Down-regulation of GRK2 after oxygen and glucose deprivation in rat hippocampal slices: role of the PI3-kinase pathway.

G protein-coupled receptor kinase 2 (GRK2) modulates G protein-coupled receptor desensitization and signaling. We previously described down-regulation of GRK2 expression in vivo in rat neonatal brain following hypoxia-ischemia. In this study, we investigated the molecular mechanisms involved in GRK2 down-regulation, using organotypic cultures of neonatal rat hippocampal slices exposed to oxygen and glucose deprivation (OGD). We observed a 40% decrease in GRK2 expression 4 h post-OGD. No changes in GRK2 protein occurred after exposure of hippocampal slices to glucose deprivation only. No significant alterations in GRK2 mRNA expression were detected, suggesting a post-transcriptional effect of OGD on GRK2 expression. Blockade of the proteasome pathway by MG132 prevented OGD-induced decrease of GRK2. It has been shown that extracellular signal-regulated kinase-dependent phosphorylation of GRK2 at Ser670 triggers its turnover via the proteasome pathway. However, despite a significant increase of pSer670-GRK2 after OGD, inhibition of the extracellular signal-regulated kinase pathway by PD98059 did neither prevent the hypoxia-ischemia-induced increase in pSer670-GRK2 nor the down-regulation of GRK2 protein. Interestingly, inhibition of phosphoinositide-3-kinase with wortmannin inhibits both OGD-induced phosphorylation of GRK2 on Ser670 and the GRK2 decrease. In conclusion, OGD-induced phosphoinositide-3-kinase-dependent phosphorylation of GRK2 on Ser670 is a novel mechanism leading to down-regulation of GRK2 protein via a proteasome-dependent pathway.

NAD(P)H:quinone oxidoreductase 1 expression in multiple sclerosis lesions.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), marked by infiltration of monocyte-derived macrophages in the brain parenchyma. Macrophages contribute to disease pathology by secretion of inflammatory mediators, such as reactive oxygen species (ROS). ROS are involved in various processes underlying MS pathology, including monocyte migration across the blood-brain barrier, phagocytosis and degradation of myelin, axonal degeneration, and oligodendrocyte damage. High concentrations of ROS cause oxidative stress, which induces transcriptional activation of phase II detoxification enzymes, such as the antioxidant protein NAD(P)H:quinone oxidoreductase 1 (NQO1). Since NQO1 expression may act as an indicator of oxidative stress and knowledge about the cellular distribution pattern of NQO1 in MS brains is lacking, we examined the expression of NQO1 in various well-characterized MS lesions. Here, we show for the first time that NQO1 is highly upregulated in active and chronic active MS lesions, particularly in hypertrophic astrocytes and myelin-laden macrophages. We hypothesize that increased NQO1 expression may reflect an endogenous defense response against ROS-mediated cellular toxicity. Compounds that induce the production of endogenous antioxidant enzymes, such as NQO1, may be potential targets for future treatment strategies in MS.

The expression of the chemorepellent Semaphorin 3A is selectively induced in terminal Schwann cells of a subset of neuromuscular synapses that display limited anatomical plasticity and enhanced vulnerability in motor neuron disease.

Neuromuscular synapses differ markedly in their plasticity. Motor nerve terminals innervating slow muscle fibers sprout vigorously following synaptic blockage, while those innervating fast-fatigable muscle fibers fail to exhibit any sprouting. Here, we show that the axon repellent Semaphorin 3A is differentially expressed in terminal Schwann cells (TSCs) on different populations of muscle fibers: postnatal, regenerative and paralysis induced remodeling of neuromuscular connections is accompanied by increased expression of Sema3A selectively in TSCs on fast-fatigable muscle fibers. To our knowledge, this is the first demonstration of a molecular difference between TSCs on neuromuscular junctions of different subtypes of muscle fibers. Interestingly, also in a mouse model for amyotrophic lateral sclerosis (ALS), Sema3A is expressed at NMJs of fast-fatigable muscle fibers. We propose that expression of Sema3A by TSCs not only suppresses nerve terminal plasticity at specific neuromuscular synapses, but may also contribute to their early and selective loss in the motor neuron disease ALS.

A phosphatidylinositol transfer protein alpha-dependent survival factor protects cultured primary neurons against serum deprivation-induced cell death.

Selective neuronal loss is a prominent feature in both acute and chronic neurological disorders. Recently, a link between neurodegeneration and a deficiency in the lipid transport protein phosphatidylinositol transfer protein alpha (PI-TPalpha) has been demonstrated. In this context it may be of importance that fibroblasts overexpressing PI-TPalpha are known to produce and secrete bioactive survival factors that protect fibroblasts against UV-induced apoptosis. In the present study it was investigated whether the conditioned medium of cells overexpressing PI-TPalpha (CMalpha) has neuroprotective effects on primary neurons in culture. We show that CMalpha is capable of protecting primary, spinal cord-derived motor neurons from serum deprivation-induced cell death. Since the conditioned medium of wild-type cells was much less effective, we infer that the neuroprotective effect of CMalpha is linked (in part) to the PI-TPalpha-dependent production of arachidonic acid metabolites. The neuroprotective activity of CMalpha is partly inhibited by suramin, a broad-spectrum antagonist of G-protein coupled receptors. Western blot analysis shows that brain cortex and spinal cord express relatively high levels of PI-TPalpha, suggesting that the survival factor may be produced in neuronal tissue. We propose that the bioactive survival factor is implicated in neuronal survival. If so, PI-TPalpha could be a promising target to be evaluated in studies on the prevention and treatment of neurological disorders.

