Neuroprotective Effects of Estradiol on Motoneurons in a Model of Rat Spinal Cord Embryonic Explants

Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disorder characterized by motoneuron death. Clinical and experimental studies in animal models of ALS have found gender differences in the incidence and onset of disease, suggesting that female hormones may play a beneficial role. Cumulative evidence indicates that 17β-estradiol (17βE2) has a neuroprotective role in the central nervous system. We have previously developed a new culture system by using rat spinal cord embryonic explants in which motoneurons have the singularity of migrating outside the spinal cord, growing as a monolayer in the presence of glial cells. In this study, we have validated this new culture system as a useful model for studying neuroprotection by estrogens on spinal cord motoneurons. We show for the first time that spinal cord motoneurons express classical estrogen receptors and that 17βE2 activates, specifically in these cells, the Akt anti-apoptotic signaling pathway and two of their downstream effectors: GSK-3β and Bcl-2. To further validate our system, we demonstrated neuroprotective effects of 17βE2 on spinal cord motoneurons when exposed to the proinflammatory cytokines TNF-α and IFN-γ. These effects of 17βE2 were fully reverted in the presence of the estrogen receptor antagonist ICI 182,780. Our new culture model and the results presented here may provide the basis for further studies on the effects of estrogens, and selective estrogen receptor modulators, on spinal cord motoneurons in the context of ALS or other motoneuron diseases.     

Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways

Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate-induced apoptosis through regulations of p38 and Akt pathways. Pre-treatment of cerebellar granule neurons (CGNs) with minocycline (10-100 microm) elicited a dose-dependent reduction of glutamate excitotoxicity and blocked glutamate-induced nuclear condensation and DNA fragmentations. Using patch-clamping and fluorescence Ca2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamate-caused rises in intracellular Ca2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38-specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3beta (GSK3beta) attenuated glutamate-induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3beta caused by glutamate, as were abolished by PI3-K inhibitors. These results demonstrate that minocycline prevents glutamate-induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3-K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.     

Early decrease of survival factors and DNA repair enzyme in spinal motor neurons of presymptomatic transgenic mice that express a mutant SOD1 gene

The primary pathogenetic mechanisms of amyotrophic lateral sclerosis (ALS) have been elusive. Some of the mechanisms would be implicated in an imbalance between death and survival factors, and impairment of DNA repair possibly caused by oxidative stress. Phosphatidylinositol 3-kinase (PI3-K) and its downstream effector, Akt/protein kinase B (PKB), have been shown to play a pivotal role in neuronal survival against apoptosis supported by neurotrophic factors. To elucidate the mechanisms of motor neuron death in ALS, we examined the expression of PI3-K, Akt, and the DNA repair enzyme redox factor-1 (Ref-1) protein in the spinal cord of transgenic mice with an ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) gene, a valuable model for human ALS. Immunoblotting and immunocytochemical analyses showed that most spinal motor neurons lost immunoreactivity for PI3-K, Akt, and Ref-1 in the presymptomatic stage that preceded a significant loss of neurons. These results suggest that an early decrease of survival signal proteins and a DNA repair enzyme in the spinal motor neurons may account for the mutant SOD1-mediated motor neuron death in this animal model of ALS.     

谷氨酸转运抑制剂对器官型培养脊髓片的影响

观察谷氨酸转运体抑制剂苏一羟天冬氨酸(Threo-hydroxyaspartate,THA)对器官型培养的脊髓片的影响,探讨谷氨酸在运动神经元损伤中的作用。取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,在培养液中加入不同浓度THA(50μmol／L、100μmol／L、5001μmol／L),用神经元的特异性免疫组化染色剂SMI-32,非磷酸化神经丝标记物,对脊髓腹角α运动神经元进行鉴定,用单克隆抗钙网膜蛋白(calretinin)抗体对背角中间神经元进行记数,测定培养液中乳酸脱氢酶(LDH)的含量,并与对照组比较。结果显示对照组α运动神经元数目恒定,THA可以引起剂量依赖性的培养液中LDH含量增高和α运动神经元数目减少,而脊髓背角的中间神经元损伤相对较轻,其中THA100μmol／L组在体外培养4周后出现类似于肌萎缩侧索硬化(ALS)的病理改变：α运动神经元数目较对照组明显减少,而脊髓背角的中间神经元数目无显著变化。细胞外谷氨酸增高主要对运动神经元造成损伤,脊髓运动神经元较感觉神经元对谷氨酸的兴奋毒作用更加敏感。     

