Neuroprotective effects of an adenoviral vector expressing the glucose transporter: a detailed description of the mediating cellular events

Considerable knowledge exists concerning the events mediating neuron death following a necrotic insult; prompted by this, there have now been successful attempts to use gene therapy approaches to protect neurons from such necrotic injury. In many such studies, however, it is not clear what sequence of cellular events connects the overexpression of the transgene with the enhanced survival. We do so, exploring the effects of overexpressing the Glut-1 glucose transporter with an adenoviral vector in hippocampal cultures challenged with the excitotoxin kainic acid (KA). Such overexpression enhanced glucose transport, attenuated the decline in ATP concentrations, decreased the release of excitatory amino acid neurotransmitters, and decreased the total free cytosolic calcium load. Commensurate with these salutary effects, neuronal survival was enhanced with this gene therapy intervention. Thus, the neuroprotective effects of this particular gene therapy occurs within the known framework of the mechanisms of necrotic neuronal injury.     

Cystamine protects from 3-nitropropionic acid lesioning via induction of nf-e2 related factor 2 mediated transcription

Systemic administration of cystamine is known to protect from both chemical and genetic models of neurotoxicity. Despite positive effects in laboratory models, cystamine has not been successfully translated to clinical application for neurodegenerative disease. Furthermore, the long held assumption that cystamine protects through tissue transglutaminase inhibition has recently been challenged. The studies described here examine other potential mechanisms of cystamine-mediated protection in an attempt to reveal molecular targets for neurodegenerative therapy. Based on previously described effects of cystamine, we examined the potential for activation of NF-E2 related factor 2 (Nrf2) mediated signaling through the antioxidant response element (ARE). We found that cystamine activates Nrf2/ARE both in cell culture and in brain tissue and then probed the mechanism of activation in cell culture. In live animals, we show that neuroprotection from 3-nitropropionic acid (3NP) toxicity is Nrf2-dependent. Therefore, these findings provide strong evidence that Nrf2 signaling may be an effective target for prevention of neurodegeneration.     

BDNF regulates BIM expression levels in 3-nitropropionic acid-treated cortical neurons

3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase that has been used to explore the primary mechanisms of cell death associated with mitochondrial dysfunction and neurodegeneration in Huntington's disease. In this study we investigated the ability of brain-derived neurotrophic factor (BDNF) to suppress mitochondrial-dependent cell death induced by 3-NP in primary cortical neurons. This neurotrophin prevented 3-NP-induced release of cytochrome c and Smac/Diablo, caspase-3-like activity and nuclear condensation/fragmentation. Furthermore, it greatly increased phosphorylation of Akt and MAPK, suggesting the involvement of these signalling pathways in BDNF neuroprotection. Interestingly, BDNF decreased the levels of the pro-apoptotic protein Bim in mitochondrial and total cell lysates through the activation of the MEK1/2 pathway. This effect was due to an increase in the degradation rates of Bim. Our data support an important role for BDNF, in protecting cortical neurons against apoptotic cell death caused by inhibition of mitochondrial complex II.     

3-nitropropionic acid induced in vivo protein oxidation in striatal and cortical synaptosomes: insights into Huntington's disease

3-nitropropionic acid (3-NP) administered systemically daily for 4 days to rats inhibits mitochondrial oxidative phosphorylation and induces selective lesions in the striatum in a manner reminiscent of Huntington's disease (HD). To investigate the potential oxidative nature of these lesions, rats were injected with 3-NP (20 mg/kg, i.p. daily for 4 days) and subsequently isolated brain synaptosomal membranes were examined for evidence of oxidative stress. Brain synaptosomal membrane proteins from rats injected with 3-NP exhibited a decreased in W/S ratio, the relevant electron paramagnetic resonance (EPR) parameter used to determine levels of protein oxidation (76% of control), and Western blot analysis for protein carbonyls revealed direct evidence of increased synaptosomal membrane protein oxidation (248% of control). Similar results were obtained in synaptosomes isolated from striatum and from cerebral cortex, demonstrating that the oxidative changes are not restricted to the lesion site. Moreover, increased oxidative stress was evident prior to the appearance of morphological lesions. These data are consistent with the hypothesis that 3-NP-induced striatal lesions, and perhaps those in HD, are associated with oxidative processes.     

