Proteins In Amyotrophic Lateral Sclerosis Research Articles

Novel Mutations in TARDBP (TDP-43) in Patients with Familial Amyotrophic Lateral Sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43–positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the ∼25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.

P3-270 Solubility characteristics of tau protein in amyotrophic lateral sclerosis with cognitive impairment (ALSCI)

P3-270 Solubility characteristics of tau protein in amyotrophic lateral sclerosis with cognitive impairment (ALSCI)

Human Glioma Cells and Undifferentiated Primary Astrocytes That Express Aberrant EAAT2 mRNA Inhibit Normal EAAT2 Protein Expression and Prevent Cell Death

Abnormal splicing of astroglial glutamate transporter EAAT2 mRNA has been suggested to account for the loss of EAAT2 protein in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). We have identified several clones of human U251 glioma cells which express varying amounts of aberrantly spliced EAAT2 mRNA; these clones do not express any detectable EAAT2 protein. When the wild-type EAAT2 cDNA was expressed in each of these clones, we found that the amount of EAAT2 protein inversely correlated with the levels of endogenous aberrant EAAT2 mRNA. We also observed that ectopic expression of normal EAAT2 protein is toxic to U251 cells as well as to undifferentiated primary astrocytes. We conclude that expression of aberrant EAAT2 mRNA may be one possible mechanism to repress normal EAAT2 protein expression. The implication of this study for the mechanisms of EAAT2 protein loss in ALS and AD is discussed.

Immunoreactivity of beta-amyloid precursor protein in amyotrophic lateral sclerosis.

We investigated the localization and extent of beta-amyloid precursor protein (beta-APP695) immunoreactivity as a sensitive marker for impairment of fast axonal transport in the spinal cords of 21 patients with amyotrophic lateral sclerosis (ALS), paying special attention to anterior horn neurons. Specimens from 18 patients without neurological disease served as controls. Increased beta-APP immunoreactivity was frequently recognized in the anterior horns of the ALS patients with short clinical courses or with mild depletion of anterior horn cells, while no beta-APP immunoreactivity was demonstrated in those with severe depletion of anterior horn neurons or with long-standing clinical courses. Increased beta-APP immunoreactivity in the anterior horn neurons was mainly confined to the perikarya and no immunoreactivity was recognized in the dendrites or proximal axons directly emanating from the somata, except some spheroids (proximal axonal swellings) which showed increased immunoreactivity of beta-APP. Increased beta-APP immunoreactivity was spotted or focally aggregated in the perikarya of normal-looking large anterior horn neurons, while it was frequently diffuse in that of degenerative neurons such as central chromatolytic cells and or those with simple atrophy. On the other hand, the controls showed no immunostaining with beta-APP in the spinal cord. These findings suggest that increased immunoreactivity of beta-APP in neuronal perikarya of the anterior horn cells and in some proximal axonal swellings is an early change of ALS, and may be a response of the increased synthesis of beta-APP resulting from neuronal damage, or the impairment of fast axonal transport.

Antibody to an abnormal protein in amyotrophic lateral sclerosis identifies Lewy body-like inclusions in ALS and Lewy bodies in Parkinson's disease

Ubiquitinated cytoplasmic inclusions are a characteristic feature of the neuronal pathology of neurodegenerative diseases. Immunocytochemical techniques have identified intermediate filaments associated with ubiquitin-immunoreactive inclusions in Alzheimer's disease (AD), Parkinson's disease (PD), and Pick's disease; however, no core protein has been detected in the ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS). The pathogenesis of these inclusions is not known, but the inclusion may result from an accumulation of an abnormal proteins. Here we report a novel protein of 32.5 kDa detected by polyacrylamide gel electrophoresis, in the spinal cord in ALS patients. A polyclonal antibody raised against this protein and used for Western blotting, suggests that the novel protein is related to actin. Immunocytochemical studies using this antibody indicate that the protein is found in Lewy body-like inclusions in anterior horn cells of ALS, and in Lewy bodies in the substantia nigra in PD.

Spinal Fluid Cells and Protein in Amyotrophic Lateral Sclerosis

The cerebrospinal fluid total protein in 385 cases of sporadic amyotrophic lateral sclerosis showed no relationship to survival, but it was related to survival time in 34 cases of familial amyotrophic lateral sclerosis. Infrequent and mild pleocytosis, and oligoclonal bands seemed to have no clinical significance in well established cases of amyotrophic lateral sclerosis.

Immunoblot analysis of circulating antibodies against muscle proteins in amyotrophic lateral sclerosis and other neurologic diseases.

Serum from patients with amyotrophic lateral sclerosis (ALS) was tested by immunoblotting for reactivity against three muscle-derived preparations: denervated chick leg muscle extracts, media conditioned by denervated rat hemidiaphragms, and a human muscle extract. For each preparation, multiple bands were present using serum from all patients and controls, and no band was unique to ALS. Against rat muscle-conditioned medium, bands in the 56,000 (56K) molecular weight range were present to an equal degree in ALS and in controls; in addition, different bands near 56K were recognized by serum from different ALS patients. These results fail to confirm a recent report suggesting that anti-56K reactivity is characteristic of ALS.

Search for antibodies to skeletal muscle proteins in amyotrophic lateral sclerosis.

We have investigated serum from 16 patients with amyotrophic lateral sclerosis (ALS) and 15 patients with other neurological diseases (OND) for antibodies which react with skeletal muscle derived antigens. Two different preparations of skeletal muscle antigens were used: i) proteins released from rat diaphragm in organ culture and ii) proteins obtained from a muscle homogenate. We also compared antigen preparations from denervated and non-denervated muscles. A rabbit anti-serum reacted strongly with three proteins in both types of protein preparation, but none of these proteins were specific to denervated muscle. None of the 16 ALS sera could be shown to react with protein antigens from any of the muscle preparations. One of the ALS patients had an IgG lambda monoclonal protein in the setting of a multiple myeloma. This monoclonal protein did not react with protein antigen from rat muscle, human grey or white matter.