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L Van Den Bosch

Publications and source records attributed to L Van Den Bosch.

At least 19 recordsLinked to original sources
PubMed2006

Role of heat shock response and Hsp27 in mutant SOD1-dependent cell death.

The fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS) is characterized by selective loss of motor neurons and mutations in the copper-zinc superoxide dismutase (SOD1) enzyme underlie one form of familial ALS. The pathogenic mechanism of these mutations is elusive but is thought to involve oxidative stress and protein aggregation. These two phenomena are known to induce heat shock proteins (Hsps) which protect stressed cells through their chaperoning and anti-apoptotic activity. In order to investigate the role of Hsp27 in mutant SOD1-dependent cell death, we used mutant and wild type SOD1 overexpressing N2a mouse neuroblastoma cells. Mutant SOD1-dependent cell death could be induced by heat shock, and by treating the cells with cyclosporine A or lactacystin. Transfection with an Hsp27 expression construct did not protect the N2a cells against mutant SOD1-dependent cell death. However, pre-conditioning N2a cells with a mild heat shock was accompanied by a significant upregulation of Hsp27 in the mutant SOD1 cells, and protected these cells against subsequent cell death induced by a more severe heat shock. Selective inhibition of the Hsp27 upregulation, through the use of Hsp27 siRNA, did not attenuate the protective effect of this treatment. These results show that activation of the heat shock response protects cells against mutant SOD1-dependent cell death, but that Hsp27 is not an essential component of the stress response leading to protection.

Animals↗
PubMed2006

The role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis.

Unfortunately and despite all efforts, amyotrophic lateral sclerosis (ALS) remains an incurable neurodegenerative disorder characterized by the progressive and selective death of motor neurons. The cause of this process is mostly unknown, but evidence is available that excitotoxicity plays an important role. In this review, we will give an overview of the arguments in favor of the involvement of excitotoxicity in ALS. The most important one is that the only drug proven to slow the disease process in humans, riluzole, has anti-excitotoxic properties. Moreover, consumption of excitotoxins can give rise to selective motor neuron death, indicating that motor neurons are extremely sensitive to excessive stimulation of glutamate receptors. We will summarize the intrinsic properties of motor neurons that could render these cells particularly sensitive to excitotoxicity. Most of these characteristics relate to the way motor neurons handle Ca(2+), as they combine two exceptional characteristics: a low Ca(2+)-buffering capacity and a high number of Ca(2+)-permeable AMPA receptors. These properties most likely are essential to perform their normal function, but under pathological conditions they could become responsible for the selective death of motor neurons. In order to achieve this worst-case scenario, additional factors/mechanisms could be required. In 1 to 2% of the ALS patients, mutations in the SOD1 gene could shift the balance from normal motor neuron excitation to excitotoxicity by decreasing glutamate uptake in the surrounding astrocytes and/or by interfering with mitochondrial function. We will discuss point by point these different pathogenic mechanisms that could give rise to classical and/or slow excitotoxicity leading to selective motor neuron death.

Amyotrophic Lateral Sclerosis↗
PubMed2005

The cellular mRNA expression of GABA and glutamate receptors in spinal motor neurons of SOD1 mice.

ALS is a fatal neurodegenerative disorder characterized by a selective loss of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord. About 10% of ALS cases are familial, in 10-20% of these, mutations in the gene coding for superoxide dismutase 1 (SOD1) can be detected. Overexpression of mutated SOD1 in mice created animal models which clinically resemble ALS. Abnormalities in glutamatergic and GABAergic neurotransmission presumably contribute to the selective motor neuron damage in ALS. By in situ hybridization histochemistry (ISH), we investigated the spinal mRNA expression of the GABAA and AMPA type glutamate receptor subunits at different disease stages on spinal cord sections of mutant SOD1 mice and control animals overexpressing wild-type SOD1 aged 40, 80, 120 days and at disease end-stage, i.e. around 140 days) (n=5, respectively). We detected a slight but statistically significant decrease of the AMPA receptor subunits GluR3 and GluR4 only in end stage disease animals.

Aging↗
PubMed2005

Excitotoxicity and amyotrophic lateral sclerosis.

