Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72

Abstract

Background: Cases of Frontotemporal Lobar Degeneration (FTLD) and Motor Neurone Disease (MND) associated with expansions in C9ORF72 gene are characterised pathologically by the presence of TDP-43 negative, but p62 positive, inclusions in granule cells of the cerebellum and in cells of dentate gyrus and area CA4 of the hippocampus.

Results: We screened 84 cases of pathologically confirmed FTLD and 23 cases of MND for the presence of p62 positive inclusions in these three brain regions, and identified 13 positive cases of FTLD and 3 of MND. All cases demonstrated expansions in C9ORF72 by Southern blotting where frozen tissues were available. The p62 positive inclusions in both cerebellum and hippocampus were immunostained by antibodies to dipeptide repeat proteins (DPR), poly Gly-Ala (poly-GA), poly Gly-Pro (poly-GP) and poly Gly-Arg (poly-GR), these arising from a putative non-ATG initiated (RAN) sense translation of the GGGGCC expansion. There was also some slight, but variable, immunostaining with poly-AP antibody implying some antisense translation might also occur, though the relative paucity of immunostaining could reflect poor antigen avidity on the part of the antisense antibodies. Of the FTLD cases with DPR, 6 showed TDP-43 type A and 6 had TDP-43 type B histology; one had FTLD-tau with the pathology of corticobasal degeneration. There were no qualitative or quantitative differences in the pattern of immunostaining with antibodies to DPR, or p62, proteins between TDP-43 type A and type B cases. Ratings for frequency of inclusions immunostained by these poly-GA, poly-GP and poly-GR antibodies broadly correlated with those for immunolabelled by p62 antibody in all three regions.

Conclusion: We conclude that DPR are a major component of p62 positive inclusions in FTLD and MND.

Figure 1

Southern blotting of FTLD and MND cases bearing expansions in C9ORF72. Lane 1: Marker, Lane 2: Negative control brain, Lane 3: ND06769, Lane 4: 01/06 FTD case #3, Lane 5: MND case #15, Lane 6: FTD/MND case #11, Lane 7: FTD case #10, Lane 8: CBD case #13, Lane 9: FTD case #1; Lane 10: control FTD case with tauopathy, Lane 11: FTD case #9, Lane 12: FTD/MND case #12, Lane 13: FTD case #2; Lane 14: MND case #14, Lane 15: FTD/MND case #7, Lane 20: MND case#16. Lanes 16-19 show expansions in other clinically diagnosed cases of MND (positive controls) where no brain tissue was available.

Figure 2

Immunostaining of the cerebellum and hippocampus for p62 proteins shows neuronal cytoplasmic inclusions in granular neurones (a), basket cells (b), Purkinje cells (c) and cells of the dentate nucleus (d) of the cerebellum, and in dentate gyrus granule cells (e) and pyramidal cells of CA4 region (f). Immunoperoxidase–haematoxylin. All × 40 microscope objective magnification.

Figure 3

Immunostaining of the cerebellum and hippocampus for poly-GA protein shows neuronal cytoplasmic inclusions in granular neurones (a), basket cells (b), Purkinje cells (c) and cells of the dentate nucleus (d) of the cerebellum, and in dentate gyrus granule cells (e) and pyramidal cells of CA4 region (f), similar to those seen on p62 immunostaining. Immunoperoxidase–haematoxylin. All × 40 microscope objective magnification.

Figure 4

Immunostaining of Pukinje cells of the cerebellum (a) and pyramidal cells of CA4 region of hippocampus (b) for poly-PR protein shows strong immunoreactivity of chromatin. Note lack of staining of NCI in either cell type. Immunoperoxidase–haematoxylin. All × 40 microscope objective magnification.