Neurotoxicological effects of 3-nitropropionic acid on the neonatal rat

An increasing amount of data provides support for the hypothesis that periventricular leukomalacia (PVL) results from pre- or perinatal hypoxia occurring and is a major cause of cerebral palsy. In this work, anoxic and hypoxic–ischemic brain injuries were observed by us, after injection of neurotoxin 3-nitropropionic acid (3-NP) in a neonatal rat model on postnatal day 5 (P5). 3-NP-induced brain injury was examined in fixed brain sections at 24 h (P6), 48 h (P7), 72 h (P8), and 9 days (P14) after 3-NP injection, respectively. Injection with 3-NP results in pathological injuries including white matter lesions, cerebral cortex destruction, callose thinness, and cerebral ventricle expansion. Numbers of immature oligodendrocytes turned to less in the model of 3-NP. Furthermore myeline basic protein expression became significantly lower after 3-NP was injected. Pathological changes after injection of 3-NP appeared also significantly among rats of postnatal day 5. The effect of the 3-NP neurotoxicity paradigm was evaluated in this study to further investigate the underlying pathology associated with PVL, which may yield a potential desirable model for clinic experiments.     

Chemical preconditioning with 3-nitropropionic acid: lack of induction of neuronal tolerance in gerbil hippocampus subjected to transient forebrain ischemia

Chemical preconditioning using the mitochondrial toxin, 3-nitropropionic acid (3-NP) has been reported to induce neuroprotection against subsequent global ischemia. To investigate the underlying mechanisms, Mongolian gerbils were pretreated with either vehicle or 3-NP at the dose of 3 or 10 mg/kg, intraperitoneal, 3 days prior to a 5-min bilateral carotid artery occlusion followed by either 48 h or 7 days of blood recirculation. Neuronal damage was assessed by a cresyl violet/fuchsin acid staining. Induction of heat shock protein 72 (HSP72) and manganese superoxide dismutase (MnSOD) expression was evaluated by Western blotting. Astroglial and microglial activation was detected by immunohistochemistry (glial fibrillary acid protein) and by histochemistry (isolectin B4 staining), respectively. Present data show that the hippocampal neuronal damage induced by ischemia were of similar extent between the vehicle- and 3-NP-treated gerbils, whatever the dose tested, indicating that 3-NP did not induce tolerance to transient forebrain ischemia under our experimental conditions. The lack of difference in the post-ischemic level of HSP72 and MnSOD protein expression and in the intensity of astroglial and microglial activation represents further indirect indications of the absence of 3-NP preconditioning effect. In conclusion, although chemical preconditioning with 3-NP is a well-established phenomenon at least in vitro and in models of focal ischemia, the relevance of 3-NP as a preconditioning molecule towards global brain ischemia remains an open question.     

A bile acid protects against motor and cognitive deficits and reduces striatal degeneration in the 3-nitropropionic acid model of Huntington's disease

There is currently no effective treatment for Huntington's disease (HD), a progressive, fatal, neurodegenerative disorder characterized by motor and cognitive deterioration. It is well established that HD is associated with perturbation of mitochondrial energy metabolism. Tauroursodeoxycholic acid (TUDCA), a naturally occurring bile acid, can stabilize the mitochondrial membrane, inhibit the mitochondrial permeability transition, decrease free radical formation, and derail apoptotic pathways. Here we report that TUDCA significantly reduced 3-nitropropionic acid (3-NP)-mediated striatal neuronal cell death in cell culture. In addition, rats treated with TUDCA exhibited an 80% reduction in apoptosis and in lesion volumes associated with 3-NP administration. Moreover, rats which received a combination of TUDCA + 3-NP exhibited sensorimotor and cognitive task performance that was indistinguishable from that of controls, and this effect persisted at least 6 months. Bile acids have traditionally been used as therapeutic agents for certain liver diseases. This is the first demonstration, however, that a bile acid can be delivered to the brain and function as a neuroprotectant and thus may offer potential therapeutic benefit in the treatment of certain neurodegenerative diseases.     

