In C9ORF72 Expansion Carriers, Protein Levels Drop in Cerebellum

C9ORF72 ALS, Gain and Loss. Hexanucleotide repeat expansion carriers had 20 percent less of the C9orf72 protein in the cerebellum of the brain than did noncarriers according to a new study, supporting the possibility that a dearth of this protein contributes to disease pathogenesis. [Courtesy of Gitler and Tsuiji, 2016, Brain Research. CC BY 4.0.]

Hexanucleotide expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet the physiological function of the protein remains a mystery. To address this gap, researchers developed a new suite of monoclonal antibodies specific to the C9ORF72 protein, and reported their initial findings in the August 3 Acta Neuropathologica. Led by Manuela Neumann of the German Center for Neurodegenerative Diseases in Tübingen, the study found that in mouse and human neurons, C9ORF72 mingled with lysosomes, other vesicles, and even resided in synapses, where it bound Rab3 proteins that release synaptic vesicles. What’s more, expansion carriers had 20 percent less of the protein in the cerebellum than did noncarriers, contrasting previous reports that found no difference despite a loss of C9 mRNA in carriers. The researchers contend that the findings support the possibility that a dearth of C9ORF72 protein contributes to disease pathogenesis.

“Novel and specific C9ORF72 antibodies are important reagents that allow for the further characterization of the protein’s endogenous function,” noted Brian Freibaum, St. Jude Children’s Research Hospital, Memphis, Tennessee.

Multiple, nonexclusive hypotheses exist to explain how C9ORF72 expansions cause disease. These include two gain-of-function mechanisms, in which RNA transcribed from the expansions, or the dipeptide repeats translated from that RNA, form toxic aggregates that sequester other RNAs and proteins. Some evidence also supports the idea that loss of C9ORF72 function could play a role, since the repeat expansions reportedly hobble transcription of the gene (see Feb 2018 news; Waite et al., 2014; van Blitterswijk et al., 2015). However, the physiological function of the wild-type C9ORF72 remains a mystery, and detecting it has been hampered by a lack of specific monoclonal antibodies. Neumann told Alzforum that commercially available antibodies only detect the protein when it is highly overexpressed, rendering efforts to measure physiological levels futile. She added that compared with other proteins, C9ORF72 happens to have very low antigenicity, presenting a high hurdle for antibody development.

First author Petra Frick and colleagues stepped up to the challenge. After screening more than 100 potential antibody clones raised against synthetic C9ORF72 peptides that were conjugated to albumin, they narrowed the choices down to a handful of hopefuls. The two monoclonals they settled on—dubbed 1C1 and 12E7—recognized exogenously expressed C9ORF72 in HEK293 cells, and also detected the protein at endogenous levels in the mouse CNS. Notably, the antibodies did not detect signals in C9ORF72 knockout mice, suggesting the antibodies are specific to the C9 protein. C9ORF72 exists in long and short isoforms, and the researchers found that while the antibodies bound both, they only detected the longer 481-amino acid isoform in western blot from mouse brain extracts, suggesting this was the predominantly expressed isoform in the CNS.

To see where C9ORF72 localized in neurons, the researchers used the antibodies to probe subcellular fractions of mouse brain, and also stained brain tissue sections by immunohistochemistry. They found that C9ORF72 primarily resided in the cytoplasm. The protein also appeared in synaptosomal fractions, suggesting a penchant for presynapses. Immunohistochemistry supported this idea; the researchers observed punctate immunoreactivity in neuropil regions that was consistent with synaptic localization. This synaptic staining pattern was most distinct in the mossy fibers emanating from neurons in the hippocampus, and also appeared in motor neurons in the spinal cord. In large motor neurons, the researchers also spotted C9ORF72 mingling with small vesicles in the cytoplasm. These vesicles were devoid of lysosomal or synaptic vesicle markers, and so far their identity is unclear.

What about in human samples? Unfortunately, the antibodies did not detect C9ORF72 in postmortem brain tissue samples. This was most likely due to sensitivity of the antibody epitopes to formalin fixation, the researchers proposed. In support of this idea, the antibodies also failed to detect C9ORF72 in mouse tissue after two days of formalin fixation. Neumann told Alzforum that the antibodies could work on postmortem brain tissue fixed for less than 24 hours, and that the researchers are seeking out those samples as they become available.

C9’s Associates. In iPSC-derived motor neurons, C9ORF72 (green) co-localized with SMCR8 (top), the lysosomal marker LAMP2 (middle), and the synaptic marker synaptophysin (bottom). [Courtesy of Frick et al., 2018, Acta Neuropathologica.]

For now, Frick and colleagues explored C9ORF72 localization in human motor neurons derived from induced pluripotent stem cells (iPSCs). The researchers spotted C9ORF72 in cytoplasmic puncta, a fraction of which co-localized with lysosomal markers LAMP1 and LAMP2. Around 11 percent of the puncta also merged with synaptic markers. C9ORF72 strongly co-localized with SMCR8, a protein previously reported to interact with C9ORF72, forming a complex that acts as a guanine nucleotide exchange factor. This GEF activated Rab proteins involved in autophagy, according to those studies (Sellier et al., 2016; Sullivan et al., 2016). Frick found that C9ORF72 co-localized with members of a different Rab protein family, Rab3, which facilitates release of synaptic vesicles into the active zone of the synapse (Binotti et al., 2016). While the role of C9ORF72 in this process remains unclear, Neumann speculated that C9ORF72 could help Rab3 proteins release vesicles.

