Neuroinflammation-Induced Memory Deficits Are Amenable to Treatment with D-Cycloserine.

Cognitive deficits, especially memory loss, are common following many types of brain insults which are associated with neuroinflammation, although the underlying mechanisms are not entirely clear. The present study aimed to characterize the long-term cognitive and behavioral impairments in a mouse model of neuroinflammation in the absence of other insults and to evaluate the therapeutic potential of D-cycloserine (DCS). DCS is a co-agonist of the NMDA receptor that ameliorates cognitive deficits in models of TBI and stroke. Using a mouse model of global neuroinflammation induced by intracisternal (i.c.) administration of endotoxin (LPS), we found long-lasting microgliosis, memory deficits, impaired LTP, and reduced levels of the obligatory NR1 subunit of the NMDA receptor. A single administration of DCS, 1 day after i.c. LPS reduced microgliosis, reversed the cognitive deficits and restored LTP and NR1 levels. These results demonstrate that neuroinflammation alone, in the absence of trauma or ischemia, can cause persistent (>6 months) memory deficits linked to deranged NNMDA receptor function and suggest a possible role for NMDA co-agonists in reducing the cognitive sequelae of neuroinflammation.

Angiotensin receptor type 2 activation induces neuroprotection and neurogenesis after traumatic brain injury.

Angiotensin II receptor type 2 (AT(2)) agonists have been shown to limit brain ischemic insult and to improve its outcome. The activation of AT(2) was also linked to induced neuronal proliferation and differentiation in vitro. In this study, we examined the therapeutic potential of AT(2) activation following traumatic brain injury (TBI) in mice, a brain pathology that displays ischemia-like secondary damages. The AT(2) agonist CGP42112A was continuously infused immediately after closed head injury (CHI) for 3 days. We have followed the functional recovery of the injured mice for 35 days post-CHI, and evaluated cognitive function, lesion volume, molecular signaling, and neurogenesis at different time points after the impact. We found dose-dependent improvement in functional recovery and cognitive performance after CGP42112A treatment that was accompanied by reduced lesion volume and induced neurogenesis in the neurogenic niches of the brain and also in the injury region. At the cellular/molecular level, CGP42112A induced early activation of neuroprotective kinases protein kinase B (Akt) and extracellular-regulated kinases ½ (ERK½), and the neurotrophins nerve growth factor and brain-derived neurotrophic factor; all were blocked by treatment with the AT(2) antagonist PD123319. Our results suggest that AT(2) activation after TBI promotes neuroprotection and neurogenesis, and may be a novel approach for the development of new drugs to treat victims of TBI.

Neuroprotection after traumatic brain injury in heat-acclimated mice involves induced neurogenesis and activation of angiotensin receptor type 2 signaling.

Long-term exposure of mice to mild heat (34°C±1°C) confers neuroprotection against traumatic brain injury (TBI); however, the underling mechanisms are not fully understood. Heat acclimation (HA) increases hypothalamic angiotensin II receptor type 2 (AT2) expression and hypothalamic neurogenesis. Accumulating data suggest that activation of the brain AT2 receptor confers protection against several types of brain pathologies, including ischemia, a hallmark of the secondary injury occurring following TBI. As AT2 activates the same pro-survival pathways involved in HA-mediated neuroprotection (e.g., Akt phosphorylation, hypoxia-inducible factor 1α (HIF-1α), and brain-derived neurotrophic factor (BDNF)), we examined the role of AT2 in HA-mediated neuroprotection after TBI. Using an AT2-specific antagonist PD123319, we found that the improvements in motor and cognitive recovery as well as reduced lesion volume and neurogenesis seen in HA mice were all diminished by AT2 inhibition, whereas no significant alternations were observed in control mice. We also found that nerve growth factor/tropomyosin-related kinase receptor A (TrkA), BDNF/TrkB, and HIF-1α pathways are upregulated by HA and inhibited on PD123319 administration, suggesting that these pathways play a role in AT2 signaling in HA mice. In conclusion, AT2 is involved in HA-mediated neuroprotection, and AT2 activation may be protective and should be considered a novel drug target in the treatment of TBI patients.

Inosine improves functional recovery after experimental traumatic brain injury.