Parenterally administered 3-nitropropionic acid and amphetamine can combine to produce damage to terminals and cell bodies in the striatum

The combined effects of amphetamine (AMPH) and 3-nitropropionic acid (3-NPA) were investigated to determine how the energy depletion proposed to be produced by AMPH interacts with an inhibitor of mitochondrial respiration to produce striatal neurotoxicity. Neither two doses (2 h apart) of 3.75 mg/kg AMPH alone nor a single dose of 30 mg/kg 3-NPA i.p. produced neurotoxicity in the striatum or lowered striatal dopamine content in rat. Administration of 40 mg/kg of 3-NPA alone almost invariably produced either lethality or did not produce neurotoxicity in the striatum of surviving animals. However, 30 mg/kg of 3-NPA administered along with 2 doses of 3.75 mg/kg AMPH to 47 animals produced striatal damage in the 31 survivors with 15 of the surviving rats showing muscle rigidity /catatonia for several days after dosing, along with decreased food consumption. Thirteen of these 15 rats showed degeneration of axons and cell bodies in the medial caudate-putamen with minimal damage to the globus pallidus. However, two rats exhibited hindlimb paralysis and signs of axonal and neuronal soma degeneration in the thalamus and cerebellar nuclei as well as striatum. Sixteen of the rats given both AMPH and 3-NPA exhibited only torpidity and loss of muscle tone 1–3 h after dosing. Such rats showed no signs of neuronal cell degeneration in the striatum, but did show significant dopamine depletions (60% of control) and reductions in tyrosine hydroxylase immunoreactivity at 14 days postexposure. The mitochondrial dysfunction produced by 3-NPA combined with activation of neuronal pathways by AMPH may have predisposed terminals, axons and cell bodies in striatum to degeneration.     

Forskolin attenuates the evoked release of [H]GABA from striatal neurons in primary culture

The actions of the diterpene forskolin, and cyclic AMP analogues, on the evoked release of [³H]GABA (γ-aminobutyric acid) was examined in intact striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to forskolin (100 μM) resulted in a 40–55% attenuation of [³H]GABA release evoked by either KCl (30 mM) or veratrine (2 μg/ml), while baseline levels of release were unaffected. The dose-dependence for forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable identical to the dose-dependent elevation of cyclic AMP levels by forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable analogues of cyclic AMP, such as p-chlorophenylthio cyclic AMP (0.5 mM) and dibutyryl cyclic AMP (1 mM), resulted in a 25 and 26% attenuation of [³H]GABA release, respectively; dibutyryl cyclic GMP (1 mM) was without effect. The similarity between the actions of forskolin and the cyclic AMP analogues suggests that, in striatal neurons in primary culture, the elevation of cyclic AMP levels results in the attenuation of the evoked release of [³H]GABA. The greater effectiveness of forskolin, compared to the cyclic AMP analogues, may be related to the recently reported, additional direct actions of forskolin on neuronal membrane ion channels.     

Cell cycle activation in striatal neurons from Huntington's disease patients and rats treated with 3-nitropropionic acid

This study was undertaken to investigate the potential role of cell cycle re-entry in an experimental model of Huntington's disease and in human brain samples. We found that after treatment of rats with the mitochondrial neurotoxin 3-nitropropionic acid, the expression of cell cycle markers of G1 phase measured by immunohistochemistry was induced in the striatal brain region. Furthermore, we detected an increase in the nuclear and also cytoplasmatic E2F-1 expression, suggesting that this protein could activate the apoptotic cascade in rat brain. Western blot analysis of post-mortem brain samples from patients also showed an increase in the expression of E2F-1 and cyclin D1 in comparison with control samples. These results indicate that cell cycle re-entry is activated in Huntington's disease and may contribute to the neurodegenerative process.     

