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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 - PubMed

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

Mahlon Collins et al. Acta Neuropathol. 2012 Nov.

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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Figures

Fig. 1
Fig. 1

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

Fig. 2
Fig. 2

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

Fig. 3
Fig. 3

RBM45 localization in the hippocampus. Representative hippocampal sections from ALS, FTLD-TDP and control subjects immunostained for RBM45 and counterstained with hematoxylin. a–c RBM45 immunoreactivity in non-neurologic disease controls. RBM45 is located in the nucleus of dentate granule cells. d–f RBM45 cytoplasmic inclusions are observed in dentate granule cells in FTLD-TDP cases (arrows in e and f). g RBM45 pathology in the hippocampus of sporadic ALS. h, i Rare CA3 pyramidal neurons in FTLD-TDP cases contained RBM45 inclusions. For panels a and d, the magnification is ×10 and the scale bars denote 60 μm, while in all other panels the magnification is ×40 and scale bars indicate 20 μm. Panels represent the following case numbers in Table 1: a 38, b 40, c 39, d 29, e 27, f 26, g 17, h 29, i 24

Fig. 4
Fig. 4

RBM45 and tau pathology do not overlap in AD cases. a, b Hippocampal sections from two AD cases stained for RBM45 (green) and phosphorylated tau (red) with nuclei (DAPI-blue) in the merged image. Abundant pTau pathology is seen in both cases, as well as RBM45 inclusions marked with arrows. No overlap of pTau pathology with RBM45 inclusions or speckled RBM45 nuclear staining is seen. Scale bar 20 μm. Panels represent the following case numbers in Table 1: a 30, b 33

Fig. 5
Fig. 5

Double-label immunofluorescence for RBM45 (green) and TDP-43 (red) in the spinal cord and hippocampus. a–c RBM45 co-localization with TDP-43 positive inclusions in ALS spinal cord motor neurons. DAPI visualizes nuclei (blue) in the merged images. RBM45 also remained in the nucleus of a motor neuron with cytoplasmic TDP-43 inclusions (c). d–f RBM45 co-localization with TDP-43 inclusions in the dentate gyrus of FTLD-TDP (d, e) and AD (f) cases. Arrows denote co-localization in intranuclear (d) and cytoplasmic (e, f) inclusions. Speckled RBM45 nuclear stain is observed in all panels and is devoid of TDP-43. Scale bars denote 20 μm in panels a–c and 30 μm in panels d–f. Each panel represents the following case numbers in Table 1: a 2, b 3, c 7, d 24, e 28, f 30

Fig. 6
Fig. 6

TDP-43 pathology can occur independent of RBM45 pathology in ALS and FTLD cases. RBM45 is labeled in green, TDP-43 denoted in red and DAPI (blue) visualizes nuclei in the merged images. a Motor neuron with nuclear RBM45 and a TDP-43 positive inclusion that labels poorly for RBM45. Several such inclusions were found throughout the lumbar spinal cord of sALS and fALS cases. b Motor neurons from C9ORF72 ALS cases exhibited nuclear depletion of RBM45. RBM45 was not contained in the TDP-43 inclusions in this motor neuron. c Several TDP-43 positive, RBM45 negative inclusions are indicated by arrowheads in the dentate gyrus of an FTLD case. While no RBM45 positive inclusions were seen, the speckled nuclear staining pattern was observed in several adjacent cells. Scale bar 20 μm. Panels represent the following case numbers in Table 1: a 7, b 2, c 27

Fig. 7
Fig. 7

Double-label immunofluorescence for RBM45 (green) and ubiquitin (red) in the spinal cord and hippocampus. a–c RBM45 co-localization with ubiquitin in ALS spinal cord motor neurons. DAPI visualizes nuclei (blue) in the merged images. Cytoplasmic ubiquitin inclusions are positive for RBM45; however, nuclear RBM45 is not labeled by ubiquitin (c). d, e Co-localization of RBM45 to ubiquitin cytoplasmic inclusions in the dentate gyrus. Arrows denote cells that exhibit co-localization of RBM45 and ubiquitin within an inclusion but retaining a speckled RBM45 nuclear immunostain. Note the lack of RBM45 labeling of an ubiquitin positive dystrophic neurite in the dentate gyrus as indicated by arrowhead (d). Scale bars denote 20 μm in panels a–d and 30 μm in panel e. Each panel represents the following case numbers in Table 1: a 10, b 7, c 22, d 27, e 26

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