Chronic elevation of extracellular glutamate due to transport blockade is innocuous for spinal motoneurons in vivo

Glutamate-mediated excitotoxicity has been considered to play an important role in the mechanism of spinal motoneuron death in amyotrophic lateral sclerosis (ALS), and some reports suggest that this excitotoxicity may be due to a decreased glutamate transport and the consequent elevation of its extracellular level. We have previously shown that short lasting increments in extracellular glutamate due to administration of the non-selective glutamate transport blocker l-2,4-trans-pyrrolidine-dicarboxylate (PDC) by microdialysis in the rat spinal cord do not induce motoneuron damage. In the present work we examined the potential involvement of chronic glutamate transport blockade as a causative factor of spinal motoneuron death and paralysis in vivo. Using osmotic minipumps, we infused directly in the spinal cord for up to 10 days PDC and another glutamate transport blocker, dl-threo-β-benzyloxyaspartate (TBOA), and we measured by means of microdialysis and HPLC the extracellular concentration of glutamate and other amino acids. We found that after the infusion of both PDC and TBOA the concentration of endogenous extracellular glutamate was 3–4-fold higher than that of the controls. Nevertheless, in spite of this elevation no motoneuron degeneration or gliosis were observed, assessed by histological examination and choline acetyltransferase and glial fibrillary acidic protein immunocytochemistry. In accord with this lack of toxic effect, no motor deficits, assessed by three motor activity tests, were observed.Because we had previously shown that under identical experimental conditions the infusion of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) induced progressive motoneuron death and paralysis, we conclude that prolonged elevation of extracellular glutamate due to its transport blockade in vivo is innocuous for spinal motoneurons and therefore that these results do not support the hypothesis that glutamate transport deficiency plays a crucial role as a causal factor of spinal motoneuron degeneration in ALS.     

Inhibitory Synaptic Regulation of Motoneurons: A New Target of Disease Mechanisms in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is the third most common adult-onset neurodegenerative disease. It causes the degeneration of motoneurons and is fatal due to paralysis, particularly of respiratory muscles. ALS can be inherited, and specific disease-causing genes have been identified, but the mechanisms causing motoneuron death in ALS are not understood. No effective treatments exist for ALS. One well-studied theory of ALS pathogenesis involves faulty RNA editing and abnormal activation of specific glutamate receptors as well as failure of glutamate transport resulting in glutamate excitotoxicity; however, the excitotoxicity theory is challenged by the inability of anti-glutamate drugs to have major disease-modifying effects clinically. Nevertheless, hyperexcitability of upper and lower motoneurons is a feature of human ALS and transgenic (tg) mouse models of ALS. Motoneuron excitability is strongly modulated by synaptic inhibition mediated by presynaptic glycinergic and GABAergic innervations and postsynaptic glycine receptors (GlyR) and GABA_A receptors; yet, the integrity of inhibitory systems regulating motoneurons has been understudied in experimental models, despite findings in human ALS suggesting that they may be affected. We have found in tg mice expressing a mutant form of human superoxide dismutase-1 (hSOD1) with a Gly93 → Ala substitution (G93A-hSOD1), causing familial ALS, that subsets of spinal interneurons degenerate. Inhibitory glycinergic innervation of spinal motoneurons becomes deficient before motoneuron degeneration is evident in G93A-hSOD1 mice. Motoneurons in these ALS mice also have insufficient synaptic inhibition as reflected by smaller GlyR currents, smaller GlyR clusters on their plasma membrane, and lower expression of GlyR1α mRNA compared to wild-type motoneurons. In contrast, GABAergic innervation of ALS mouse motoneurons and GABA_A receptor function appear normal. Abnormal synaptic inhibition resulting from dysfunction of interneurons and motoneuron GlyRs is a new direction for unveiling mechanisms of ALS pathogenesis that could be relevant to new therapies for ALS.     

