thapsigargin and Amyotrophic-Lateral-Sclerosis

The neuroblastoma-spinal motor neuron fusion cell line, NSC-34, in its differentiated form, NSC-34D, permits examining the effects of riluzole, a proven treatment for amyotrophic lateral sclerosis (ALS) on cell death induction by staurosporine (STS), thapsigargin (Thaps), hydrogen peroxide (H(2)O(2)) and homocysteine (HCy). These neurotoxins, applied exogenously, have mechanisms of action related to the various proposed molecular pathogenetic pathways in ALS and are differentiated from endogenous cell death that is associated with cytoplasmic aggregate formation in motor neurons. Nuclear morphology, caspase-3/7 activation and high content imaging were used to assess toxicity of these neurotoxins with and without co-treatment with riluzole, a benzothiazole compound with multiple pharmacological actions. STS was the most potent neurotoxin at killing NSC-34D cells with a toxic concentration at which 50% of maximal cell death is achieved (TC(50)=0.01μM), followed by Thaps (TC(50)=0.9μM) and H(2)O(2) (TC(50)=15μM) with HCy requiring higher concentrations to kill at the same level (TC(50)=2200μM). Riluzole provided neurorescue with a 20% absolute reduction (47.6% relative reduction) in apoptotic cell death against Thaps-induced NSC-34D cell (p≤0.05), but had no effect on STS-, H(2)O(2)- and HCy-induced NSC-34D cell death. This effect of riluzole on Thaps induction of cell death was independent of caspase-3/7 activation. Riluzole mitigated a toxin that can cause intracellular calcium dysregulation associated with endoplasmic reticulum (ER) stress but not toxins associated with other cell death mechanisms.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Calcium; Caspase 3; Caspase 7; Cell Line; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Homocysteine; Hybrid Cells; Hydrogen Peroxide; Mice; Motor Neurons; Neuroblastoma; Neuroprotective Agents; Neurotoxins; Riluzole; Staurosporine; Thapsigargin

TDP-43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis and other neurodegenerative diseases. Here we demonstrate, using neuronal and spinal cord organotypic culture models, that chronic excitotoxicity, oxidative stress, proteasome dysfunction and endoplasmic reticulum stress mechanistically induce mislocalization, phosphorylation and aggregation of TDP-43. This is compatible with a lack of function of this protein in the nucleus, specially in motor neurons. The relationship between cell stress and pathological changes of TDP-43 also includes a dysfunction in the survival pathway mediated by mitogen-activated protein kinase/extracellular signal-regulated kinases (ERK1/2). Thus, under stress conditions, neurons and other spinal cord cells showed cytosolic aggregates containing ERK1/2. Moreover, aggregates of abnormal phosphorylated ERK1/2 were also found in the spinal cord in amyotrophic lateral sclerosis (ALS), specifically in motor neurons with abnormal immunoreactive aggregates of phosphorylated TDP-43. These results demonstrate that cellular stressors are key factors in neurodegeneration associated with TDP-43 and disclose the identity of ERK1/2 as novel players in the pathogenesis of ALS.

Topics: Aged; Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Case-Control Studies; Cell Line, Transformed; DNA-Binding Proteins; Enzyme Inhibitors; Female; Gene Expression Regulation; Humans; Hydrogen Peroxide; Male; Middle Aged; Mitogen-Activated Protein Kinases; Motor Neurons; Neurons; Oligopeptides; Organ Culture Techniques; Oxidants; Oxidative Stress; Rats; Signal Transduction; Spinal Cord; Thapsigargin; Transfection

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by loss of motor neurons in the brain and spinal cord, leading to muscle weakness and eventually death from respiratory failure. ALS is familial in about 10% of cases, with SOD1 mutations accounting for 20% of familial cases. Here we describe a consanguineous family segregating juvenile ALS in an autosomal recessive pattern and describe the genetic variant responsible for the disorder.. We performed homozygosity mapping and direct sequencing to detect the genetic variant and tested the effect of this variant on a motor neuron-like cell line model (NSC34) expressing the wild-type or mutant gene.. We identified a shared homozygosity region in affected individuals that spans ~120 kbp on chromosome 9p13.3 containing 9 RefSeq genes. Sequencing the SIGMAR1 gene revealed a mutation affecting a highly conserved amino acid located in the transmembrane domain of the encoded protein, sigma-1 receptor. The mutated protein showed an aberrant subcellular distribution in NSC34 cells. Furthermore, cells expressing the mutant protein were less resistant to apoptosis induced by endoplasmic reticulum stress.. Sigma-1 receptors are known to have neuroprotective properties, and recently Sigmar1 knockout mice have been described to have motor deficiency. Our findings emphasize the role of sigma-1 receptors in motor neuron function and disease.

Topics: Amyotrophic Lateral Sclerosis; Animals; Apoptosis; Cell Line; Child; Child, Preschool; Chromosome Mapping; Chromosomes, Human, Pair 9; Cloning, Molecular; Enzyme Inhibitors; Family Health; Female; Genetic Predisposition to Disease; Glutamic Acid; Glutamine; Humans; In Situ Nick-End Labeling; Infant; Male; Mice; Motor Neurons; Mutagenesis, Site-Directed; Phenotype; Polymorphism, Single Nucleotide; Receptors, sigma; Saudi Arabia; Sigma-1 Receptor; Thapsigargin; Transfection