Despite years of research, no effective therapy is yet available for the treatment of traumatic brain injury (TBI). The most prevalent and debilitating features in survivors of TBI are cognitive deficits and motor dysfunction. A potential therapeutic method for improving the function of patients following TBI would be to restore, at least in part, plasticity to the CNS in a controlled way that would allow for the formation of compensatory circuits. Inosine, a naturally occurring purine nucleoside, has been shown to promote axon collateral growth in the corticospinal tract (CST) following stroke and focal TBI. In the present study, we investigated the effects of inosine on motor and cognitive deficits, CST sprouting, and expression of synaptic proteins in an experimental model of closed head injury (CHI). Treatment with inosine (100 mg/kg i.p. at 1, 24 and 48 h following CHI) improved outcome after TBI, significantly decreasing the neurological severity score (NSS, p<0.04 vs. saline), an aggregate measure of performance on several tasks. It improved non-spatial cognitive performance (object recognition, p<0.016 vs. saline) but had little effect on sensorimotor coordination (rotarod) and spatial cognitive functions (Y-maze). Inosine did not affect CST sprouting in the lumbar spinal cord but did restore levels of the growth-associated protein GAP-43 in the hippocampus, though not in the cerebral cortex. Our results suggest that inosine may improve functional outcome after TBI.

Establishment and characterization of pheochromocytoma tumor models expressing different levels of trkA receptors.

To date experimental in vivo pheochromocytoma (PC) models have not been available. A major in vitro PC model consists of PC12 cells that respond to nerve growth factor (NGF) by differentiation, mediated by the trkA receptor. We report the establishment of PC12 tumor models expressing low and high levels of trkA receptor in CD1 nude mice. The tumors are characterized by their responsiveness to NGF, karyotype, presence of enolase, and chromaffin granules, as well as dopamine release. These novel PC models facilitate research on the role of the trkA receptor in cancer and the development of trkA-selective anti-cancer agents.

Characterization of nerve growth factors (NGFs) from snake venoms by use of a novel, quantitative bioassay utilizing pheochromocytoma (PC12) cells overexpressing human trkA receptors.

Snake venoms are a very abundant source of nerve growth factors (NGF). NGFs of Elapidae showing 65% sequence homology with mouse or human NGF, while the Viperidae NGF shows N-glycosylation (Asn-21) typical of these mammalian NGFs. Snake NGF-induced neurite outgrowth (neurotropic activity) was measured in the past by using PC12 cell or dorsal root ganglion bioassays. The present study was aimed at comparing, by dose-response experiments, the neurotropic activity of cobra and vipera versus mammalian NGFs, by using a novel bioassay involving PC12 cells genetically engineered to overexpress NGF-trkA receptors of human origin. These cells respond to NGF by differentiation (morphologically expressed as neurite outgrowth) by a process mediated by NGF-trkA receptors. This process was evaluated by two different criteria: (1) elongation of neurites (E), and (2) Percentage of responsive cells (PRC) determined by digital acquisition of data and computer analysis. We found that snake venom NGFs were less potent than mouse NGF, and that cobra NGF was more potent than vipera NGF. These data indicate the following order of NGF activity towards recombinant human trkA receptors: recombinant human NGF>mouse NGF>cobra NGF>vipera NGF. The neurotropic efficacy of these NGFs was found to be similar, reaching 80-90% of maximal activity obtained with all NGF forms. Interestingly, cobra (but not vipera) NGF demonstrated prolonged neurotropic activity compared with mouse NGF. The results of the present study indicate that cobra and vipera venom NGFs represent natural agonists of human trkA-receptor of a lower potency, but of similar efficacy, compared with mammalian NGFs. These compounds are important pharmacological tools to characterize the trkA receptor structure-function relationship, and to develop novel neurotropic drugs.

A quantitative bioassay for nerve growth factor, using PC12 clones expressing different levels of trkA receptors.

Nerve growth factor (NGF) is a neurotrophin required for differentiation, development, and survival of the sympathetic nervous system, with many of its biological effects being mediated via trkA receptors. There is a need for a standard quantitative bioassay for NGF, to be used in basic research and in pharmaceutical studies. The objective of the present research was to develop a selective, quantitative, and reliable bioassay for NGF, using a morphological criterion: neurite cell outgrowth. In addition, we aimed to apply the aforementioned bioassay to measure NGF administered to mice. Pheochromocytoma PC12 cell variants including wild-type cultures, and a trkA-overexpressing stable transfectant PC12-6.24-I, PC12nnr5, and PC12EN lacking trkA receptors, were used. Dose-response curves were generated with NGF beta-subunit (2.5S) purified from mouse submaxillary glands. Our results demonstrated that the bioassay was sensitive to 0.3-20 ng/mL, and selective, as neurite outgrowth was not seen by any other growth factor other than NGF. In addition, variant clones PC12nnr5 and PC12EN, lacking trkA receptors, did not respond to NGF. The bioassay detected NGF in serum of mice injected with NGF. This novel developed bioassay can serve as a model system for various neuroscience purposes.