Enhanced sensitivity of dopaminergic neurons to rotenone-induced toxicity with aging.

Rotenone has been reported to induce various degrees of Parkinsonism in rats. We tested whether advancing age alters the sensitivity of dopaminergic neurons to rotenone. A low, systemic dose of rotenone had no effect on young rats, but led to a 20-30% reduction of tyrosine hydroxylase-positive neurons in the substantia nigra of older rats. The effect was specific to nigral dopaminergic neurons and may be associated with the increase of glial cell activation in older rats. These data suggest that age enhances the sensitivity of dopaminergic neurons to rotenone and should be considered when assessing models of Parkinson's disease.

The Nrf2-ARE Signalling pathway: promising drug target to combat oxidative stress in neurodegenerative disorders.

A large body of evidence indicates that oxidative stress is a salient pathological feature in many neurodegenerative diseases, including Amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. In addition to signs of systemic oxidative stress, at the biochemical and neuropathological level, neuronal degeneration in these disorders has been shown to coincide with several markers of oxidative damage to lipids, nucleic acids, and proteins in affected brain regions. Neuroinflammatory processes, often associated with the induction of free radical generating enzymes and the accumulation of reactive astrocytes and microglial cells, are considered as a major source of oxidative stress. Given the pathogenic impact of oxidative stress and neuroinflammation, therapeutic strategies aimed to blunt these processes are considered an effective way to confer neuroprotection. Recently, the nuclear transcription factor Nrf2, that binds to the antioxidant response element (ARE) in gene promoters, has been reported to constitute a key regulatory factor in the co-ordinate induction of a battery of endogenous cytoprotective genes, including those encoding for both antioxidant- and anti-inflammatory proteins. In the present review, besides discussing recent evidence underscoring the thesis that the Nrf2-ARE signalling pathway is an attractive therapeutic target for neurodegenerative diseases, we advocate the view that chemopreventive agents might be suitable candidates to serve as lead compounds for the development of a new class of neuroprotective drugs.

CGP 3466B has no effect on disease course of (G93A) mSOD1 transgenic mice.

BACKGROUND: There is an accumulating body of evidence that apoptosis is involved in the motor neuron death that occurs in ALS, and in the (G93A) mSOD1 transgenic mouse model (mSOD1 mice). CGP 3466B, a tricyclic propargylamine structurally related to (-)-deprenyl, was found to inhibit apoptosis in a wide variety of in vitro and in vivo models. We therefore studied the effect of CGP 3466B in mSOD1 mice. METHODS: As the effect of CGP 3466B was previously reported to have a bell-shaped curve, we performed a dose-ranging study. High-copy G93A mSOD1 mice were treated subcutaneously from the age of 50 days until death with four concentrations of CGP 3466B (0.39 microg kg(-1), 3.9 microg kg(-1), 39 microg kg(-1), and 390 microg kg(-1)). Behavioural tests were performed daily to determine disease onset, disease progression and survival. At the age of 110 days, two mice per group were sacrificed for histopathological analysis of the lumbar ventral horn and for semiquantitative analysis of motor neuron number. RESULTS: We observed no effect on disease onset, disease progression, or survival of the mice. We also did not observe a significant effect on the number of motor neurons due to CGP 3466B. CONCLUSIONS: We conclude that in high-copy G93A mSOD1 mice, chronic subcutaneous treatment with CGP 3466B offers no clinical benefit.

Cells over-expressing EAAT2 protect motoneurons from excitotoxic death in vitro.

Amyotrophic lateral sclerosis is an incurable disease in which cerebral and spinal motoneurons degenerate, causing paralysis and death within 2-5 years. One of the pathogenic factors of motoneuron death is a chronic excess of glutamate, which exceeds its removal by astrocytes, i.e. excitotoxicity. Extra glutamate uptake in the spinal cord may slow down or prevent motoneuron death. We have engineered cells over-expressing the main glutamate transporter and tested their potential to rescue motoneurons exposed to high levels of glutamate in vitro. The engineered cells protected motoneurons in a motoneuron-astrocyte co-culture at glutamate concentrations when astrocytes were no longer capable of removing glutamate. This suggests that engineered cells, introduced into the spinal column, can help remove glutamate, thereby preventing motoneuron death.

The neuroprotective antioxidant alpha-lipoic acid induces detoxication enzymes in cultured astroglial cells.

alpha-Lipoic acid (LA), an antioxidant with broad neuroprotective capacity, is thought to act by scavenging reactive oxygen species and stimulation of glutathione synthesis. LA shows structural resemblance to dithiolethiones, like anethole dithiolethione (ADT). ADT protects against oxidative damage, primarily by induction of phase II detoxication enzymes, in particular NAD(P)H:quinone oxidoreductase (NQO1) and glutathione-S-transferase (GST). Therefore, we investigated whether LA, like ADT, is capable also of inducing these protective enzymes. Our data show that LA, like ADT, induces a highly significant, time- and concentration dependent, increase in the activity of NQO1 and GST in C6 astroglial cells. The LA or ADT mediated induction of NQO1 was further confirmed by quantitative PCR and western blot analysis. This work for the first time unequivocally demonstrates LA mediated upregulation of phase II detoxication enzymes, which may highly contribute to the compounds' neuroprotective potential. Moreover, the data support the notion of a common mechanism of action of LA and ADT.