor or photoreceptors. It is well established that conventional photoreceptors, rods and cones, are not required for circadian photoreception, suggesting that the inner retinal layer might contribute to circadian photoreception. Melanopsin, a novel photo pigment expressed in retinal ganglion cells (RGCs), has been proposed recently as a candidate circadian photoreceptor. Melanopsin-containing RGCs are intrinsically photosensitive, form part of the RHT, and contain neurotransmitters known to play a critical role in the circadian response to light. Furthermore, melanopsin-containing RGCs do not depend on inputs from rods and cones to transmit light signals to the SCN. However, based on a review of the available information about melanopsin and on new data from our laboratory, we propose that melanopsin, in itself, is not necessary for circadian photoreception. In fact, it appears that of the known photoreceptor systems, none, in and of itself, is necessary for circadian photoreception. Instead, it appears that within the photoreceptive systems there is some degree of redundancy, each contributing in some way to photic entrainment.

ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury.

The neurotrophin receptor p75 is induced by various injuries to the nervous system, but its role after injury has remained unclear. Here, we report that p75 is required for the death of oligodendrocytes following spinal cord injury, and its action is mediated mainly by proNGF. Oligodendrocytes undergoing apoptosis expressed p75, and the absence of p75 resulted in a decrease in the number of apoptotic oligodendrocytes and increased survival of oligodendrocytes. ProNGF is likely responsible for activating p75 in vivo, since the proNGF from the injured spinal cord induced apoptosis among p75(+/+), but not among p75(-/-), oligodendrocytes in culture, and its action was blocked by proNGF-specific antibody. Together, these data suggest that the role of proNGF is to eliminate damaged cells by activating the apoptotic machinery of p75 after injury.