Meta-analysis of vascular endothelial growth factor variations in amyotrophic lateral sclerosis: increased susceptibility in male carriers of the -2578AA genotype.

BACKGROUND: Targeted delivery of the angiogenic factor, vascular endothelial growth factor (VEGF), to motor neurons prolongs survival in rodent models of amyotrophic lateral sclerosis (ALS), while mice expressing reduced VEGF concentrations develop motor neuron degeneration reminiscent of ALS, raising the question whether VEGF contributes to the pathogenesis of ALS. An initial association study reported that VEGF haplotypes conferred increased susceptibility to ALS in humans, but later studies challenged this initial finding. METHODS AND FINDINGS: A meta-analysis was undertaken to critically reappraise whether any of the three common VEGF gene variations (-2578C/A, -1154G/A and -634G/C) increase the risk of ALS. Over 7000 subjects from eight European and three American populations were included in the analysis. Pooled odds ratios were calculated using fixed-effects and random-effects models, and four potential sources of heterogeneity (location of disease onset, gender, age at disease onset and disease duration) were assessed. After correction, none of the genotypes or haplotypes was significantly associated with ALS. Subgroup analysis by gender revealed, however, that the -2578AA genotype, which lowers VEGF expression, increased the risk of ALS in males (OR = 1.46 males vs females; 95% CI = 1.19 to 1.80; p = 7.8 10E-5), even after correction for publication bias and multiple testing. CONCLUSIONS: This meta-analysis does not support the original conclusion that VEGF haplotypes increase the risk of ALS in humans, but the significant association of the low-VEGF -2578AA genotype with increased susceptibility to ALS in males reappraises the link between reduced VEGF concentrations and ALS, as originally revealed by the fortuitous mouse genetic studies.

Protective effect of metabotropic glutamate receptor inhibition on amyotrophic lateral sclerosis-cerebrospinal fluid toxicity in vitro.

Conflicting results have been reported concerning the toxicity of cerebrospinal fluid from patients with amyotrophic lateral sclerosis (ALS-CSF) when added to neuronal cultures. The possible toxic factor(s) and the exact mode of action (e.g. requirement of glial cells) have not been identified so far. Glutamate is a potential candidate for this toxic effect, since antagonists of ionotropic glutamate receptors have been shown to attenuate ALS-CSF toxicity. We studied the effects of ALS-CSF on mixed and motoneuron-enriched chick embryonic spinal cord cultures. We found a toxic action of ALS-CSF in both culture types which could not be attenuated by 5 kDa-filtration or 15 min 90 degrees C heating. Nevertheless, the metabotropic glutamate receptor (mGluR) group I antagonist 1-aminoindan-1,5-dicarboxylic acid, but also the group I agonist (s)-3,5-dihydroxyphenylglycine (DHPG) exerted protective effects against ALS-CSF toxicity. In this experimental setting, DHPG may functionally act via a receptor blockade due to sustained activation. No protective effect was seen with the mGluR group III inhibitor (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG). Addition of DHPG did not increase the protective action of the AMPA inhibitor 6-chloro-4-hydroxyquinoline-2-carboxylic acid (6-CKU). Addition of l-glutamate did not mimic these toxic ALS-CSF effects in motoneuron-enriched cultures. Our experiments demonstrate that ALS-CSF toxicity is mediated by a small heat-resistant molecule which may act directly on neurons. Since blockade of group I mGluRs exerts a protective effect, the possibility of targeting these mGluRs pharmacologically in motoneuron disease should be kept in mind.

Activation of metabotropic glutamate receptors delays apoptosis of chick embryonic motor neurons in vitro.

Glutamatergic excitotoxicity has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, activation of metabotropic glutamate receptors (mGluRs) is neuroprotective in several paradigms. We therefore tested the effect of selective mGluR agonists on cultured chick embryonic motor neurons. Activation of group I mGluRs with (s)-3,5-dihydroxyphenylglycine (DHPG) and group III mGluRs with L-2-amino-4-phosphono-butanoate (L-AP4) promoted a modest but significant, dose-dependent delay of apoptosis, which could be blocked by specific mGluR antagonists. Group II or selective mGluR5 stimulations were ineffective. Correspondingly, in situ hybridization experiments showed only expression of mGluR1 (group I) and mGluR4 and 7 (group III) in human motor neurons. Dissection of the pathways involved in this survival effect may help to elucidate the pathogenesis of ALS.

