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.
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Cardiff University
Since the discovery of the C9ORF72 hexanucleotide repeat expansion as a causative mutation in ALS-FTD, a number of studies have used various cellular and animal models to explore the normal cellular roles of C9ORF72 protein isoforms and to determine if and how haploinsufficiency of C9ORF72 contributes to disease pathogenesis. However, studies of endogenous C9ORF72 protein isoforms have been hampered by the lack of commercial antibodies with high specificity and sensitivity. A small number of antibodies have also been developed by academic groups, including our group (Waite et al., 2014; Koppers et al., 2015), but there is still a lack of knockout validated C9ORF72 antibodies that are suitable for biochemical, immunocytochemical, and immunohistochemical studies. The study reported here describes the generation, characterization, and preliminary use of novel mouse and rat anti-C9ORF72 monoclonal antibodies generated against both long and short protein isoforms. The authors performed detailed characterization of the monoclonal antibodies using analysis of biochemical, immunocytochemical, and immunohistochemical methods including the use of C9ORF72 knockout mouse tissue to verify specificity. A parallel characterization of commercially available antibodies demonstrated their lack of specificity in biochemical and immunohistochemical analyses (in agreement with our published study of commercial antibodies using siRNA-mediated knockdown of C9ORF72 in a human cell line (Waite et al., 2014). They used their own validated mouse 1C1 and rat 12E7 antibodies (that can detect both long and short C9ORF72 protein isoforms) in further analyses of mouse tissue, hiPSC-derived motor neurons and human postmortem tissue.
The authors used the 1C1 and 12E7 monoclonal antibodies to demonstrate that the long C9ORF72 protein isoform is predominantly detected in mouse and human tissue by western blot (in agreement with our own findings in human cells and tissue (Waite et al., 2014) with multiple tissue western blot analysis showing highest levels of protein detected in the CNS and spleen. Immunohistochemistry analysis of mouse tissue using a knockout validated protocol revealed a punctate neuropil staining pattern throughout the CNS reminiscent of synaptic localisation (showing an overlapping immunostaining pattern with the presynaptic marker synaptoporin) with highest levels of reactivity in the hippocampal mossy fiber system and globus pallidus, with weaker signals in the neuropil of the cortex, caudate-putamen, and molecular layer of the cerebellum. C9ORF72 was also localised to cytoplasmic punctae (identity unknown) in the cell bodies of large motor neurons in the brain stem and spinal cord. No immunoreactivity was detected in white matter or glial cells. Subcellular fractionation experiments also demonstrated the cytosolic localisation of C9ORF72 with an enrichment in synaptosomes (enriched in synaptic vesicles).
Extending the study to immunocytochemical analysis of hiPSC-derived motor neurons demonstrated a punctate-staining pattern with high co-localization with the SMCR8 protein, a robust interaction partner of C9ORF72 (Sellier et al., 2016), and partial co-localization with the lysosomal marker LAMP2, in agreement with previous studies (Amick et al., 2016) and the synaptic vesicle marker synaptophysin. Furthermore, the authors demonstrate an association of C9ORF72 with the RAB3 protein family of synaptic vesicle localized GTPases, which are essential for normal neurotransmitter release, via co-immunoprecipitation of recombinant and endogenous material, and they found evidence of partial co-localization in hiPSC-derived motor neurons. Therefore, several lines of evidence suggest that a pool of C9ORF72 may be involved in synaptic vesicle function, however, this requires further mechanistic studies in animal and cellular models, as noted by the authors.
Finally, the authors use their validated antibodies to analyze C9ORF72 protein levels by western blot of postmortem brain tissue of C9ORF72 mutation cases and neurologic controls. They demonstrate that decreasing C9ORF72 protein levels correlate with general neuronal loss thus, complicating the analysis of tissues undergoing neurodegeneration as shown in frontal cortex samples from a neurodegeneration panel (AD, ALS, FTD, and hypoxia). As an alternative, they perform western blot analyses of cerebellum tissue that escapes overt neurodegeneration in ALS-FTD. C9ORF72 showed a reduction of approximately 20 percent in hexanucleotide repeat expansion cases versus controls and displayed no alteration in protein solubility. In contrast to western blotting, the antibodies could not be validated for use in immunohistochemical analysis of postmortem brain tissue. The authors propose that this could be due to the extended formalin fixation times for human tissue versus mouse tissue. Further studies of human postmortem tissue will require modified fixation conditions for improved detection of C9ORF72 using immunohistochemistry.
This study provides additional support for reduced C9ORF72 protein as a consequence of the hexanucleotide repeat expansion, however it remains to be conclusively determined if and how reduced protein levels contribute to disease pathogenesis. Published studies of human ALS-FTD postmortem material, and C9ORF72 and Smcr8 deficient mouse models (Koppers et al., 2015; Sudria-Lopez et al., 2016; Atanasio et al., 2016; Burberry et al., 2016; Zhang et al., 2018) suggest that haploinsufficiency is insufficient to cause ALS-FTD. However, it is possible that a combination of reduced C9ORF72 function, hexanucleotide repeat-associated gain-of-function mechanisms, and other cellular stressors ultimately lead to disease progression and may explain the phenotypic heterogeneity associated with the C9ORF72 repeat expansion (see Shi et al., 2018, and recent review in Balendra and Isaacs, 2018). The availability of knockout-validated monoclonal antibodies provides an abundant resource for future studies of clinical material, to study cellular and animal models aimed to further investigate the context-dependent expression, for the localization and function of C9ORF72, and to elucidate the contribution of loss-of-function to ALS-FTD pathogenesis.
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