Phosphatidylinositol 3-Kinase: Increased Activity and Protein Level in Amyotrophic Lateral Sclerosis

Abstract: Enzyme activities and protein levels of several protein and lipid kinases were measured in postmortem tissue from patients who died with amyotrophic lateral sclerosis (ALS) as well as from control subjects. Patients who died with ALS had increased activities and protein levels of phosphatidylinositol 3-kinase (PI 3-K) in particulate fractions of spinal cord tissue compared with control subjects. The PI 3-K activity increased with PI 3-K protein level, indicating no change in specific PI 3-K activity in ALS. No differences in PI 3-K activities were found in cytosolic fractions of spinal cord, or in motor and visual cortices, from ALS patients compared with those from controls. PI 3-K activities and protein levels were unchanged in brain tissue from patients who died with Alzheimer's disease compared with those from controls. PI 3-K is a lipid kinase that is important for cell survival and is activated in response to many growth factors. Increased PI 3-K activities in particulate fractions of spinal cord from ALS patients may be related to the increase of PI 3-K protein levels found in this tissue. The protein kinases Erk2, protein kinase B (PKB), and p70 ribosomal S6 kinase (S6K) showed no differences in activities in spinal cord tissue between ALS patients and controls. However, the amounts of PKB and S6K protein were significantly higher in ALS patients, whereas Erk2 protein amount was unchanged compared with controls. Protein kinase C activity was increased in spinal cord tissue from ALS patients, which is consistent with our previous report. The increased activity of PI 3-K in spinal cord tissue from patients with ALS implicates the involvement or activation of PI 3-K in ALS, as either a cause or a consequence of the neuron loss. The lack of up-regulation in the activities of PKB and S6K in ALS tissue supports an impairment in signal transduction cascades mediated by PI 3-K in this neurodegenerative disease.     

Early decrease of survival signal-related proteins in spinal motor neurons of presymptomatic transgenic mice with a mutant SOD1 gene

The mechanisms of motor neuronal death in amyotrophic lateral sclerosis (ALS) remain to be unclear. Phosphatidy-linositol 3-kinase (PI3-K) and its main downstream effector, Akt/protein kinase B (PKB) have been shown to play a central role in neuronal survival against apoptosis supported by neurotrophic factors. In order to investigate a possible impairment of survival signaling, we examined expressions of PI3-K and Akt in the spinal cord of the transgenic mice overexpressing a mutant Cu/Zn superoxide dismutase (SOD1) gene, a valuable model for human ALS. Immunoblotting and immunohistochemical analyses showed that the majority of spinal motor neurons lost the immunoreactivities for both PI3-K and Akt in the early and presymptomatic stage that preceded significant loss of the neurons. The present results suggest that an early decrease of survival signal proteins in the spinal motor neurons may account for the subsequent motor neuronal loss in this animal model of ALS.     

The C‐terminal domain of tetanus toxin protects motoneurons against acute excitotoxic damage on spinal cord organotypic cultures

The C‐terminal domain of tetanus toxin (Hc‐TeTx) has been suggested to act as a neuroprotective agent by activating signaling pathways related to neurotrophins and also to exert anti‐apoptotic effects. Here, we show the beneficial properties of the recombinant protein Hc‐TeTx to protect spinal motoneurons against excitotoxic damage. In vitro spinal cord organotypic cultures were used to assess acute glutamate excitotoxic damage. Our results indicate that Hc‐TeTx treatment improves motoneuron survival within a short therapeutical window (the first 2 h post‐injury). Within this interval, we found that p44/p42 MAP kinase (ERK1/2) and glycogen synthase kinase‐3 (GSK3β) signaling pathways play a crucial role in the neuroprotective effect. Moreover, we demonstrated that Hc–TeTx treatment initiate autophagy which is ERK1/2‐ and GSK3β‐dependent. These findings suggest a possible therapeutical tool to improve motoneuron survival immediately after excitotoxic insults or during the secondary injury phase that occurs after spinal cord trauma.     

PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis

Vascular endothelial growth factor (VEGF), a specific pro-angiogenic peptide, has shown neuroprotective effects in the Parkinson’s disease (PD) models, but the underlying mechanisms remain elusive. In this study, the neuroprotective properties of VEGF on 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced neurotoxicity in primary cerebellar granule neurons were investigated. Pretreatment of VEGF prevented MPP⁺-induced neuronal apoptosis in a concentration- and time-dependent manner. And this prevention was blocked by PTK787/ZK222584, a VEGF receptor-2 specific inhibitor. Both inhibition of the Akt pathway and activation of the extracellular signal-regulated kinase (ERK) pathway contribute to MPP⁺-induced neuronal apoptosis. VEGF reversed the inhibition of phosphoinositide 3-kinase (PI3-K)/Akt pathway caused by MPP⁺, but further enhanced the activation of ERK induced by MPP⁺. Interestingly, VEGF and PD98059 (an ERK kinase inhibitor) play a synergistic role in protecting neurons from MPP⁺-induced toxicity. Collectively, these findings suggest that the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP⁺-induced neuronal apoptosis. This finding offers not only a new and clinically significant modality as to how VEGF exerts its neuroprotective effects but also a novel therapeutic strategy for PD by differentially regulating PD-associated signaling pathways.     

Activation of Phosphatidylinositol 3-Kinase, but Not Extracellular-Regulated Kinases, Is Necessary to Mediate Brain-Derived Neurotrophic Factor-Induced Motoneuron Survival

Chick embryo spinal cord motoneurons develop a trophic response to some neurotrophins when they are maintained in culture in the presence of muscle extract. Thus, after 2 days in culture, brain-derived neurotrophic factor (BDNF) promotes motoneuron survival. In the present study we have analyzed the intracellular pathways that may be involved in the BDNF-induced motoneuron survival. We have observed that BDNF activated the extracellular-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and the phosphatidylinositol (PI) 3-kinase pathways. To examine the contribution of these pathways to the survival effect triggered by BDNF, we used PD 98059, a specific inhibitor of MAP kinase kinase, and LY 294002, a selective inhibitor of PI 3-kinase. PD 98059, at doses that significantly reduced the phosphorylation of ERKs, did not show any prominent effect on neuronal survival. However, LY 294002 at doses that inhibited the phosphorylation of Akt, a down-stream element of the PI 3-kinase, completely abolished the motoneuron survival effects of BDNF. Moreover, cell death triggered by LY 294002 treatment exhibited features similar to those observed after muscle extract deprivation. Our results suggest that the PI 3-kinase pathway plays an important role in the survival effect triggered by BDNF on motoneurons, whereas activation of the ERK MAP kinase pathway is not relevant.     

Alzheimer's disease: synaptic dysfunction and Aβ

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause, characterized by the selective and progressive death of both upper and lower motoneurons, leading to a progressive paralysis. Experimental animal models of the disease may provide knowledge of the pathophysiological mechanisms and allow the design and testing of therapeutic strategies, provided that they mimic as close as possible the symptoms and temporal progression of the human disease. The principal hypotheses proposed to explain the mechanisms of motoneuron degeneration have been studied mostly in models in vitro, such as primary cultures of fetal motoneurons, organotypic cultures of spinal cord sections from postnatal rodents and the motoneuron-like hybridoma cell line NSC-34. However, these models are flawed in the sense that they do not allow a direct correlation between motoneuron death and its physical consequences like paralysis. In vivo, the most widely used model is the transgenic mouse that bears a human mutant superoxide dismutase 1, the only known cause of ALS. The major disadvantage of this model is that it represents about 2%–3% of human ALS. In addition, there is a growing concern on the accuracy of these transgenic models and the extrapolations of the findings made in these animals to the clinics. Models of spontaneous motoneuron disease, like the wobbler and pmn mice, have been used aiming to understand the basic cellular mechanisms of motoneuron diseases, but these abnormalities are probably different from those occurring in ALS. Therefore, the design and testing of in vivo models of sporadic ALS, which accounts for >90% of the disease, is necessary. The main models of this type are based on the excitotoxic death of spinal motoneurons and might be useful even when there is no definitive demonstration that excitotoxicity is a cause of human ALS. Despite their difficulties, these models offer the best possibility to establish valid correlations between cellular alterations and motor behavior, although improvements are still necessary in order to produce a reliable and integrative model that accurately reproduces the cellular mechanisms of motoneuron degeneration in ALS.     