Since its description by Charcot more than 130 years ago, the pathogenesis of selective motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remains unsolved. Over the years, many pathogenic mechanisms have been proposed. Amongst others these include: oxidative stress, excitotoxicity, aggregate formation, inflammation, growth factor deficiency and neurofilament disorganization. This multitude of contributing factors indicates that ALS is a complex disease and also suggests that ALS is a multifactorial disorder. Excitotoxicity is not the newest and most spectacular hypothesis in the ALS field, but it is undoubtedly one of the most robust pathogenic mechanisms supported by an impressive amount of evidence. Moreover, the therapeutic efficacy of riluzole, the only drug proven to slow disease progression in ALS, is most likely related to its anti-excitotoxic properties. In this review, we will give an overview of the arguments in favor of the involvement of excitotoxicity in ALS and of the possible mechanisms leading to motor neuron death. We will also summarize the intrinsic properties of motor neurons that render these cells particularly vulnerable to excitotoxicity and could explain the selective vulnerability of motor neurons in ALS. All this information could help to develop new and better therapeutic strategies that could protect motor neurons from excitotoxicity.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Chloride influx aggravates Ca2+-dependent AMPA receptor-mediated motoneuron death.

AMPA receptor-mediated excitotoxicity has been implicated in the pathogenesis of stroke, neurotrauma, epilepsy, and many neurodegenerative diseases such as motoneuron disease. We studied the role of Cl- in AMPA receptor-mediated Ca2+-dependent excitotoxicity in cultured rat spinal motoneurons. Using the gramicidin perforated patch-clamp technique, the intracellular Cl- concentration could be calculated from the reversal potential of the GABA-induced current. The membrane depolarization caused by AMPA receptor stimulation resulted in Cl- influx through 5-nitro-2(3-phenylpropyl-amino) benzoic acid- and niflumic acid-sensitive Cl- channels. Cl- influx during AMPA receptor stimulation aggravated excitotoxic motoneuron death by two mechanisms: an increase of AMPA receptor conductance and an elevation of the Ca2+ driving force through a partial repolarization. The Cl- influx during AMPA receptor stimulation was enhanced by coadministration of GABA. This resulted in an increased Ca2+ influx and an enhanced cell death, suggesting that concomitant GABAergic stimulation may aggravate excitotoxic motoneuron death.

Animals↗
PubMed2002

Protective effect of parvalbumin on excitotoxic motor neuron death.

The mechanism responsible for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is poorly understood. Several lines of evidence indicate that susceptibility of motor neurons to Ca(2+) overload induced by excitotoxic stimuli is involved. In this study, we investigated whether the high density of Ca(2+)-permeable AMPA receptors on motor neurons gives rise to higher Ca(2+) transients in motor neurons compared to dorsal horn neurons. Dorsal horn neurons were chosen as controls as these cells do not degenerate in ALS. In cultured spinal motor neurons, the rise of the cytosolic Ca(2+) concentration induced by kainic acid (KA) and mediated by the AMPA receptor was almost twice as high as in spinal neurons from the dorsal horn. Furthermore, we investigated whether increasing the motor neuron's cytosolic Ca(2+)-buffering capacity protects them from excitotoxic death. To obtain motor neurons with increased Ca(2+) buffering capacity, we generated transgenic mice overexpressing parvalbumin (PV). These mice have no apparent phenotype. PV overexpression was present in the central nervous system, kidney, thymus, and spleen. Motor neurons from these transgenic mice expressed PV in culture and were partially protected from KA-induced death as compared to those isolated from nontransgenic littermates. PV overexpression also attenuated KA-induced Ca(2+) transients, but not those induced by depolarization. We conclude that the high density of Ca(2+)-permeable AMPA receptors on the motor neuron's surface results in high Ca(2+) transients upon stimulation and that the low cytosolic Ca(2+)-buffering capacity of motor neurons may contribute to the selective vulnerability of these cells in ALS. Overexpression of a high-affinity Ca(2+) buffer such as PV protects the motor neuron from excitotoxicity and this protective effect depends upon the mode of Ca(2+) entry into the cell.