Immunosuppressant FK506 does not exert beneficial effects in symptomatic G93A superoxide dismutase-1 transgenic mice.

The immunosuppressant drug FK506 has been shown to exert neuroprotective effects in various model systems via inhibition of the protein phosphatase calcineurin (CN). The enzyme Cu/Zn-superoxide dismutase (SOD1), which is mutated in a familial form of amyotrophic lateral sclerosis (ALS), is an endogenous regulator of CN. Altered function of CN may therefore be involved in the pathogenesis of ALS. We tested FK506 in a transgenic mouse model expressing mutated SOD1 for potential beneficial effects. This treatment, initiated after onset of symptoms, did not cause a reduction in the decline of motor function nor did it prolong survival. These results argue against a crucial role of CN in the process leading to motoneuronal degeneration in SOD1-mutated mice.

CEP-1347/KT7515, a JNK pathway inhibitor, supports the in vitro survival of chick embryonic neurons.

Developing neurons depend on target-derived trophic factors for survival in vivo and in vitro, which also decrease the activity of c-Jun N-terminal kinase (JNK). We have recently described a survival-promoting effect of inhibitors of cyclin-dependent kinases and JNK on chick peripheral embryonic neurons. Here, we report that the small trophic molecule CEP-1347/KT7515, which has been shown to inhibit the JNK signalling pathway, can promote long term-survival of cultured chick embryonic dorsal root ganglion, sympathetic, ciliary and motor neurons. Because of their pharmacological properties, small trophic molecules such as CEP-1347/KT7515 might be of interest for the treatment of neurodegenerative disorders.

Axotomy of the sciatic nerve differentially affects expression of metabotropic glutamate receptor mRNA in adult rat motoneurons.

Previous studies indicated that axotomy exposes motoneurons to glutamatergic excitotoxic stress and protection from glutamatergic overactivation might be crucial for survival. Depending on the experimental model and the subtype involved, activation of metabotropic glutamate receptors (mGluRs) may either enhance excitotoxicity or exert protective effects. To investigate a possible involvement of mGluRs in neuronal rescue mechanisms after axotomy we have monitored the distribution of mGluR mRNA with in situ hybridization in adult rat motoneurons 1, 2, 3, and 4 weeks after sciatic nerve transection. Motoneurons in sham-operated control animals expressed mGluR 1, 4, and 7 mRNA. The mGluR1 mRNA signal was reduced to 49.6+/-6.9% as compared to the contralateral side 2 weeks after axotomy and 31.2+/-8.3% after 4 weeks. The mGluR4 signal declined to 22.1+/-5.1% after 1 week and 10.2+/-1.6% after 2 weeks, remaining stable thereafter. During the entire observation period the mRNA for mGluR7 was not significantly altered. Axotomy did not change the overall number of motoneurons on the ipsi- or contralateral side. The differential regulation of mGluR subtypes may be part of an adaptive cell program that helps to rescue adult motoneurons from excitotoxic cell death during the stress induced by peripheral denervation.

Distribution and developmental changes in metabotropic glutamate receptor messenger RNA expression in the rat lumbar spinal cord.

Using in situ hybridisation, the regional distribution of primary transcripts and splice variants of all metabotropic glutamate receptor subtypes (mGluR) currently known to be expressed in the spinal cord have been studied in the lumbar enlargement of the rat spinal cord. In adult animals, the messenger RNA of the mGluR subtypes 1, 5, 3, 4 and 7 were differentially expressed. The transcripts of mGluR1 and 5 were most abundant with mGluR5 messenger RNA being concentrated in the superficial dorsal horn. In contrast, the mGluR2 transcript was not detectable with the sensitivity of the method. Secondly, age related changes (postnatal days 1, 7, 12, 21) in the postnatal expression of mGluR1-5 and 7 transcripts have been investigated. mGluR1 and 7 messenger RNA showed a general decrease in spinal expression from postnatal day 1 to day 21. Quantitative densitometry showed high mGluR3 and 5 messenger RNA levels especially in the superficial dorsal horn at birth, however these levels decreased with age. In addition to changes in density, the regional distribution of mGluR3 messenger RNA was altered with postnatal development. Up to postnatal day 12, mGluR3 messenger RNA expression was almost exclusively restricted to the spinal grey matter, but with postnatal day 21 a strong additional expression in the white matter occurred. Distribution of mGluR4 messenger RNA showed little change in the dorsal horn, however motoneuronal expression emerged during development. These changes may suggest different roles for mGluRs in the maturation of spinal transmission of the rat nervous system.