Vascular endothelial growth factor prevents G93A‐SOD1‐induced motor neuron degeneration

Amyotrophic lateral sclerosis (ALS) is an adult‐onset neurodegenerative disorder characterized by selective loss of motor neurons (MNs). Twenty percent of familial ALS cases are associated with mutations in Cu²⁺/Zn²⁺ superoxide dismutase (SOD1). To specifically understand the cellular mechanisms underlying mutant SOD1 toxicity, we have established an in vitro model of ALS using rat primary MN cultures transfected with an adenoviral vector encoding a mutant SOD1, G93A‐SOD1. Transfected cells undergo axonal degeneration and alterations in biochemical responses characteristic of cell death such as activation of caspase‐3. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can increase axonal outgrowth, block neuronal apoptosis, and promote neurogenesis. Decreased VEGF gene expression in mice results in a phenotype similar to that seen in patients with ALS, thus linking loss of VEGF to the pathogenesis of MN degeneration. Decreased neurotrophic signals prior to and during disease progression may increase MN susceptibility to mutant SOD1‐induced toxicity. In this study, we demonstrate a decrease in VEGF and VEGFR2 levels in the spinal cord of G93A‐SOD1 ALS mice. Furthermore, in isolated MN cultures, VEGF alleviates the effects of G93A‐SOD1 toxicity and neuroprotection involves phosphatidylinositol 3‐kinase/protein kinase B (PI3K/Akt) signaling. Overall, these studies validate the usefulness of VEGF as a potential therapeutic factor for the treatment of ALS and give valuable insight into the responsible signaling pathways and mechanisms involved. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

The Metabolic Disturbances of Motoneurons Exposed to Glutamate

Glutamate-induced excitotoxicity is considered as one of the major pathophysiological factors of motoneuron death in amyotrophic lateral sclerosis and other motoneuron diseases. In order to expand our knowledge on mechanisms of glutamate-induced excitotoxicity, the present study proposes to determine the metabolic consequences of glutamate and astrocytes in primary enriched motoneuron culture. Using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), we showed that the presence of astrocytes and glutamate profoundly modified the metabolic profile of motoneurons. Our study highlights for the first time that crosstalk between astrocytes and enriched motoneuron culture induced alterations in phenylalanine, tryptophan, purine, arginine, proline, aspartate, and glutamate metabolism in motoneurons. We observed that astrocytes modulate the sensitivity of motoneurons to glutamate, since metabolites altered by glutamate in motoneurons cultured alone were different (except 5-hydroxylysine) from those altered in co-cultured motoneurons. Our findings provide new insight into the metabolic alterations associated to astrocytes and glutamate in motoneurons and provide opportunities to identify novel therapeutic targets.     