Amyotrophic Lateral Sclerosis↗
PubMed2002

Na(+) entry through AMPA receptors results in voltage-gated k(+) channel blockade in cultured rat spinal cord motoneurons.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents, evoked with the agonist kainate, were studied with the gramicidin perforated-patch-clamp technique in cultured rat spinal cord motoneurons. Kainate-induced currents could be blocked by the AMPA receptor antagonist LY 300164 and displayed an apparent strong inward rectification. This inward rectification was not a genuine property of AMPA receptor currents but was a result of a concomitant decrease in outward current at potentials positive to -40.5 +/- 1.3 mV. The AMPA receptor current itself was nearly linear (rectification index 0.91). The kainate-inhibited outward current had a reversal potential close to the estimated K(+) equilibrium potential and was blocked by 30 mM tetraethylammonium. When voltage steps were applied, it was found that kainate inhibited both the delayed rectifier K(+) current K(V) and the transient outward K(+) current, K(A). The kainate-induced inhibition of K(+) currents was dependent on ion flux through the AMPA receptor, because no change in the membrane conductance was noticed in the presence of LY 300164. Removing extracellular Ca(2+) had no effect, whereas replacing extracellular Na(+) or clamping the membrane close to the estimated Na(+) equilibrium potential during kainate application attenuated the inhibition of the K(+) current. Sustained Na(+) influx induced by application of the Na(+) ionophore monensin could mimic the effect of kainate on K(+) conductance. These findings demonstrate that Na(+) influx through AMPA receptors results in blockade of voltage-gated K(+) channels.

Animals↗
PubMed2002

GluR2-dependent properties of AMPA receptors determine the selective vulnerability of motor neurons to excitotoxicity.

AMPA receptor-mediated excitotoxicity has been implicated in the selective motor neuron loss in amyotrophic lateral sclerosis. In some culture models, motor neurons have been shown to be selectively vulnerable to AMPA receptor agonists due to Ca(2+) influx through Ca(2+)-permeable AMPA receptors. Because the absence of GluR2 in AMPA receptors renders them highly permeable to Ca(2+) ions, it has been hypothesized that the selective vulnerability of motor neurons is due to their relative deficiency in GluR2. However, conflicting evidence exists about the in vitro and in vivo expression of GluR2 in motor neurons, both at the mRNA and at the protein level. In this study, we quantified electrophysiological properties of AMPA receptors, known to be dependent on the relative abundance of GluR2: sensitivity to external polyamines, rectification index, and relative Ca(2+) permeability. Cultured rat spinal cord motor neurons were compared with dorsal horn neurons (which are resistant to excitotoxicity) and with motor neurons that survived an excitotoxic insult. Motor neurons had a higher sensitivity to external polyamines, a lower rectification index, and a higher relative Ca(2+) permeability ratio than dorsal horn neurons. These findings confirm that motor neurons are relatively deficient in GluR2. The AMPA receptor properties correlated well with each other and with the selective vulnerability of motor neurons because motor neurons surviving an excitotoxic event had similar characteristics as dorsal horn neurons. These data indicate that the relative abundance of GluR2 in functional AMPA receptors may be a major determinant of the selective vulnerability of motor neurons to excitotoxicity in vitro.

Animals↗
PubMed2001

Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration.

Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.

Amyotrophic Lateral Sclerosis↗
PubMed2000

Ca(2+)-permeable AMPA receptors and selective vulnerability of motor neurons.

To evaluate the role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis (ALS), we compared the sensitivity of motor neurons and that of dorsal horn neurons to kainic acid (KA). Short exposure to KA resulted in the death of motor neurons, while dorsal horn neurons were unaffected. This selective motor neuron death was completely dependent on extracellular Ca(2+) and insensitive to inhibitors of voltage-operated Ca(2+) or Na(+) channels. It was also completely inhibited by the specific AMPA antagonist LY300164 and by Joro spider toxin (JSTx), a selective blocker of AMPA receptors that lack the edited GluR2 subunit. KA selectively killed those motor neurons that stained positive for the Co(2+) histochemical staining, a measure for the presence of Ca(2+)-permeable AMPA receptors. These results suggest that Ca(2+) entry via Ca(2+)-permeable AMPA receptors is responsible for the selective motor neuron death. As the Ca(2+) permeability of the AMPA receptor is regulated by its GluR2 subunit, we stained motor neurons for GluR2. Immunoreactivity was present in all motor neurons, albeit to a variable degree. However, double-staining experiments demonstrated that motor neurons clearly expressing GluR2, also expressed Ca(2+)-permeable AMPA receptors. This indicates that despite the abundant expression of GluR2, this subunit is excluded from a subset of AMPA receptors and that the activation of these receptors is responsible for the selective motor neuron death.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2000

Different receptors mediate motor neuron death induced by short and long exposures to excitotoxicity.