NMR spectroscopy study of the effect of 3-nitropropionic acid on glutamate metabolism in cultured astrocytes

3-Nitropropionic acid (3-NPA) is a selective and irreversible inhibitor of succinate dehydrogenase. The effect of this compound on the metabolism of [U-¹³C]glutamate was studied in astrocytes using ¹³C nuclear magnetic resonance spectroscopy. The appearance of [1,2,3-¹³C]glutamate in cell extracts and [1,2,3-¹³C]glutamine and [U-¹³C]lactate in cell media demonstrated the metabolism of labeled glutamate via the tricarboxylic acid cycle. Such labeling was observed in the control situation and also in cells treated with 3 mM 3-NPA. In the cells treated with 3 mM 3-NPA, however, the labeling was significantly reduced, and with 10 mM 3-NPA no such labeling was observed. Labeled aspartate was observed in untreated cells only. Labeled succinate was not detectable under control conditions, but increased dose dependently in the presence of 3-NPA. Glutamate uptake and conversion of [U-¹³C]glutamate to U-¹³C]glutamine was largely unaffected by 3-NPA, and ATP content was unchanged. In a previous study using cerebellar neurons, tricarboxylic acid cycle metabolism was blocked with 3 mM 3-NPA. The present results show that astrocyte metabolism is more adaptable to blockade of the tricarboxylic acid cycle by 3-NPA than neuronal metabolism. J. Neurosci. Res. 47:650–654, 1997. © 1997 Wiley-Liss, Inc.     

Differential regulation of 5' splice variants of the glutamate transporter EAAT2 in an in vivo model of chemical hypoxia induced by 3-nitropropionic acid

Defective glutamate uptake has been implicated as a pathogenic event of neuronal damage related to cerebral ischemia and hypoxia. In several models of ischemia-hypoxia, a reduced immunoreactivity and altered RNA expression of excitatory amino acid transporter 2 (EAAT2), the major excitatory amino acid transporter, have been reported. However, the gene regulation of EAAT2 under these conditions is incompletely understood. In this study, we investigated alternative splicing of EAAT2 in an in vivo mouse model of chemical hypoxia as induced by 3-nitropropionic acid (3-NP). The neurotoxin 3-NP is an inhibitor of mitochondrial energy production. Furthermore, it is known to inhibit glutamate reuptake directly, representing at least one of the mechanisms responsible for 3-NP-induced neurodegeneration. Here we report an expression analysis of five known (mEAAT2/5UT1-5) and two novel (mEAAT2/5UT6, -7) 5' splice variants of EAAT2 using semiquantitative PCR. The RNA expression was studied at 2, 12, 24, 48, and 72 hr and 7 days after 3-NP administration. mEAAT2/5UT4 and mEAAT2/5UT5 were up-regulated in the frontal cortex and down-regulated in the hippocampus 12-72 hr after chemical hypoxia. In the cerebellum, there was an increased expression of mEAAT2/5UT4 and a down-regulation of mEAAT2/5UT5. mEAAT2/5UT3 show a different regional expression pattern, being regulated in the cerebellum only. mEAAT2/5UT1-7 encoded distinct 5' regulatory sequences, including conserved elements of translational control. It is easily conceivable that expression alterations of 5' splice variants of EAAT2 are related to glutamate transporter malfunction after chemical hypoxia. Our findings contribute to the hypothesis that RNA splicing events can serve as a molecular mechanism of posthypoxic gene regulation.     