Finally, the researchers wanted to know whether the presence of hexanucleotide expansions in C9ORF72 affected expression of the protein. To test this, they acquired freshly frozen cerebellar tissue from 18 expansion carriers with ALS, ALS/FTD, or FTD, as well as 33 noncarriers, who had been neurologically healthy, had Alzheimer’s disease, or had non-C9ORF72-related type ALS or FTD. Because the cerebellum does not degenerate in ALS/FTD, the researchers reasoned that measuring C9ORF72 protein there would avoid confounds of neuronal loss in more vulnerable regions, such as the frontal cortex or spinal cord. They found that compared with noncarriers, expansion carriers had about 20 percent less of the protein in the cerebellum. However, the cerebellar C9ORF72 concentrations did not correlate with the clinical phenotype of the carriers. Nevertheless, Freibaum thought that the loss might be important. “Though the 20 percent reduction in protein quantity seems small, this could have dramatic effects over the lifespan of the neurons, especially coupled with gain-of-function mechanisms or impairment of autophagy,” he wrote.

The cerebellum data contradicts some previous reports, which found no reduction in protein levels in the region in expansion carriers, despite less mRNA there, even as the protein plummeted in the cortex (see Aug 2015 news; Waite et al., 2014). The researchers attributed these differences to the higher sensitivity of their antibodies, claiming that the drop in C9ORF72 protein levels in degenerating regions of the brain may have been easier to pick up with less sensitive antibodies. Neumann is currently sharing the new antibodies with interested collaborators, and told Alzforum she has initiated the process of making them available commercially as well.

Featured Paper

Frick P, Sellier C, Mackenzie IR, Cheng CY, Tahraoui-Bories J, Martinat C, Pasterkamp RJ, Prudlo J, Edbauer D, Oulad-Abdelghani M, Feederle R, Charlet-Berguerand N, Neumann M. Novel antibodies reveal presynaptic localization of C9orf72 protein and reduced protein levels in C9orf72 mutation carriers. Acta Neuropathol Commun. 2018 Aug 3;6(1):72. PubMed.


Waite AJ, Bäumer D, East S, Neal J, Morris HR, Ansorge O, Blake DJ. Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion. Neurobiol Aging. 2014 Jul;35(7):1779.e5-1779.e13. Epub 2014 Jan 17 PubMed.

van Blitterswijk M, Gendron TF, Baker MC, DeJesus-Hernandez M, Finch NA, Brown PH, Daughrity LM, Murray ME, Heckman MG, Jiang J, Lagier-Tourenne C, Edbauer D, Cleveland DW, Josephs KA, Parisi JE, Knopman DS, Petersen RC, Petrucelli L, Boeve BF, Graff-Radford NR, Boylan KB, Dickson DW, Rademakers R. Novel clinical associations with specific C9ORF72 transcripts in patients with repeat expansions in C9ORF72. Acta Neuropathol. 2015 Dec;130(6):863-76. Epub 2015 Oct 5 PubMed.

Sellier C, Campanari ML, Julie Corbier C, Gaucherot A, Kolb-Cheynel I, Oulad-Abdelghani M, Ruffenach F, Page A, Ciura S, Kabashi E, Charlet-Berguerand N. Loss of C9ORF72 impairs autophagy and synergizes with polyQ Ataxin-2 to induce motor neuron dysfunction and cell death. EMBO J. 2016 Jun 15;35(12):1276-97. Epub 2016 Apr 21 PubMed.

Sullivan PM, Zhou X, Robins AM, Paushter DH, Kim D, Smolka MB, Hu F. The ALS/FTLD associated protein C9orf72 associates with SMCR8 and WDR41 to regulate the autophagy-lysosome pathway. Acta Neuropathol Commun. 2016 May 18;4(1):51. PubMed.

Binotti B, Jahn R, Chua JJ. Functions of Rab Proteins at Presynaptic Sites. Cells. 2016 Feb 6;5(1) PubMed.

Further Reading

Gitler AD, Tsuiji H. There has been an awakening: Emerging mechanisms of C9orf72 mutations in FTD/ALS. Brain Res. 2016 Sep 15;1647:19-29. PubMed.

Zhang Y, Burberry A, Wang JY, Sandoe J, Ghosh S, Udeshi ND, Svinkina T, Mordes DA, Mok J, Charlton M, Li QZ, Carr SA, Eggan K. The C9orf72-interacting protein Smcr8 is a negative regulator of autoimmunity and lysosomal exocytosis. Genes Dev. 2018 Jul 1;32(13-14):929-943. PubMed.

Shi Y, Lin S, Staats KA, Li Y, Chang WH, Hung ST, Hendricks E, Linares GR, Wang Y, Son EY, Wen X, Kisler K, Wilkinson B, Menendez L, Sugawara T, Woolwine P, Huang M, Cowan MJ, Ge B, Koutsodendris N, Sandor KP, Komberg J, Vangoor VR, Senthilkumar K, Hennes V, Seah C, Nelson AR, Cheng TY, Lee SJ, August PR, Chen JA, Wisniewski N, Hanson-Smith V, Belgard TG, Zhang A, Coba M, Grunseich C, Ward ME, van den Berg LH, Pasterkamp RJ, Trotti D, Zlokovic BV, Ichida JK. Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons. Nat Med. 2018 Mar;24(3):313-325. PubMed.

O’Rourke JG, Bogdanik L, Yáñez A, Lall D, Wolf AJ, Muhammad AK, Ho R, Carmona S, Vit JP, Zarrow J, Kim KJ, Bell S, Harms MB, Miller TM, Dangler CA, Underhill DM, Goodridge HS, Lutz CM, Baloh RH. C9orf72 is required for proper macrophage and microglial function in mice. Science. 2016 Mar 18;351(6279):1324-9. PubMed.

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