We compared the effect of short and long exposures of cultured motor neurons to glutamate and kainate (KA) and studied the receptors involved in these two types of excitotoxicity. There was no difference in the receptor type used between short and long glutamate exposures as activation of the N-methyl-D-asparate (NMDA) receptor was in both cases responsible for the motor neuron death. Cell death through activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors only became apparent when desensitization of these receptors was prevented. In such conditions, motor neurons became much more sensitive to excitotoxicity, and activation of different types of AMPA receptors mediated motor neuron death after short, compared to long, exposures to the non-desensitizing AMPA receptor agonist, KA. Short KA exposures selectively affected motor neurons containing Ca(2+)-permeable AMPA receptors, as the KA effect was completely inhibited by Joro spider toxin and only motor neurons that were positive for the histochemical Co(2+) staining were killed. A long exposure to KA affected motor neurons through both Ca(2+)-permeable and Ca(2+)-impermeable AMPA receptors. The selective death of motor neurons vs. dorsal horn neurons was observed after short KA exposures indicating that the selective vulnerability of motor neurons to excitotoxicity is related to the presence of Ca(2+)-permeable AMPA receptors.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1999

Calcium handling proteins in isolated spinal motoneurons.

Amyotrophic lateral sclerosis is characterized by motoneuron degeneration, in which glutamate-induced cell death is thought to play a pathogenic role. This excitotoxic process is mediated by cytosolic Ca2+ overload. The glutamatergic ionotropic channel molecules, which constitute a major route of Ca2+ entry, were present on cultured spinal motoneurons. Using ratio RT-PCR, the relative presence in isolated motoneurons of the GluR subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor was evaluated. GluR1 and GluR2 mRNAs were present abundantly, while GluR3 and GluR4 mRNAs were much less abundant. The relative amount of mRNAs encoding the different protein isoforms responsible for Ca2+ uptake into the internal stores and for controlled release of Ca2+ from these stores was also determined. For the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), only the SERCA2b class 4 splice variant was found. The inositol 1,4,5-trisphosphate receptor (IP3R) mRNAs were mainly transcribed from the IP3RI and IP3RII genes. Heterogeneity was also observed for the ryanodine receptors (RyR) as the RyR1, RyR2 and RyR3 mRNAs were present.

Animals↗
PubMed1998

Glial cells potentiate kainate-induced neuronal death in a motoneuron-enriched spinal coculture system.

AMPA/kainate receptor-mediated excitotoxicity is believed to play a pathogenic role in amyotrophic lateral sclerosis. To further characterize the mechanisms involved in AMPA/kainate receptor-mediated motoneuron injury, we investigated the influence of spinal glial cells on kainate-induced motoneuron death in vitro. A motoneuron-enriched neuronal population was obtained from embryonic mouse spinal cord by metrizamide density centrifugation. This population was cultured either on a pre-established glial feeder layer of ventral spinal origin (coculture) or in glia-free conditions (monoculture). Glial feeder layers significantly enhanced basal survival of neurons, and supported neuronal differentiation as judged by neuronal morphology and expression of the motoneuron markers peripherin and SMI-32. Neuronal vulnerability to kainate was two- to three-fold higher in coculture than in monoculture, and increased significantly with time in coculture. The effects of glial feeder layers on neuronal basal survival, differentiation and kainate vulnerability were not mimicked by conditioned medium from glial cells. The increase in neuronal kainate vulnerability with time in coculture was associated with a marked rise in the proportion of cocultured neurons possessing Ca2+-permeable AMPA/kainate receptors, as determined by kainate-activated Co2+-uptake. Neurons in monoculture were unstained by kainate-activated Co2+-uptake. Neurons were immunoreactive to specific antibodies against the AMPA receptor subunits GluR1 and GluR2 both in monoculture and coculture. This study indicates that motoneuron differentiation in coculture is associated with increased vulnerability to kainate and increased expression of Ca2+-permeable AMPA/kainate receptors. In this paradigm glial cells support basal survival and differentiation of neurons, but potentiate kainate-induced neuronal death.

Animals↗
PubMed1998

Tissue-type plasminogen activator is not required for kainate-induced motoneuron death in vitro.

