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Drosha inclusions are new components of dipeptide-repeat protein aggregates in FTLD-TDP and ALS C9orf72 expansion cases.

Porta S, Kwong LK, Trojanowski JQ, Lee VM - J. Neuropathol. Exp. Neurol. (2015)

Bottom Line: Moreover, through a repeat-associated non-ATG translation mechanism, G4C2 repeats translation leads to dipeptide-repeat protein aggregation in the cytoplasm of neurons.Further characterization of Drosha-positive neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum revealed colocalization with p62 and ubiquilin-2, 2 pathognomonic signatures of c9FTLD-TDP and c9ALS cases; however, Drosha inclusions rarely colocalized with TDP-43 pathology.We conclude that Drosha may play a unique pathogenic role in the onset or progression of FTLD-TDP/ALS in patients with the C9orf72 mutation.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania.

ABSTRACT
Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are 2 neurodegenerative disorders that share clinical, genetic, and neuropathologic features. The presence of abnormal expansions of GGGGCC repeats (G4C2 repeats) in a noncoding region of the Chromosome 9 open reading frame 72 (C9orf72) gene is the major genetic cause of both FTLD and ALS. Transcribed G4C2 repeats can form nuclear RNA foci and recruit RNA-binding proteins, thereby inhibiting their normal function. Moreover, through a repeat-associated non-ATG translation mechanism, G4C2 repeats translation leads to dipeptide-repeat protein aggregation in the cytoplasm of neurons. Here, we identify Drosha protein as a new component of these dipeptide-repeat aggregates. In C9orf72 mutation cases of FTLD-TDP (c9FTLD-TDP) and ALS (c9ALS), but not in FTLD or ALS cases without C9orf72 mutation, Drosha is mislocalized to form neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum. Further characterization of Drosha-positive neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum revealed colocalization with p62 and ubiquilin-2, 2 pathognomonic signatures of c9FTLD-TDP and c9ALS cases; however, Drosha inclusions rarely colocalized with TDP-43 pathology. We conclude that Drosha may play a unique pathogenic role in the onset or progression of FTLD-TDP/ALS in patients with the C9orf72 mutation.

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Colocalization of Drosha-positive inclusions with p62 and ubiquilin-2. Double immunofluorescence (IF) results of Drosha, p62, and ubiquilin-2 in the hippocampus of c9FTLD-TDP cases. Double labeling of Drosha (A, D, red) and p62 (B) or ubiquilin-2 (E) in dentate granule neurons shows colocalization of Drosha-positive inclusions with p62 protein (C, arrowhead) and ubiquilin-2 pathology (F, arrowhead). Asterisks show some cytoplasmic aggregates positive for p62 (B, C) or ubiquilin-2 (E, F) protein that did not contain mislocalized Drosha protein (A, D). Cell nuclei are lightly stained with DAPI (blue) to enable visualization of the double label IF results. Scale bar = 10 μm.
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Figure 4: Colocalization of Drosha-positive inclusions with p62 and ubiquilin-2. Double immunofluorescence (IF) results of Drosha, p62, and ubiquilin-2 in the hippocampus of c9FTLD-TDP cases. Double labeling of Drosha (A, D, red) and p62 (B) or ubiquilin-2 (E) in dentate granule neurons shows colocalization of Drosha-positive inclusions with p62 protein (C, arrowhead) and ubiquilin-2 pathology (F, arrowhead). Asterisks show some cytoplasmic aggregates positive for p62 (B, C) or ubiquilin-2 (E, F) protein that did not contain mislocalized Drosha protein (A, D). Cell nuclei are lightly stained with DAPI (blue) to enable visualization of the double label IF results. Scale bar = 10 μm.

Mentions: The presence of p62-positive/TDP-43–negative NCIs in the hippocampus and cerebellum has been described as a key pathologic feature in c9FTLD-TDP and c9ALS cases (36, 40–47). In addition, NCIs and DNs immunoreactive for the ubiquilin-2 have been found in several brain regions of c9FTLD-TDP and c9ALS cases and are diagnostic signatures of the presence of C9orf72 mutations in these disorders (36). To determine if Drosha, TDP-43, p62, and/or ubiquilin-2 colocalize in the inclusions found in C9orf72 mutation cases, we examined pathologic NCIs in the hippocampus, frontal cortex, and cerebellum for the presence of these proteins. Analysis of double IF of Drosha-positive NCIs in the hippocampus, frontal cortex, and cerebellum of both c9FTLD-TDP and c9ALS cases showed that high percentages of them were also immunopositive for p62: 89.3% ± 11.9%, 97.1% ± 9.2%, and 92.8% ± 6.2%, respectively (Fig. 4A–C). Similar colocalization of Drosha and ubiquilin-2 was also found: 89.1% ± 14.1% in the hippocampus, 94.9% ±18.8% in the frontal cortex, and 99.5% ±1.43% in the cerebellum (Fig. 4D–F). Nonsignificant differences were observed in the percentage of colocalization between the 3 brain areas analyzed. Interestingly, only rare Drosha NCIs were found to colocalize with phosphorylated TDP-43, a hallmark of FTLD-TDP and ALS pathology (data not shown). Moreover, we noted that not all p62- and ubiquilin-2–immunoreactive inclusions contained mislocalized Drosha.