S-100β protects cultured neurons against glutamate- and staurosporine-induced damage and is involved in the antiapoptotic action of the 5 HT1A-receptor agonist, Bay x 3702

The serotonin (5-HT)_1A receptor agonists have already been shown to protect cultured neurons from excitotoxic as well as from apoptotic damage [B. Ahlemeyer, J. Krieglstein, Stimulation of 5-HT_1A receptors inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo, Brain Res. 777 (1997) 179–186.; B. Ahlemeyer, A. Glaser, C. Schaper, I. Semkova, J. Krieglstein, The 5-HT_1A receptor agonist, Bay x 3702, inhibited apoptosis induced by serum deprivation in cultured neurons, Eur. J. Pharmacol. 370 (1999) 211–216.; J.H.M. Prehn, M. Welsch, C. Backhauß, J. Nuglisch, F. Ausmeier, C. Karkoutly, J. Krieglstein, Effects of serotonergic drugs in experimental brain ischemia: evidence for a protective role of serotonin in cerebral ischemia, Brain Res. 630 (1993) 110–120.; I. Semkova, P. Wolz, J. Krieglstein, Neuroprotective effect of 5-HT_1A receptor agonist, Bay x 3702, demonstrated in vitro and in vivo, Eur. J. Pharmacol. 359 (1998) 251–260.; B. Suchanek, H. Struppeck, T. Fahrig, The 5-HT_1A receptor agonist, Bay x 3702, prevents staurosporine-induced apoptosis, Eur. J. Pharmacol. 355 (1998) 95–101.] and to increase the release of the neurotrophic protein, S-100β [P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, Stimulation of astroglial 5-HT_1A receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology, Brain Res. 497 (1989) 80–86.; P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, S-100 protein is released from astroglial cells by stimulation of 5-HT_1A receptors, Brain Res. 528 (1990) 155–158.]. In this study, we tried to find out whether S-100β can protect cultured neurons from glutamate- and staurosporine-induced damage and whether the neuroprotective activity of the highly selective 5-HT_1A receptor agonist, Bay x 3702, is mediated by an induction of S-100β. Extracellularly added S-100β (1–10 ng/ml) reduced staurosporine-induced damage in pure neuronal cultures from chick embryo telencephalon as well as in mixed neuronal/glial cultures from neonatal rat hippocampus. In addition, S-100β (1 ng/ml) reduced neuronal death induced by exposure to glutamate (0.25 mM, 30 min) in mixed neuronal/glial cultures from neonatal rat hippocampus. In cultured rat cortical astrocytes, a 24 h-treatment with Bay x 3702 (1 nM) increased the S-100β content in the culture medium from 2.2±0.3 (controls) to 6.2±0.7 ng/ml. In the adult rat, a 4 h-infusion of 4 μg/kg Bay x 3702 (i.v.) was found to increase the S-100β content in the striatum 6 h after the beginning of the infusion to 153±37 μg/g compared with 60±20 μg/g in vehicle-treated rats. Bay x 3702 had no effect on the S-100β content in the rat hippocampus. Finally, we tried to block the protective effect of Bay x 3702 against staurosporine-induced damage in mixed neuronal/glial cultures from rat neonatal hippocampus by anti-S-100β antibodies. We found only a partial blockade, although the antibodies fully blocked the antiapoptotic effect of S-100β itself demonstrating that the antibody was effective in blocking neuroprotection by S-100β. Thus, we conclude that S-100β was able to protect cultured neurons against glutamate- and staurosporine-induced damage. Furthermore, S-100β mediated partially the protective effect of the 5-HT_1A receptor agonist, Bay x 3702, against staurosporine-induced apoptosis in mixed neuronal/glial cultures from neonatal rat hippocampus.     