Apelin deficiency accelerates the progression of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Recent studies have implicated that chronic hypoxia and insufficient vascular endothelial growth factor (VEGF)-dependent neuroprotection may lead to the degeneration of motor neurons in ALS. Expression of apelin, an endogenous ligand for the G protein-coupled receptor APJ, is regulated by hypoxia. In addition, recent reports suggest that apelin protects neurons against glutamate-induced excitotoxicity. Here, we examined whether apelin is an endogenous neuroprotective factor using SOD1(G93A) mouse model of ALS. In mouse CNS tissues, the highest expressions of both apelin and APJ mRNAs were detected in spinal cord. APJ immunoreactivity was observed in neuronal cell bodies located in gray matter of spinal cord. Although apelin mRNA expression in the spinal cord of wild-type mice was not changed from 4 to 18 weeks age, that of SOD1(G93A) mice was reduced along with the paralytic phenotype. In addition, double mutant apelin-deficient and SOD1(G93A) displayed the disease phenotypes earlier than SOD1(G93A) littermates. Immunohistochemical observation revealed that the number of motor neurons was decreased and microglia were activated in the spinal cord of the double mutant mice, indicating that apelin deficiency pathologically accelerated the progression of ALS. Furthermore, we showed that apelin enhanced the protective effect of VEGF on H(2)O(2)-induced neuronal death in primary neurons. These results suggest that apelin/APJ system in the spinal cord has a neuroprotective effect against the pathogenesis of ALS.     

Exogenous testosterone prevents motoneuron atrophy induced by contralateral motoneuron depletion

Gonadal steroids exhibit neuroprotective and neurotherapeutic effects. The lumbar spinal cord of male rats contains a highly androgen-sensitive population of motoneurons, the spinal nucleus of the bulbocavernosus (SNB), whose morphology and function are dependent on testosterone in adulthood. Unilateral SNB motoneuron depletion induces dendritic atrophy in contralateral SNB motoneurons, but this atrophy is reversed in previously castrated males treated with testosterone. In the present experiment we test the hypothesis that the morphology of SNB motoneurons is protected from atrophy after contralateral motoneuron depletion by exogenous testosterone alone (i.e., with no delay between castration and testosterone replacement). We unilaterally depleted SNB motoneurons by intramuscular injection of cholera toxin conjugated saporin. Simultaneously, some saporin-injected rats were castrated and immediately given replacement testosterone. Four weeks later, contralateral SNB motoneurons were labeled with cholera toxin conjugated HRP, soma sizes were measured, and dendritic arbors were reconstructed. Contralateral SNB motoneuron depletion induced somal atrophy and dendritic retraction, but testosterone treatment prevented both of these effects. Thus, the presence of high-normal levels of testosterone prevents motoneuron atrophy induced by contralateral motoneuron depletion. These data support a therapeutic role for testosterone in preventing atrophy induced by motoneuron injury.     

Lectin from Canavalia brasiliensis (ConBr) protects hippocampal slices against glutamate neurotoxicity in a manner dependent of PI3K/Akt pathway

The excitotoxicity induced by excessive activation of the glutamatergic neurotransmission pathway is involved in several neuropathologies. In this sense, molecules that prevent the release of glutamate or the excessive activation of its receptors can be useful in preventing the neuronal cell death observed in these diseases. Lectins are proteins capable of reversible binding to the carbohydrates in glycoconjugates, and some have been used in the study and purification of glutamate receptors. ConBr is a mannose/glucose-binding lectin purified from Canavalia brasiliensis seeds. In the present study, we aimed to evaluate the neuroprotective activity of ConBr against glutamate-induced excitotoxicity. Hippocampal slices were isolated from adult male mice and incubated for 6 h in Krebs saline/DMEM buffer alone (control), in the presence of glutamate or glutamate plus ConBr. The phosphorylation of Akt and mitogen activated protein kinases (MAPKs) such as ERK1/2, p38^MAPK and JNK1/2/3 was evaluated with western blotting. The results indicate that glutamate provoked a reduction in the hippocampal slice viability (−25%), diminished the phosphorylation of Akt and augmented p38^MAPK and ERK1 phosphorylation. No changes were observed in the phosphorylation of JNK1/2/3 or ERK2. Notably, ConBr, through a mechanism dependent on carbohydrate interaction, prevented the reduction of cell viability and Akt phosphorylation induced by glutamate. Furthermore, in the presence of the PI3K inhibitor LY294002, ConBr was unable to reverse glutamate neurotoxicity. Taken together, our data suggest that the neuroprotective effect of ConBr against glutamate neurotoxicity requires oligosaccharide interaction and is dependent on the PI3K/Akt pathway.     

