Rinat Tabakman

Interactions between the cells of the immune and nervous system: neurotrophins as neuroprotection mediators in CNS injury.

Inflammatory processes in the central nervous system (CNS) are considered neurotoxic, although recent studies suggest that they also can be beneficial and confer neuroprotection (neuroprotective autoimmunity). Cells from the immune system have been detected in CNS injury and found to produce and secrete a variety of neurotrophins such as NGF, BDNF, NT-3 and NT-4/5, and to express (similarly to neuronal cells), members of the tyrosine kinase (Trk) receptor family such as TrkA, TrkB and TrkC. Indeed, autocrine and paracrine interactions are observed at the site of CNS injury, resulting in a variety of homologic-heterologic modulations of immune and neuronal cell function. The end result of the inflammatory process, neurotoxicity and/or neuroprotection, is a function of the fine balance between the two cellular systems, i.e., of the complex signaling relationships between anti-inflammatory neuroprotective factors (neurotrophins and other chemical mediators) and proinflammatory neurotoxic factors (TNF, free radicals, certain cytokines, etc.). Autoimmune neuroprotection is a novel therapeutic approach aimed at shifting the balance between the immune and neuronal cells towards survival pathways in a variety of CNS injuries. This review focuses on data supporting this concept and its future therapeutical implications for optic nerve injury and multiple sclerosis.

Neuroprotective and neurotoxic effects of monoamine oxidase-B inhibitors and derived metabolites under ischemia in PC12 cells

Selegiline and rasagiline are selective and irreversible monoamine oxidase-B inhibitors that exert neuroprotective effects in various preclinical models. The aim of the present study was to examine the effect of selegiline and its major metabolite, L-methamphetamine in comparison to rasagiline and its major metabolite, 1-R-aminoindan on oxygen-glucose deprivation induced cell death in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells. Our results show that selegiline reduces oxygen-glucose deprivation induced cell death by 30%. When the cultures were treated with rasagiline at similar concentrations, cell death induced by oxygen-glucose deprivation was reduced by 45-55%. L-methamphetamine, a major selegiline metabolite, but not 1-R-aminoindan, the major rasagiline metabolite, enhanced oxygen-glucose deprivation-induced cell death by 70%. Under normoxic conditions, both metabolites lack neurotoxicity. Concomitant exposure of the cultures under oxygen-glucose deprivation, to a combination of either selegiline and L-methamphetamine or rasagiline and 1-R-aminoindan, indicated that L-methamphetamine, but not 1-R-aminoindan, blocked the neuroprotective effect of the parental drug. These results suggest there may be a neuroprotective advantage of rasagiline over selegiline.

Neuroprotection by cord blood neural progenitors involves antioxidants, neurotrophic and angiogenic factors

Human umbilical cord blood (HUCB) is a valuable source for cell therapy since it confers neuroprotection in stroke animal models. However, the responsible sub-populations remain to be established and the mechanisms involved are unknown. To explore HUCB neuroprotective properties in a PC12 cell-based ischemic neuronal model, we used an HUCB mononuclear-enriched population of collagen-adherent cells, which can be differentiated in vitro into a neuronal phenotype (HUCBNP). Upon co-culture with insulted-PC12 cells, HUCBNP conferred approximately 30% neuroprotection, as evaluated by decreased lactate dehydrogenase and caspase-3 activities. HUCBNP decreased by 95% the level of free radicals in the insulted-PC12 cells, in correlation with the appearance of antioxidants, as measured by changes in the oxidation-reduction potential of the medium using cyclic-voltammetry. An increased level of nerve growth factor (NGF), vascular endothelial growth factor and basic fibroblast growth factor in the co-culture medium was temporally correlated with a -medium neuroprotection effect, which was partially abolished by heat denaturation. HUCBNP-induced neuroprotection was correlated with changes in gene expression of these neurotrophic factors, while blocked by K252a, an antagonist of the TrkA/NGF receptor. These findings indicate that HUCBNP-induced neuroprotection involves antioxidant(s) and neurotrophic factors, which, by paracrine and/or autocrine interactions between the insulted-PC12 and the HUCBNP cells, conferred neuroprotection.