The new indirubin derivative inhibitors of glycogen synthase kinase-3, 6-BIDECO and 6-BIMYEO, prevent tau phosphorylation and apoptosis induced by the inhibition of protein phosphatase-2A by okadaic acid in cultured neurons

Alterations in glycogen synthase kinase‐3β (GSK3β) and protein phosphatase‐2A (PP2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation linked to Alzheimer's disease (AD). Interconnections between GSK3β and PP2A signaling pathways are well established. Targeting tau kinases was proposed to represent a therapeutic strategy for AD. However, which tau kinases should be blocked and to what extent, keeping in mind that kinases have physiological roles? Because most kinase inhibitors are relatively specific and many of them interfere with the cell cycle, it is necessary to develop more specific tau kinase inhibitors devoid of cell toxicity. Here, we used the PP2A inhibition by okadaic acid (OKA) in primary cultured cortical neurons as an in vitro model of increased tau phosphorylation and apoptosis. We tested the effects of two newly characterized indirubin derivative inhibitors of GSK3, 6‐BIDECO (6‐bromoindirubin‐3′‐[O‐(N,N‐diethylcarbamyl)‐oxime] and 6‐BIMYEO (6‐bromoindirubin‐3′‐[O‐(2‐morpholin‐1‐ylethyl)‐oxime] hydrochloride) on OKA‐induced tau phosphorylation and neuronal apoptosis. Both compounds exhibit higher selectivity toward GSK3 compared with other tau kinases (for 6‐BIDECO, IC50 is 0.03 μM for GSK3, >10 μM for CDK1, and 10 μM for CDK5; for 6‐BIMYEO, IC50 is 0.11 μM for GSK3, 1.8 μM for CDK1, and 0.9 μM for CDK5). We show that 6‐BIDECO and 6‐BIMYEO used at micromolar concentrations are not neurotoxic and potently reversed tau phosphorylation and apoptosis induced by OKA. The neuroprotection by these compounds should be further validated in animal models of AD. © 2011 Wiley‐Liss, Inc.     

Delayed grafting of BDNF and NT-3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration

Ex vivo gene therapy, utilizing modified fibroblasts that deliver BDNF or NT-3 to the acutely injured spinal cord, has been shown to elicit regeneration and recovery of function in the adult rat. Delayed grafting into the injured spinal cord is of great clinical interest as a model for treatment of chronic injury but may pose additional obstacles that are not present after acute injury, such as the need to remove an established scar, increased retrograde cell loss and/or atrophy, and diminished capacity for regeneration by neurons which may be doubly injured. The purpose of the present study was to determine if delayed grafting of neurotrophin secreting fibroblasts would have anatomical effects similar to those seen in acute grafting models. We grafted a mixture of BDNF and NT-3 producing fibroblasts or control fibroblasts into a complete unilateral cervical hemisection after a 6-week delay. Fourteen weeks after delayed grafting we found that both the neurotrophin secreting fibroblasts and control fibroblasts survived, but that only the neurotrophin secreting grafts provided a permissive environment for host axon growth, as indicated by immunostaining for RT-97, a marker for axonal neurofilaments, GAP-43, a marker for elongating axons, CGRP, a marker for dorsal root axons, and 5-HT, a marker for raphe spinal axons, within the graft. Anterograde tracing of the uninjured vestibulospinal tract showed growth into neurotrophin producing transplants but not into control grafts, while anterograde tracing of the axotomized rubrospinal tract showed a small number of regenerating axons within the genetically modified grafts, but none in control grafts. The neurotrophin expressing grafts, but not the control grafts, significantly reduced retrograde degeneration and atrophy in the injured red nucleus. Grafts of BDNF + NT-3 expressing fibroblasts delayed 6 weeks after injury therefore elicit growth from intact segmental and descending spinal tracts, stimulate modest regenerative growth by rubrospinal axons, and partially rescue axotomized supraspinal neurons and protect them from atrophy. The regeneration of rubrospinal axons into delayed transplants was much less than has been observed when similar transplants were placed acutely into a lateral funiculus or, after a 4-week delay, into a hemisection lesion. This suggests that the regenerative capacity of chronically injured red nucleus neurons was markedly diminished. The increased GAP43 reactivity in the corticospinal tracts ipsilaterally and contralaterally to the combination grafts suggests that these axons remain responsive to the neurotrophins, that the neurotrophins may stimulate both regenerative and sprouting responses, and that the grafted cells continue to secrete the neurotrophins.