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Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis

Acta Neuropathologica volume 135, pages 459–474 (2018)Cite this article

Abstract

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically related and clinically unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically related areas compared to unrelated areas (p < 0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p < 0.0001). While most poly-GR dendritic inclusions were pTDP-43 positive, only 4% of pTDP-43 dendritic inclusions were poly-GR positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin β1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to dendrites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites.

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Acknowledgements

This research was supported by Grants from ALS Association (5356S3), Target ALS (20134792), National Institute of Neurological Diseases and Stroke (NIH R01NS088578 and NS047101), and Pam Golden. JJ is a recipient of career development Grant from Muscular Dystrophy Association (479769) and was supported by postdoctoral training Grant (T32 AG00216) and postdoctoral fellowship (F32 NS087842) from the NIH. MB is supported by NIH NIGMS Award T32GM008666.

Author information

Author notes

  1. Shahram Saberi

    Present address: Department of Pathology, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy, Place Box 1194, New York, NY, 10029, USA

  2. Jennifer E. Stauffer

    Present address: Jackson Laboratory, 600 Main St, Bar Harbor, ME, 04609, USA

  3. Derek Schulte

    Present address: NeuroPace, Inc, 455 N. Bernardo Ave, Mountain View, CA, 94043, USA

Authors and Affiliations

  1. Department of Neurosciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0624, USA

    Shahram Saberi, Jennifer E. Stauffer, Jie Jiang, Sandra Diaz Garcia, Amy E. Taylor, Derek Schulte, Takuya Ohkubo, Maria J. Rodriguez & John Ravits

  2. Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA

    Michael Baughn & Don Cleveland

  3. Laboratory for Cell Biology, Ludwig Institute for Cancer Research, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0670, USA

    Jie Jiang, Cheyenne L. Schloffman, Marcus Maldonado & Don Cleveland

  4. Department of Pathology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA

    Don Pizzo

Authors
  1. Shahram Saberi
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  2. Jennifer E. Stauffer
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  3. Jie Jiang
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  4. Sandra Diaz Garcia
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  5. Amy E. Taylor
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  6. Derek Schulte
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  7. Takuya Ohkubo
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  8. Cheyenne L. Schloffman
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  9. Marcus Maldonado
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  10. Michael Baughn
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  11. Maria J. Rodriguez
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  12. Don Pizzo
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  13. Don Cleveland
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  14. John Ravits
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Corresponding author

Correspondence to John Ravits.

Additional information

Shahram Saberi, Jennifer E. Stauffer and Jie Jiang contributed equally.

Electronic supplementary material

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401_2017_1793_MOESM1_ESM.pdf

Supplemental Figure 1 Dipeptide repeat protein distribution in different CNS regions. (a-h) Burden of all DPRs in different layers of cortex. Motor cortex (a) and frontal cortex (b) are disease-related parts of cortex, while occipital cortex (c) and parietal cortex (d) are disease-unrelated parts. Retrosplenial granular cortex, cerebellum, hippocampus and olfactory bulb are of uncertain relation to disease. All DPRs are more abundant in layers of brain with prominent neuronal population (layers 2-6). No DPRs could be seen in subcortical white matter with mostly glial and oligodendrocyte population. (i) Burden of DPRs in different parts of lumbar spinal cord. No DPRs could be seen in white matter (columns) of spinal cord with mostly glial and oligodendrocyte population. (j) Specificity of poly-GR immunoreactivity is demonstrated by quenching antibody with recombinant GST-(GR)10 proteins. (k-l) When analyzed by actual percentage as opposed to grading of neurons that contain DPRs, poly-GR is still uniquely significantly more abundant in disease-related areas of CNS (PDF 12142 kb)

401_2017_1793_MOESM2_ESM.pdf

Supplemental Figure 2 RanGap, Lamin B1, Nup205 and Importin β1 immunohistochemical (IHC) staining in C9 ALS and controls. (a-d) IHC identifying RanGap in Betz cells (a-b) and spinal motor neurons (c-d) of C9 ALS (a and c) and controls (b and d). (e-h) IHC identifying Lamin B1 in Betz cells (e-f) and spinal motor neurons (g-h) of C9 ALS (e and g) and controls (f and h). Patterns of expression of Lamin B1 is very similar as RanGap. (i-l) IHC identifying Nup205 in Betz cells (i-j) and spinal motor neurons (k-l) of C9 ALS (i and k) and controls (j and l). Despite the prominent nuclear signal seen with RanGap and Lamin B1, the signal with Nup205 is mostly diffuse in the cytoplasm. (m-n) There is no significant differences between controls, SALS and C9-ALS cases in patterns of expression of Lamin B1 in spinal motor neurons. (o-p) IHC identifying Importin β1 in spinal motor neurons shows smooth, intermediate or irregular patterns (o) and there are no significant differences between controls, SALS and C9-ALS in patterns of expression (p) (PDF 9690 kb)

401_2017_1793_MOESM3_ESM.pdf

Supplemental Figure 3 Diffuse nuclear pattern of RanGap is a three-dimensional effect. (a–d) Examples of diffuse nuclear RanGap signal in the motor neurons of spinal cord in C9 ALS cases when confocal images are stacked. On the left side and at the bottom of each panel are the transversal views of each sample. Below (red line) and to the left (yellow line) are the corresponding horizontal and transversal sections of each neuron. (a´–d´) show nuclear ring pattern seen by a three-dimensional plane in which image is cut through the cell (a´, b´, c´ and d´ are different co-focal planes through a, b, c and d, respectively) (PDF 9846 kb)

401_2017_1793_MOESM4_ESM.pdf

Supplemental Figure 4 Aberrant nuclear shapes in C9 ALS and SALS patients. (a–i) Nuclear morphologies were evaluated based on RanGap IF staining. We classified them as normal (a–c), moderately aberrant (d–f), and severely aberrant (g–i). (j) Evaluating the shape of the spinal motor neuron nucleus with RanGap shows slightly more moderately and severely aberrant nucleus in ALS cases (both SALS and C9 ALS) in comparison to controls (PDF 16387 kb)

401_2017_1793_MOESM5_ESM.pdf

Supplemental Figure 5 Short and long C9orf72 mRNA and proteins. (a–b) C9orf72 protein detection with immunohistochemical staining shows significant decrease after quenching with recombinant C9orf72 proteins in human tonsil epithelial lining. (c) Western blotting shows that C9orf72 protein expression is not decreased in cerebellum of C9 ALS compared to control and SALS cases. (d–e) Short (d) and long (e) mRNA foci (small black dot-like signals) could be seen in the epithelium of human tonsil, using CISH (chromogenic in situ hybridization), but not in the same tissue without applying probes (d and e-insert). (f) No predicted short isoform C9orf72 protein is detected using antibodies generated with amino acid 1-169, which should recognize both long and the predicted short isoform C9orf72 protein (PDF 24307 kb)

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Saberi, S., Stauffer, J.E., Jiang, J. et al. Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis. Acta Neuropathol 135, 459–474 (2018). https://doi.org/10.1007/s00401-017-1793-8

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  • DOI: https://doi.org/10.1007/s00401-017-1793-8

Keywords

  • C9orf72
  • Hexanucleotide repeat expansions
  • Amyotrophic lateral sclerosis
  • Dipeptide repeat proteins
  • Arginine-containing dipeptide repeat proteins
  • Poly-GR
  • Sense strand
  • Antisense strand
  • Nuclear pore complex and nucleocytoplasmic transport