Survival-promoting activity of pituitary adenylate cyclase-activating polypeptide in the presence of phosphodiesterase inhibitors on rat motoneurons in culture: cAMP-protein kinase A-mediated survival

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to be neurotrophic or neuroprotective in various neurons in culture. It is expressed in spinal motoneurons in vivo and its expression is increased markedly after axotomy, suggesting a neuroprotective role via an autocrine mechanism. However, neurotrophic activity of PACAP has not been reported for motoneurons. In the present study, we investigated the effects of PACAP on rat motoneurons in culture. In the presence of a phosphodiesterase inhibitor, PACAP showed significant neurotrophic activity at concentrations as low as 0.01 nM. Previously, we found that glutamate was excitotoxic to motoneurons even in the presence of brain-derived neurotrophic factor, which is neurotrophic for motoneurons. PACAP with a phosphodiesterase inhibitor protected motoneurons against this excitotoxicity. The activity of PACAP was inhibited by the protein kinase A inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, as was the case with the activity of forskolin, suggesting downstream involvement of a cAMP-protein kinase A signaling pathway. The present results may suggest a physiological role of PACAP in vivo, and implicate the PACAP-cAMP signaling pathway for the possible therapeutic target of amyotrophic lateral sclerosis as glutamate excitotoxicity was suggested in sporadic amyotrophic lateral sclerosis.     

Cellular basis of steroid neuroprotection in the wobbler mouse, a genetic model of motoneuron disease

1. The Wobbler mouse suffers an autosomal recessive mutation producing severe motoneuron degeneration and astrogliosis in the spinal cord. It has been considered a suitable model of human motoneuron disease, including the sporadic form of amyotrophic lateral sclerosis (ALS).2. Evidences exist demonstrating increased oxidative stress in the spinal cord of Wobbler mice, whereas antioxidant therapy delayed neurodegeneration and improved muscle trophism. 21-Aminosteroids are glucocorticoid-derived hydrophobic compounds with antioxidant potency 3 times higher than vitamin E and 100 times higher than methylprednisolone. They do not bind to intracellular receptors, and prevent lipid peroxidation by insertion into membrane lipid bilayers.3. In common with the spinal cord of ALS patients, Wobbler mice present astrocytosis with hyperexpression of glial fibrillary acidic protein (GFAP), and increased expression of nitric oxide synthase (NOS) and growth-associated protein (GAP-43) in motoneurons. Here, we review our studies on the effects of a 21-aminosteroid on GFAP, NOS, and GAP-43.4. First, we showed that 21-aminosteroid treatment further increased GFAP-expressing astrocytes in gray matter of the Wobbler spinal cord. This effect may provide neuroprotection if one considers a trophic and beneficial function of astrocytes during the course of degeneration. Other neuroprotectans used in Wobbler mice (T-588) also increased preexisting astrocytosis.5. Second, histochemical determination of NADPH-diaphorase, a parameter indicative of neuronal NOS activity, showed that the 21-aminosteroid down-regulated the high activity of this enzyme in ventral horn motoneurons. Therefore, suppression of nitric oxide by decreasing NADPH-diaphorase (NOS) activity may provide neuroprotection considering that excess NO is highly toxic to motoneurons.6. Finally, 21-aminosteroid treatment significantly attenuated the aberrant expression of both GAP-43 protein and mRNA in Wobbler motoneurons. Hyperexpression of GAP-43 possibly indicated abnormal synaptogenesis, denervation, and muscle atrophy, parameters which may return to normal following antioxidant steroid treatment.7. Besides 21-aminosteroids, other steroids also behave as neuroprotectans. In this regard, degenerative diseases may constitute potential targets of these hormones, based on the fact that the spinal cord expresses in a regional and cell-specific fashion, receptors for androgens, progesterone, adrenal steroids, and estrogens.