Neuroprotection by NGF in the PC12 In Vitro OGD Model

Abstract: Neurodegenerative disorders and chronic disability due to stroke in the brain or spinal cord afflict a large sector of the population. To investigate the mechanism involved in ischemic stroke and to develop neuroprotective drugs/therapies, in vivo and in vitro, pharmacological models are needed. To investigate the cellular and molecular neuroprotective mechanisms of nerve growth factor (NGF), a member of the nervous system neurotrophin family of growth factors, under ischemia, we used an oxygen‐glucose‐deprivation (OGD) device and pheochromocytoma PC12 cells exposed to a paradigm of ischemic insult. Pretreatment of the cultures with 50 ng/mL of NGF, 18 h prior to OGD insult, conferred 30% of neuroprotection. Time‐course experiments showed marked activation of the ERK, JNK, and p‐38 MAPK isoforms during the OGD phase, but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL of NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD‐induced activation of JNK 1, and 20% and 50% attenuation of OGD‐induced activation of p‐38 α and β, respectively. The effect of NGF on gene expression in the PC12 ischemic model using Affymatrix Rat DNA‐Microarray technology indicates that only 6% of the genes are differentially regulated (induced/suppressed) by OGD insult and/or NGF. These findings support the notion that pretreatment with NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms and differential gene expression. This ischemic model may be useful to investigate molecular mechanisms of OGD‐induced neurotoxicity and NGF‐induced neuroprotection, and to generate novel therapeutic concepts for stroke treatment.

Apoptotic characteristics of cell death and the neuroprotective effect of homocarnosine on pheochromocytoma PC12 cells exposed to ischemia

We recently improved an in vitro ischemic model, using PC12 neuronal cultures exposed to oxygen‐glucose deprivation (OGD) for 3 hr in a special device, followed by 18 hr of reoxygenation. The cell death induced in this ischemic model was evaluated by a series of markers: lactate dehydrogenase (LDH) release, caspase‐3 activation, presence of cyclin D1, cytochrome c leakage from the mitochondria, BAX cellular redistribution, cleavage of poly (ADP‐ribose) polymerase (PARP) to an 85‐kDa apoptotic fragment, and DNA fragmentation. The OGD insult, in the absence of reoxygenation, caused a strong activation of the mitogen‐activated protein kinase (MAPK) isoforms extracellular regulated kinase (ERK), c‐Jun NH2‐terminal kinase (JNK), and stress‐activated protein kinase (SAPK), also known as p‐38. The detection of apoptotic markers and activation of MAPKs during the ischemic insult strongly suggest that apoptosis plays an important role in the PC12 cell death. Homocarnosine, a neuroprotective histidine dipeptide, present in high concentrations in the brain, was found to provide neuroprotection, as expressed by a 40% reduction in LDH release and caspase‐3 activity at 1 mM. Homocarnosine reduced OGD activation of ERK 1, ERK 2, JNK 1, and JNK 2 by 40%, 46%, 55%, and 30%, respectively. These results suggest that apoptosis is an important characteristic of OGD‐induced neuronal death and that antioxidants, such as homocarnosine, may prevent OGD‐induced neuronal death by inhibiting the apoptotic process and/or in relation to the differential attenuation of activity of MAPKs.

Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38α and p38β activation and confers neuroprotection in the pheochromocytoma PC12 model

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by addition of glucose and oxygen (OGD reperfusion). Neuronal cell death induced in this model was measured by the release of lactate dehydrogenase (LDH), activation of caspase-3 and mitogen-activated protein kinases (MAPKs), measured with specific anti-phospho-antibodies. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, conferred 30% neuroprotection. However, treatment of the cultures with NGF concomitantly with the OGD insult did not result in neuroprotection. Time-course experiments showed marked activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK), and p38 MAPK isoforms during the OGD phase but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK1, and 20% and 50% attenuation of OGD-induced activation of p38α and β, respectively. These findings support the notion that NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms, and provide the PC12 model as an in vitro OGD system to investigate molecular mechanisms of neurotoxicity and neuroprotection.

