Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot-Marie-Tooth neuropathy.

Charcot-Marie-Tooth (CMT) neuropathies are common disorders of the peripheral nervous system caused by demyelination or axonal degeneration, or a combination of both features. We previously assigned the locus for autosomal dominant intermediate CMT neuropathy type C (DI-CMTC) to chromosome 1p34-p35. Here we identify two heterozygous missense mutations (G41R and E196K) and one de novo deletion (153-156delVKQV) in tyrosyl-tRNA synthetase (YARS) in three unrelated families affected with DI-CMTC. Biochemical experiments and genetic complementation in yeast show partial loss of aminoacylation activity of the mutant proteins, and mutations in YARS, or in its yeast ortholog TYS1, reduce yeast growth. YARS localizes to axonal termini in differentiating primary motor neuron and neuroblastoma cultures. This specific distribution is significantly reduced in cells expressing mutant YARS proteins. YARS is the second aminoacyl-tRNA synthetase found to be involved in CMT, thereby linking protein-synthesizing complexes with neurodegeneration.

Ablation of proliferating microglia does not affect motor neuron degeneration in amyotrophic lateral sclerosis caused by mutant superoxide dismutase.

Microglial activation is a hallmark of all neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here, a detailed characterization of the microglial cell population within the spinal cord of a mouse model of familial ALS was performed. Using flow cytometry, we detected three distinct microglial populations within the spinal cord of mice overexpressing mutant superoxide dismutase (SOD1): mature microglial cells (CD11b(+), CD45(low)), myeloid precursor cells (CD11b(+), CD45(int)), and macrophages (CD11b(+), CD45(high)). Characterization of cell proliferation within the CNS of SOD1(G93A) mice revealed that the expansion in microglial cell population is mainly attributable to the proliferation of myeloid precursor cells. To assess the contribution of proliferating microglia in motor neuron degeneration, we generated CD11b-TK(mut-30); SOD1(G93A) doubly transgenic mice that allow the elimination of proliferating microglia on administration of ganciclovir. Surprisingly, a 50% reduction in reactive microglia specifically in the lumbar spinal cord of CD11b-TK(mut-30); SOD1(G93A) doubly transgenic mice had no effect on motor neuron degeneration. This suggests that proliferating microglia-expressing mutant SOD1 are not central contributors of the neurodegenerative process in ALS caused by mutant SOD1.

Role of mitochondria in kainate-induced fast Ca2+ transients in cultured spinal motor neurons.

Motor neuron death in amyotrophic lateral sclerosis (ALS) has been linked to selective vulnerability towards AMPA receptor-mediated excitotoxicity. We investigated intracellular mechanisms leading to impairment of motor neuron Ca2+ homeostasis with near physiological AMPA receptor activation. Using fast solution exchange on patch-clamped cultured neurons, kainate (KA) was applied for 2s. This induced a transient increase in the cytosolic Ca2+ concentration ([Ca2+]c) for seconds. Inhibition of the mitochondrial uniporter by RU-360 abolished the decay of the Ca2+ transient and caused immediate [Ca2+]c overload. Repetitive short KA stimulation caused a slowing of the decay of the Ca2+ transient and a gradual increase in peak and baseline [Ca2+]c in motor neurons, but not in other neurons, indicating saturation of the mitochondrial buffer. Furthermore, mitochondrial density was lower in motor neurons and, in a network of neurons with physiological synaptic AMPA receptor input, RU-360 acutely induced an increase in Ca2+ transients. We conclude that motor neurons have an insufficient mitochondrial capacity to buffer large Ca2+ elevations which is partly due to a reduced mitochondrial density per volume compared to non-motor neurons. This may exert deleterious effects in motor neuron disease where mitochondrial function is thought to be compromised.

Microglia in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a neurodegenerative disorder that results in the selective death of motor neurons in the central nervous system. This progressive motor neuron degeneration leads to death of the patient on average three to five years after onset of the disease. To date, no therapy is available. Many hypotheses have been formulated to explain the selective degeneration of motor neurons. One of the most studied hypotheses is the putative role of the inflammatory response that accompanies motor neuron death. The proliferation of microglia and astrocytes has been considered to be a secondary phenomenon, but recently, evidence is accumulating in favour of a contributory role of the non-neuronal cell populations to the pathogenesis of the disease. In this review, we will introduce the characteristics of microglial cells in the central nervous system. We will summarize the evidence of the expansion and the activation of the microglial cell population that accompanies motor neuron degeneration. Finally, an overview will be given of the different therapeutic strategies that targeted the inflammatory process in amyotrophic lateral sclerosis.

Role of matrix metalloproteinase-9 in a mouse model for amyotrophic lateral sclerosis.

