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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 distribution in spinal cord by light microscopy. Representative sections are shown from lumbar spinal cord sections from control, ALS and FTLD-TDP patients stained for RBM45 and counterstained with hematoxylin. a, b Motor neurons from control subjects show a punctate staining of the nucleus and cytoplasm. c–e Motor neurons from ALS patients, including a C9ORF72 case in e, contain RBM45-positive inclusions with globular, skein-like and neuritic morphology. Arrow in e indicates a glial inclusion. f Two glial inclusions from a sporadic ALS patient are shown (arrows). g RBM45 positive inclusions were also detected in the motor neurons of non-SOD1, non-C9ORF72 fALS cases. h Glial inclusions are also observed in fALS. i Spinal cord motor neuron of FTLD-TDP case containing skein-like RBM45 inclusion. All images are taken at ×40 magnifications. Scale bars equal 30 μm. Panels represent the following case numbers in Table 1: a 37, b 39, c 2, d 2, e 3, f 13, g 23, h 21, i 24
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Fig2: RBM45 distribution in spinal cord by light microscopy. Representative sections are shown from lumbar spinal cord sections from control, ALS and FTLD-TDP patients stained for RBM45 and counterstained with hematoxylin. a, b Motor neurons from control subjects show a punctate staining of the nucleus and cytoplasm. c–e Motor neurons from ALS patients, including a C9ORF72 case in e, contain RBM45-positive inclusions with globular, skein-like and neuritic morphology. Arrow in e indicates a glial inclusion. f Two glial inclusions from a sporadic ALS patient are shown (arrows). g RBM45 positive inclusions were also detected in the motor neurons of non-SOD1, non-C9ORF72 fALS cases. h Glial inclusions are also observed in fALS. i Spinal cord motor neuron of FTLD-TDP case containing skein-like RBM45 inclusion. All images are taken at ×40 magnifications. Scale bars equal 30 μm. Panels represent the following case numbers in Table 1: a 37, b 39, c 2, d 2, e 3, f 13, g 23, h 21, i 24

Mentions: We next examined the cell type-specific expression patterns and subcellular localization of RBM45 in ALS and control subjects using immunohistochemistry. Non-neurologic disease control subjects exhibited a punctate staining pattern for RBM45 in the nucleus and cytoplasm of motor neurons in the lumbar spinal cord, with limited staining of nuclei within glial cells (Fig. 2a, b). Examination of sporadic and non-SOD1 familial ALS spinal cord tissue revealed RBM45-positive inclusion pathology bearing a striking resemblance to that seen with TDP-43 or FUS in ALS motor neurons (Fig. 2c–e). Several distinct morphologies were observed, including skein-like (Fig. 2d), globular (Fig. 2c, e), and neuritic (Fig. 2d, e) inclusions. Prior studies have demonstrated a clearance of TDP-43 from the nucleus of neurons that harbor cytoplasmic inclusions [23, 36]. However, we observed motor neurons with cytoplasmic RBM45 inclusions that retained RBM45 in a speckled or diffuse staining pattern in the nucleus (Fig. 2d). In addition, we were able to detect glial inclusions that stained positive for RBM45 (Fig. 2e, f). We also detected RBM45 inclusions in the spinal cord of familial ALS and FTLD-TDP cases (Fig. 2g–i). A semi-quantitative assessment of RBM45 and TDP-43 pathology is shown in Table 2. Within the spinal cord, we observed nuclear cytoplasmic RBM45 inclusions in 78 % of ALS patients (n = 18 of 23 cases). RBM45 glial cytoplasmic inclusions were observed in the spinal cord from 74 % of ALS patients (n = 17 of 23 cases). Two cases displayed RBM45 glial inclusions but no motor neuron inclusions (cases 18 and 21). Conversely, no RBM45-containing inclusions were observed in either cell type in the spinal cord of control subjects. From these data, there appeared to be a relationship between the RBM45 and TDP-43 pathology that was further examined by double-label confocal microscopy as described below.Fig. 2