Drosha inclusions are new components of dipeptide-repeat protein aggregates in FTLD-TDP and ALS C9orf72 expansion cases.

Porta S, Kwong LK, Trojanowski JQ, Lee VM - J. Neuropathol. Exp. Neurol. (2015)

Colocalization of Drosha-positive inclusions with p62 and ubiquilin-2. Double immunofluorescence (IF) results of Drosha, p62, and ubiquilin-2 in the hippocampus of c9FTLD-TDP cases. Double labeling of Drosha (A, D, red) and p62 (B) or ubiquilin-2 (E) in dentate granule neurons shows colocalization of Drosha-positive inclusions with p62 protein (C, arrowhead) and ubiquilin-2 pathology (F, arrowhead). Asterisks show some cytoplasmic aggregates positive for p62 (B, C) or ubiquilin-2 (E, F) protein that did not contain mislocalized Drosha protein (A, D). Cell nuclei are lightly stained with DAPI (blue) to enable visualization of the double label IF results. Scale bar = 10 μm.
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Figure 4: Colocalization of Drosha-positive inclusions with p62 and ubiquilin-2. Double immunofluorescence (IF) results of Drosha, p62, and ubiquilin-2 in the hippocampus of c9FTLD-TDP cases. Double labeling of Drosha (A, D, red) and p62 (B) or ubiquilin-2 (E) in dentate granule neurons shows colocalization of Drosha-positive inclusions with p62 protein (C, arrowhead) and ubiquilin-2 pathology (F, arrowhead). Asterisks show some cytoplasmic aggregates positive for p62 (B, C) or ubiquilin-2 (E, F) protein that did not contain mislocalized Drosha protein (A, D). Cell nuclei are lightly stained with DAPI (blue) to enable visualization of the double label IF results. Scale bar = 10 μm.
Mentions: The presence of p62-positive/TDP-43–negative NCIs in the hippocampus and cerebellum has been described as a key pathologic feature in c9FTLD-TDP and c9ALS cases (36, 40–47). In addition, NCIs and DNs immunoreactive for the ubiquilin-2 have been found in several brain regions of c9FTLD-TDP and c9ALS cases and are diagnostic signatures of the presence of C9orf72 mutations in these disorders (36). To determine if Drosha, TDP-43, p62, and/or ubiquilin-2 colocalize in the inclusions found in C9orf72 mutation cases, we examined pathologic NCIs in the hippocampus, frontal cortex, and cerebellum for the presence of these proteins. Analysis of double IF of Drosha-positive NCIs in the hippocampus, frontal cortex, and cerebellum of both c9FTLD-TDP and c9ALS cases showed that high percentages of them were also immunopositive for p62: 89.3% ± 11.9%, 97.1% ± 9.2%, and 92.8% ± 6.2%, respectively (Fig. 4A–C). Similar colocalization of Drosha and ubiquilin-2 was also found: 89.1% ± 14.1% in the hippocampus, 94.9% ±18.8% in the frontal cortex, and 99.5% ±1.43% in the cerebellum (Fig. 4D–F). Nonsignificant differences were observed in the percentage of colocalization between the 3 brain areas analyzed. Interestingly, only rare Drosha NCIs were found to colocalize with phosphorylated TDP-43, a hallmark of FTLD-TDP and ALS pathology (data not shown). Moreover, we noted that not all p62- and ubiquilin-2–immunoreactive inclusions contained mislocalized Drosha.

Bottom Line: Moreover, through a repeat-associated non-ATG translation mechanism, G4C2 repeats translation leads to dipeptide-repeat protein aggregation in the cytoplasm of neurons.Further characterization of Drosha-positive neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum revealed colocalization with p62 and ubiquilin-2, 2 pathognomonic signatures of c9FTLD-TDP and c9ALS cases; however, Drosha inclusions rarely colocalized with TDP-43 pathology.We conclude that Drosha may play a unique pathogenic role in the onset or progression of FTLD-TDP/ALS in patients with the C9orf72 mutation.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, Pennsylvania.

ABSTRACT
Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are 2 neurodegenerative disorders that share clinical, genetic, and neuropathologic features. The presence of abnormal expansions of GGGGCC repeats (G4C2 repeats) in a noncoding region of the Chromosome 9 open reading frame 72 (C9orf72) gene is the major genetic cause of both FTLD and ALS. Transcribed G4C2 repeats can form nuclear RNA foci and recruit RNA-binding proteins, thereby inhibiting their normal function. Moreover, through a repeat-associated non-ATG translation mechanism, G4C2 repeats translation leads to dipeptide-repeat protein aggregation in the cytoplasm of neurons. Here, we identify Drosha protein as a new component of these dipeptide-repeat aggregates. In C9orf72 mutation cases of FTLD-TDP (c9FTLD-TDP) and ALS (c9ALS), but not in FTLD or ALS cases without C9orf72 mutation, Drosha is mislocalized to form neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum. Further characterization of Drosha-positive neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum revealed colocalization with p62 and ubiquilin-2, 2 pathognomonic signatures of c9FTLD-TDP and c9ALS cases; however, Drosha inclusions rarely colocalized with TDP-43 pathology. We conclude that Drosha may play a unique pathogenic role in the onset or progression of FTLD-TDP/ALS in patients with the C9orf72 mutation.

Show MeSH
Related in: MedlinePlus