The pathogenesis of amyotrophic lateral sclerosis remains poorly understood, but microglial and astroglial activation are thought to contribute to motor neuron death. Evidence suggests that matrix metalloproteinase-9 (MMP-9) is a mediator of this deleterious effect. In this study, we evaluated the effect of MMP-9 on the pathogenesis of amyotrophic lateral sclerosis. Although marked microglial and astroglial proliferation was seen in the spinal cord and in-vitro studies proved MMP-9 to be produced by these cells, deletion of the MMP-9 gene in SOD1(G93A) mice accelerated rather than delayed the motor neuron disease and significantly reduced survival. Our results suggest that the effect of MMP-9 on mutant superoxide dismutase-1 (SOD1)-induced motor neuron disease is protective rather than hazardous. Therefore, the effect of pharmacological inhibition of MMP-9 activity is unlikely to be of therapeutical benefit in amyotrophic lateral sclerosis.

Effects of electrical stimulation or lesion in nucleus accumbens on the behaviour of rats in a T-maze after administration of 8-OH-DPAT or vehicle.

Electrical brain stimulation may be a therapeutic alternative for irreversible lesions in treatment-resistant patients with obsessive-compulsive disorder (OCD). We compared the effects of electrical stimulation and lesion in the nucleus accumbens (n acc) on the behaviour of rats in a model for OCD. Rats were tested for spontaneous alternation behaviour (AB) in a T-maze and assigned to four groups: an electrode implant group with stimulation 'ON' (stimON) or 'OFF' (stimOFF), a lesion or a sham group. Postoperatively, the number of arm visits and AB were tested after 8-hydroxy-2-(di-n-propylamino)-tetralin hydrobromide (8-OH-DPAT; 2 mg/kg) or saline administration. After 8-OH-DPAT administration, more arm visits were counted in the stimON (92.2%) and lesion groups (79.3%) than in both control groups (stimOFF 54.2; sham 61.2%). AB was significantly decreased in the stimON (10.5%) and lesion groups (10.2%) relative to the sham (22.0%) but not to the stimOFF group (14.7%). After saline administration, rats performed more arm visits in the stimON (81.5% non-significant) and lesion groups (93.6% significant) relative to the stimOFF (70.8%) and the sham groups (74.5%). No significant differences, however, were observed for AB. In conclusion, both treatments resulted in a decreased AB after 8-OH-DPAT administration (modelling an increase in compulsions) and more arm visits.

Inhibition of p38 mitogen activated protein kinase activation and mutant SOD1(G93A)-induced motor neuron death.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective loss of motor neurons. Stress activated protein kinases (SAPK) have been suggested to play a role in the pathogenesis of ALS. We studied the relevance of p38 MAPK for motor neuron degeneration in the mutant SOD1 mouse. Increased levels of phospho-p38 MAPK were present in the motor neurons and microglia of the ventral spinal cord. The p38 MAPK-inhibitor, SB203580, completely inhibited mutant SOD1-induced apoptosis of motor neurons and blocked LPS-induced activation of microglia. Semapimod, a p38 MAPK inhibitor suitable for clinical use, prolonged survival of mutant SOD1 mice to a limited extent, but largely protected motor neurons and proximal axons from mutant SOD1-induced degeneration. Our data confirm the abnormal activation of p38 MAPK in mutant SOD1 mice and the involvement of p38 MAPK in mutant SOD1-induced motor neuron death. We demonstrate the effect of p38 MAPK inhibition on survival of mutant SOD1 mice and reveal a dissociation between the effect on survival of motor neurons and that on survival of the animal, the latter likely depending on the integrity of the entire motor axon.

Prospective exploration of biochemical tissue composition via imaging mass spectrometry guided by principal component analysis.

MALDI-based Imaging Mass Spectrometry (IMS) is an analytical technique that provides the opportunity to study the spatial distribution of biomolecules including proteins and peptides in organic tissue. IMS measures a large collection of mass spectra spread out over an organic tissue section and retains the absolute spatial location of these measurements for analysis and imaging. The classical approach to IMS imaging, producing univariate ion images, is not well suited as a first step in a prospective study where no a priori molecular target mass can be formulated. The main reasons for this are the size and the multivariate nature of IMS data. In this paper we describe the use of principal component analysis as a multivariate pre-analysis tool, to identify the major spatial and mass-related trends in the data and to guide further analysis downstream. First, a conceptual overview of principal component analysis for IMS is given. Then, we demonstrate the approach on an IMS data set collected from a transversal section of the spinal cord of a standard control rat.

Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity.

Influx of Ca(2+) ions through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca(2+) permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca(2+) ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo. Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.