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 distribution in spinal cord by light microscopy. Representative sections are shown from lumbar spinal cord sections from control, ALS and FTLD-TDP patients stained for RBM45 and counterstained with hematoxylin. a, b Motor neurons from control subjects show a punctate staining of the nucleus and cytoplasm. c–e Motor neurons from ALS patients, including a C9ORF72 case in e, contain RBM45-positive inclusions with globular, skein-like and neuritic morphology. Arrow in e indicates a glial inclusion. f Two glial inclusions from a sporadic ALS patient are shown (arrows). g RBM45 positive inclusions were also detected in the motor neurons of non-SOD1, non-C9ORF72 fALS cases. h Glial inclusions are also observed in fALS. i Spinal cord motor neuron of FTLD-TDP case containing skein-like RBM45 inclusion. All images are taken at ×40 magnifications. Scale bars equal 30 μm. Panels represent the following case numbers in Table 1: a 37, b 39, c 2, d 2, e 3, f 13, g 23, h 21, i 24
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Fig2: RBM45 distribution in spinal cord by light microscopy. Representative sections are shown from lumbar spinal cord sections from control, ALS and FTLD-TDP patients stained for RBM45 and counterstained with hematoxylin. a, b Motor neurons from control subjects show a punctate staining of the nucleus and cytoplasm. c–e Motor neurons from ALS patients, including a C9ORF72 case in e, contain RBM45-positive inclusions with globular, skein-like and neuritic morphology. Arrow in e indicates a glial inclusion. f Two glial inclusions from a sporadic ALS patient are shown (arrows). g RBM45 positive inclusions were also detected in the motor neurons of non-SOD1, non-C9ORF72 fALS cases. h Glial inclusions are also observed in fALS. i Spinal cord motor neuron of FTLD-TDP case containing skein-like RBM45 inclusion. All images are taken at ×40 magnifications. Scale bars equal 30 μm. Panels represent the following case numbers in Table 1: a 37, b 39, c 2, d 2, e 3, f 13, g 23, h 21, i 24
Mentions: We next examined the cell type-specific expression patterns and subcellular localization of RBM45 in ALS and control subjects using immunohistochemistry. Non-neurologic disease control subjects exhibited a punctate staining pattern for RBM45 in the nucleus and cytoplasm of motor neurons in the lumbar spinal cord, with limited staining of nuclei within glial cells (Fig. 2a, b). Examination of sporadic and non-SOD1 familial ALS spinal cord tissue revealed RBM45-positive inclusion pathology bearing a striking resemblance to that seen with TDP-43 or FUS in ALS motor neurons (Fig. 2c–e). Several distinct morphologies were observed, including skein-like (Fig. 2d), globular (Fig. 2c, e), and neuritic (Fig. 2d, e) inclusions. Prior studies have demonstrated a clearance of TDP-43 from the nucleus of neurons that harbor cytoplasmic inclusions [23, 36]. However, we observed motor neurons with cytoplasmic RBM45 inclusions that retained RBM45 in a speckled or diffuse staining pattern in the nucleus (Fig. 2d). In addition, we were able to detect glial inclusions that stained positive for RBM45 (Fig. 2e, f). We also detected RBM45 inclusions in the spinal cord of familial ALS and FTLD-TDP cases (Fig. 2g–i). A semi-quantitative assessment of RBM45 and TDP-43 pathology is shown in Table 2. Within the spinal cord, we observed nuclear cytoplasmic RBM45 inclusions in 78 % of ALS patients (n = 18 of 23 cases). RBM45 glial cytoplasmic inclusions were observed in the spinal cord from 74 % of ALS patients (n = 17 of 23 cases). Two cases displayed RBM45 glial inclusions but no motor neuron inclusions (cases 18 and 21). Conversely, no RBM45-containing inclusions were observed in either cell type in the spinal cord of control subjects. From these data, there appeared to be a relationship between the RBM45 and TDP-43 pathology that was further examined by double-label confocal microscopy as described below.Fig. 2

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