A role for the urokinase-type plasminogen activator system in amyotrophic lateral sclerosis.

There is substantial evidence, implicating extracellular matrix (ECM) regulating enzymes in the pathogenesis of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The most important ECM-degrading proteases are serine proteases (plasminogen activators, PA) and matrix metalloproteinases (MMPs). Since the role of MMPs in ALS has been addressed recently, we investigated the expression of the serine protease urokinase-type plasminogen activator (uPA) and its receptor in ALS. Employing rtPCR, zymography and immunohistochemistry we analyzed the expression of uPA and its receptor uPAR in spinal cord tissue of ALS cases and in the G93A SOD1 transgenic mouse. In the ventral horn of the spinal cord of ALS cases we found increased uPAR staining of motor neurons. In G93A mice, the expression profile of uPA and uPAR mRNA was significantly increased starting at the age of 90 days as compared to non-transgenic littermates. The uPA-dependent plasminogen activation in G93A mice at endstage increased markedly compared with controls and immunostaining of the spinal cord from G93A mice revealed increased uPAR immunostaining in neurons. To determine the functional role of uPA, we investigated the effect of intraperitoneal (i.p.) administration of the uPA inhibitor WX-340 (10 mg/kg), starting at the age of 30 days (n=18). Treatment with WX-340 prolonged (p<0.05) survival of the animals (135+/-2 vs. 126+/-3) as well as improving rotarod performance. Our experiments demonstrate that uPA and its receptor are expressed in ALS patients and in an animal model of ALS. Early inhibition with a synthetic uPA inhibitor prolonged the life of the transgenic animals. These findings indicate that the urokinase-type plasminogen activator system may play a role in the complex pathogenesis of ALS.

Possible gender-dependent association of vascular endothelial growth factor (VEGF) gene and ALS.

Individuals homozygous for haplotypes -2578-A/-1154-A/-634-G or -2578-A/-1154-G/-634-G in the promoter/5'UTR of the VEGF gene have a 1.8-fold increased risk of ALS in several European populations. We did not observe any significant association with single markers, or haplotype pairs, in a German sample of 580 sporadic ALS patients and 628 controls. However, the promoter SNP-1154 (rs1570360) was associated with affection status in women (p = 0.036), suggesting that the VEGF effect may be dependent on the sex ratio of the sample.

Mutations in the acid alpha-glucosidase gene (M. Pompe) in a patient with an unusual phenotype.

Glycogenosis type II (Pompe disease) is a lysosomal storage disease caused by deficiency of acid alpha-glucosidase (acid maltase). The disease is autosomal recessive inherited and is clinically and genetically heterogenous. The authors describe a 30-year-old woman affected by late-onset Pompe disease with vascular affection resembling atherosclerotic angiopathy of the elderly. Genetic analysis revealed two novel mutations (Ala237Val and Gly293Arg) in the acid alpha-glucosidase gene in this patient.

Molecular basis of treatment in motor neurone disease.

The pathways leading to motorneuron degeneration in amyotrophic lateral sclerosis (ALS) are complex. Excitotoxicity, oxidative damage and, maybe, abnormal aggregation of neurofilaments are key events on which therapeutical strategies can be designed. This paper reviews current knowledge on these strategies. Even though we should be aware that appropriate management of disease symptoms remains the most effective therapeutical intervention, understanding the pathophysiology of ALS is essential for developing new therapies.