Spinal motoneurons are highly vulnerable to kainate both in vivo and in vitro. Tissue-type plasminogen activator (tPA) and plasmin have recently been shown to mediate kainate-induced neuronal death in the mouse hippocampus in vivo. The aim of the present study was to determine whether tPA also mediates the kainate-induced death of motoneurons in vitro. A motoneuron-enriched neuronal population was isolated from the ventral spinal cord of wild-type (WT) and tPA-deficient (tPA-/-) mouse embryos. WT and tPA-/- neurons were cultured on WT and tPA-/- spinal glial feeder layers, respectively. WT and tPA-/- co-cultures were morphologically indistinguishable. Expression of tPA in WT co-cultures was demonstrated using RT-PCR. WT and tPA-/- co-cultures were exposed to kainate for 24 h. The neurotoxic effect of kainate did not differ significantly between WT and tPA-/- cultures. The plasmin inhibitor alpha2-antiplasmin did not protect WT neurons against kainate-induced injury. These results indicate that the plasmin system is not a universal mediator of kainate-induced excitotoxicity.

Animals↗
PubMed1998

Increased sensitivity of fibroblasts from amyotrophic lateral sclerosis patients to oxidative stress.

Although the cause of amyotrophic lateral sclerosis (ALS) is unkNown, free radical toxicity is thought to play a pathogenic role. We investigated whether cells from ALS patients are more vulnerable to exogenously induced oxidative stress than cells from controls. We therefore studied the sensitivity of fibroblasts from patients with sporadic ALS (SALS), and from patients with familial ALS (FALS) associated with copper/zinc superoxide dismutase (Cu/ZnSOD) mutations (SOD1-FALS), to the free radical-generating agents 3-morpholinosydnonimine (SIN-1) and hydrogen peroxide (H2O2), and to serum withdrawal. SOD1-FALS and SALS fibroblasts were significantly more sensitive than controls to SIN-1 but not to serum withdrawal. In addition, SOD1-FALS fibroblasts were more sensitive to H2O2 than SALS fibroblasts and than fibroblasts of controls. These results suggest that the mechanism underlying both SOD1-FALS and SALS jeopardizes the cell's defense against free radical stress, and that SOD1-FALS cells are particularly sensitive to H2O2. The latter finding is compatible with biochemical data on the increased affinity of the mutated Cu/ZnSOD for H2O2.

Amyotrophic Lateral Sclerosis↗
PubMed1997

Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript.

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) requires activation of an inefficient optional splice process at the 3' end of the primary gene transcript. The sequence elements required for this regulated splice event were studied by modifying a minigene containing the 3' end of the SERCA2 gene. An important requirement appears to be a strong muscle-specific acceptor site, as replacing it by a weak one prevented the induction of muscle-type splicing during myogenic differentiation. The induction of muscle-type splicing did not depend on positive cis-active sequences in the muscle-specific exon. On the other hand, replacement of a broad region around the acceptor site dramatically deregulated the expression pattern, as this modification strongly induced muscle-type splicing in undifferentiated muscle cells and in fibroblasts. This cis-active region is also involved in the suppression of the neuronal type of splicing. Furthermore selective replacement of the acceptor site as well as deletions or replacements in the muscle-specific exon induced muscle-type splicing to various extents in undifferentiated myogenic cells. Therefore sequence elements in the distal part of the optional intron and in the muscle-specific exon contribute to the suppression of muscle-specific SERCA2 splicing.

Alternative Splicing↗
PubMed1996

Kinetics of the non-specific calcium leak from non-mitochondrial calcium stores in permeabilized A7r5 cells.

We have investigated the detailed kinetics of the passive Ca2+ leak from non-mitochondrial Ca2+ stores in permeabilized A7r5 cells. The decrease in the content of stored Ca2+ in the presence of 2 microM thapsigargin deviated from a single-exponential curve in the initial phase of the efflux. The deviation persisted after correcting this efflux for passively bound Ca2+. The non-single-exponential nature of the spontaneous release also occurred when the initial store Ca2+ content was reduced to 40% of its original value by pretreatment with 200 nM inositol 1,4,5-trisphosphate (InsP3). The passive Ca2+ leak could be modelled by two exponential curves with discrete rate constants of 0.06 min-1 and 0.98 min-1, and not by any other type of non-exponential decay. We concluded that individual store units are heterogeneous with respect to their passive Ca2+ permeability. This non-exponential nature of the passive Ca2+ release is unrelated to the non-single-exponential InsP3-induced Ca2+ release.

Animals↗