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The RNA-binding motif 45 (RBM45) protein accumulates in inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) patients.

Collins M, Riascos D, Kovalik T, An J, Krupa K, Krupa K, Hood BL, Conrads TP, Renton AE, Traynor BJ, Bowser R - Acta Neuropathol. (2012)

Bottom Line: These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus.By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies.In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin.

View Article: PubMed Central - PubMed

Affiliation: Departments of Neurobiology and Pathology, University of Pittsburgh, PA, USA.

ABSTRACT
RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer's disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.

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RBM45 immunoblot analysis. a Representative immunoblot comparing the level of RBM45 between 9 control and 9 ALS CSF samples. Equal amount of protein (10 μg) was loaded on each gel lane. Right panel is a densitometric analysis for all cases and comparison between ALS and controls. The difference between ALS and controls was not statistically significant by Mann–Whitney t test (p = 0.28). b Immuoblot analysis of lumbar spinal cord tissue extracts. The band representative of RBM45 is similar across all samples. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. The right panel is a densitometric analysis for all cases normalized to the level of GAPDH in each sample. No significant difference was seen between ALS and controls (p = 0.89). AU Arbitrary units
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Fig1: RBM45 immunoblot analysis. a Representative immunoblot comparing the level of RBM45 between 9 control and 9 ALS CSF samples. Equal amount of protein (10 μg) was loaded on each gel lane. Right panel is a densitometric analysis for all cases and comparison between ALS and controls. The difference between ALS and controls was not statistically significant by Mann–Whitney t test (p = 0.28). b Immuoblot analysis of lumbar spinal cord tissue extracts. The band representative of RBM45 is similar across all samples. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. The right panel is a densitometric analysis for all cases normalized to the level of GAPDH in each sample. No significant difference was seen between ALS and controls (p = 0.89). AU Arbitrary units

Mentions: We first verified the presence of RBM45 in the CSF and spinal cord tissue extracts of ALS and control subjects by immunoblot. Using an affinity-purified antibody that recognizes amino acids 216–267 of RBM45 that lacks homology to TDP-43 or FUS, RBM45 protein was evident in all CSF samples, with a modest increase in ALS patients (Fig. 1a). We next prepared total protein extracts from spinal cord tissue of ALS and control subjects and detected similar levels of RBM45 protein in all samples (Fig. 1b). Therefore, we verified the presence of RBM45 in both CSF and spinal cord tissue, with a trend towards increased CSF levels in ALS patients.Fig. 1


The RNA-binding motif 45 (RBM45) protein accumulates in inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) patients.

Collins M, Riascos D, Kovalik T, An J, Krupa K, Krupa K, Hood BL, Conrads TP, Renton AE, Traynor BJ, Bowser R - Acta Neuropathol. (2012)

RBM45 immunoblot analysis. a Representative immunoblot comparing the level of RBM45 between 9 control and 9 ALS CSF samples. Equal amount of protein (10 μg) was loaded on each gel lane. Right panel is a densitometric analysis for all cases and comparison between ALS and controls. The difference between ALS and controls was not statistically significant by Mann–Whitney t test (p = 0.28). b Immuoblot analysis of lumbar spinal cord tissue extracts. The band representative of RBM45 is similar across all samples. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. The right panel is a densitometric analysis for all cases normalized to the level of GAPDH in each sample. No significant difference was seen between ALS and controls (p = 0.89). AU Arbitrary units
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472056&req=5

Fig1: RBM45 immunoblot analysis. a Representative immunoblot comparing the level of RBM45 between 9 control and 9 ALS CSF samples. Equal amount of protein (10 μg) was loaded on each gel lane. Right panel is a densitometric analysis for all cases and comparison between ALS and controls. The difference between ALS and controls was not statistically significant by Mann–Whitney t test (p = 0.28). b Immuoblot analysis of lumbar spinal cord tissue extracts. The band representative of RBM45 is similar across all samples. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were used as a loading control. The right panel is a densitometric analysis for all cases normalized to the level of GAPDH in each sample. No significant difference was seen between ALS and controls (p = 0.89). AU Arbitrary units
Mentions: We first verified the presence of RBM45 in the CSF and spinal cord tissue extracts of ALS and control subjects by immunoblot. Using an affinity-purified antibody that recognizes amino acids 216–267 of RBM45 that lacks homology to TDP-43 or FUS, RBM45 protein was evident in all CSF samples, with a modest increase in ALS patients (Fig. 1a). We next prepared total protein extracts from spinal cord tissue of ALS and control subjects and detected similar levels of RBM45 protein in all samples (Fig. 1b). Therefore, we verified the presence of RBM45 in both CSF and spinal cord tissue, with a trend towards increased CSF levels in ALS patients.Fig. 1

Bottom Line: These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus.By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies.In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin.

View Article: PubMed Central - PubMed

Affiliation: Departments of Neurobiology and Pathology, University of Pittsburgh, PA, USA.

ABSTRACT
RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer's disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.

Show MeSH
Related in: MedlinePlus