Differential expression of group I metabotropic glutamate receptors in rat spinal cord somatic and autonomic motoneurons: possible implications for the pathogenesis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of somatic, but not autonomic, motoneurons. The reason for this selective vulnerability is unknown. The pathogenesis of ALS is thought to involve glutamatergic excitotoxic mechanisms. While overactivation of ionotropic glutamate receptors may trigger excitotoxicity, we have previously shown that stimulation of group I metabotropic glutamate receptors (mGluRs) can exert neuroprotective effects on cultured motoneurons. Using in situ hybridization, we found a differential expression of group I mGluRs (mGluR1 and 5) in rat spinal cord. Autonomic motoneurons from the sacral parasympathetic Onuf's nucleus and thoracic sympathetic neurons, which are spared in ALS, express high levels of mGluR5, while somatic motoneurons do not. In addition, mGluR1 mRNA is found only in smaller somatic motoneurons, which seem to be less vulnerable in ALS. Thus, differential mGluR expression might provide a possible clue to the selective vulnerability of different motoneuronal subpopulations in ALS.

Glial cells of the spinal cord and subcortical white matter up-regulate neuronal nitric oxide synthase in sporadic amyotrophic lateral sclerosis.

Several studies have suggested that excessive generation of nitric oxide (NO) may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). Recently, a selective induction of the neuronal isoform of nitric oxide synthase (nNOS) in glial cells has been reported in an animal model of familial ALS. We therefore examined in postmortem tissue the expression of nNOS in patients with sporadic ALS and patients without any history of neurological disease. Using immunohistochemistry, we found an up-regulation of nNOS in glial cells of the spinal cord and subcortical white matter in ALS patients compared to controls. The enhanced glial nNOS expression seen in ALS patients could conceivably contribute to motoneuronal degeneration through NO-mediated cytotoxic effects.

Apoptosis of central and peripheral neurons can be prevented with cyclin-dependent kinase/mitogen-activated protein kinase inhibitors.

Previous studies have indicated that certain members of the cyclin-dependent kinase/mitogen-activated protein kinase superfamily are involved in apoptosis of neuronal cells. Here, we have examined programmed cell death induced by withdrawal of neurotrophic support from CNS (rat retinal) and PNS (chick sympathetic, sensory, and ciliary) neurons. All four neuron types were equally rescued by the purine analogues olomoucine and roscovitine. Olomoucine inhibits multiple cyclin-dependent and mitogen-activated protein kinases with similar potency. Roscovitine is a more selective cyclin-dependent kinase inhibitor; but, so is butyrolactone I, which did not prevent retinal ganglion cell death. The specific p38MAPK inhibitor SB-203580 did not prevent apoptosis in retinal ganglion cells. Death of these cells in the absence of neurotrophic factors was accompanied by morphological changes indicative of apoptosis, including nuclear condensation and fragmentation. Treatment with olomoucine or roscovitine not only prevented these apoptotic changes in retinal ganglion cells but also blocked neurite outgrowth. The survival-promoting activity of olomoucine correlated with its in vitro IC50 for c-Jun N-terminal kinase-1 and its potency to repress c-jun induction in live PC12 cells. Roscovitine was more potent in rescuing neurons than in inhibiting Jun kinase. Thus, the antiapoptotic action of roscovitine might be due to inhibition of additional kinases.

Survival-promoting activity of inhibitors of cyclin-dependent kinases on primary neurons correlates with inhibition of c-Jun kinase-1.

Cyclin-dependent kinases and mitogen-activated protein kinases have been implicated in the regulation of cellular survival and apoptosis. We tested the effect of two mitogen-activated/cyclin-dependent kinase inhibitors, olomoucine and butyrolactone I, on the in vitro survival of chick embryonic neurons. Sensory, sympathetic, and ciliary neurons, when prepared at their respective time point of programmed cell death, could be rescued from apoptosis by both inhibitors in a dose-dependent fashion. In contrast, dividing sympathetic precursors underwent apoptosis when treated with olomoucine, but not butyrolactone I, at the same range of concentration. With similar potency, olomoucine and butyrolactone I inhibited immunocomplex c-Jun kinase activity. Both substances inhibited neurite outgrowth in a dose-dependent manner; developmentally younger neurons were more sensitive to this effect than older ones. These results suggest that certain mitogen-activated/cyclin-dependent kinases associated with cell division in neuronal precursors (i) may become essential components of the apoptotic machinery by the time neurons reach their phase of naturally occurring cell death and (ii) may be necessary for neurite outgrowth during development.