Neuroprotective effects of nimodipine and nifedipine in the NGF-differentiated PC12 cells exposed to oxygen-glucose deprivation or trophic withdrawal

The goal of this study was to compare the neuroprotective properties of the l‐type Ca2+ channel blockers, nimodipine and nifedipine, using nerve growth factor (NGF)‐differentiated PC12 neuronal cultures exposed to oxygen‐glucose deprivation (OGD) and trophic withdrawal‐induced cell death. Nimodipine (1–100 μM) conferred 65 ± 13% neuroprotection upon exposure to OGD and 35 ± 6% neuroprotection towards different trophic withdrawal‐induced cell death measured by lactate dehydrogenase and caspase 3 activities. The time window of nimodipine conferred neuroprotection was detected during the first 5 h but not at longer OGD exposures. Nifedipine (1–100 μM), to a lower potency than nimodipine, conferred 30–55 ± 8% neuroprotection towards OGD in PC12 cells and 29 ± 5% in rat hypocampal slices, and 10 ± 3% neuroprotection at 100 μM towards trophic withdrawal‐induced PC12 cell death. The ability to demonstrate that nimodipine conferred neuroprotection in a narrow therapeutic time‐window indicates that the OGD PC12 model mimics the in vivo models and therefore suitable for neuroprotective drug discovery and development.

The induction of tuftelin expression in PC12 cell line during hypoxia and NGF-induced differentiation

The tuftelin protein isoforms undergo post‐translation modifications, and are ubiquitously expressed in various tissues in embryos, adults, and tumors. Developmental and pathological studies suggested an apparent correlation between oxygen deprivation and tuftelin expression. The aim of the study was therefore to investigate the effect of a pathological insult (hypoxia) and a physiological growth factor (NGF), which antagonistically regulate HIF1 expression, on tuftelin expression using the neuronal PC12 cell model. In the present study, we first demonstrated the expression of tuftelin in PC12 cells, providing an experimental system to investigate the pathophysiological role of tuftelin. Furthermore, we demonstrated the induction of tuftelin during hypoxia by oxygen deprivation and during chemical hypoxia by cobalt chloride. Down‐regulation of HIF1α mRNA blocked hypoxia‐induced HIF1α expression, and reduced by 89% hypoxia‐induced tuftelin expression. In mice, intraperitoneal injection of cobalt chloride significantly induced tuftelin mRNA and protein expression in the brain. During NGF‐mediated PC12 differentiation, tuftelin expression was significantly induced in correlation with neurite outgrowth. This induction was partially blocked by K252a, a selective antagonist of the NGF receptor TrkA, indicating the involvement of the TrkA‐signaling pathways in tuftelin induction by NGF. Revealing the physiological role of tuftelin will clarify mechanisms related to the “hypoxic genome,” and NGF‐induced neurotrophic and angiogenic effects. J. Cell. Physiol. 226: 165–172, 2010.

Improved muscle strength and mobility in the dy2J/dy2J mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate

The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder.

Dexamethasone-Induced Down-Regulation of Nerve Growth Factor Receptor p75NTR is Mediated by Glucocorticoid Type II Receptor in PC12 Cell Model

PC12 clones are established neuronal models to investigate mechanisms involved in the cross-talk between the neurotrophins and drugs. Chronic treatment of PC12 cells with the glucocorticoid agonist drug, dexamethasone (Dex), elicited a 50% decrease in the selective binding of 125 I-NGF along with a reduction in the NGF receptor p75 NTR mRNA and protein levels, suggesting a transcriptional mechanism. This down regulation of p75 NTR was antagonized by the glu- cocorticoid type II receptor (GR-2), RU-38486 but not by the minerallocorticoid receptor, RU-28318, antagonists. This process was associated with increased autophosphorylation of the NGF receptor, TrkA. Chronic treatment of PC12 with Dex abolished the NGF-induced proliferation of the cells after 20 hours and inhibited by 45% the neurite elongation after 96 hours. RU-38486 blocked Dex-induced shift of PC12 cells from dopaminergic to noradrenergic phenotype. Dex- induced down regulation of p75 NTR receptor is mediated by GR-2 and is correlated with disruption of NGF induced prolif-