Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS

Abstract

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. Hexanucleotide (GGGGCC) repeat expansions in a noncoding region of C9ORF72 are the major genetic cause of FTD and ALS (c9FTD/ALS). The RNA structure of GGGGCC repeats renders these transcripts susceptible to an unconventional mechanism of translation-repeat-associated non-ATG (RAN) translation. Antibodies generated against putative GGGGCC repeat RAN-translated peptides (anti-C9RANT) detected high molecular weight, insoluble material in brain homogenates, and neuronal inclusions throughout the CNS of c9FTD/ALS cases. C9RANT immunoreactivity was not found in other neurodegenerative diseases, including CAG repeat disorders, or in peripheral tissues of c9FTD/ALS. The specificity of C9RANT for c9FTD/ALS is a potential biomarker for this most common cause of FTD and ALS. These findings have significant implications for treatment strategies directed at RAN-translated peptides and their aggregation and the RNA structures necessary for their production.

Figure 1. Anti-C9RANT immunoreactivity is specific to c9FTD/ALS

(A) Schematic representation of the possible protein products generated by RAN translation of expanded GGGGCC repeats in the three alternate reading frames. (B, C) Immunoreactivity of each anti-C9RANT antibody (Rb5822 and RB5823) towards indicated peptides was measured by adsorbing peptides onto carbon electrodes in 96-well MSD plates, and co-incubating wells with anti-C9RANT and a SULFO-tagged anti-rabbit secondary antibody. Anti-C9RANT binding to respective peptides was quantified by measuring the intensity of emitted light upon electrochemical stimulation of the plate using the MSD Sector Imager 2400. The amino acid sequence for (GX)₅ is Gly-Met-Gly-Asp-Gly-Ser-Gly-Leu-Gly-Thr. (D) Western blot analysis of cerebellar tissue urea fractions from C9ORF72 repeat expansion and non-expansion FTLD cases using anti-C9RANT. Note the high molecular weight product (Arrow). (E) Anti-C9RANT immunoreactivity in cerebellar urea fractions from FTLD-TDP and ALS cases with or without expanded GGGGCC repeats, as assessed by dot blot. Each dot represents one case. See also Figure S1. (F-I) Immunohistochemistry with each anti-C9RANT antibody revealed that abundant neuronal inclusion in the cerebellum of c9FTD (F, H), but not in sporadic FTLD-TDP (G, I). C9RANT-immunoreactive lesions were granular neuronal cytoplasmic inclusions (seen clearly in the Purkinje cell shown in inset of F, H). Scale Bar=50 μm in main images, 20 μm in insets.

Figure 2. Regional neuropathology of C9RANT

C9RANT-immunoreactive neuronal inclusions were observed throughout the central nervous system, including cerebellum (A– cerebellar molecular layer, B–cerebellar Purkinje cell layer, C–cerebellar internal granular layer), neocortex (D–frontal cortex, E–temporal cortex, F–motor cortex), subcortical gray matter (G– amygdala, H–dentate gyrus of the hippocampus, I–CA3 of the hippocampus, J–lateral geniculate nucleus, K–lateral thalamus, L–medial geniculate nucleus, N–globus pallidus, O– hypothalamus, P–nucleus basalis of Meynert), and to a lesser extent the brainstem (M– substantia nigra). (Q) Semi-quantitative pathology scoring in these aforementioned regions, as well as in the entorhinal cortex, medulla, putamen, midbrain, and spinal cord reveals variable, C9RANT immunoreactive inclusions throughout the central nervous system. Cbl=cerebellum; ctx= cortex; Hp=hippocampus; MB=midbrain; S=substantia; SC=spinal cord; Subthal=subthalamic; Thal=thalamus.

Figure 3. C9RANT-immunoreactive inclusions are present in neurons but not glia

Double-label immunofluorescence was performed on hippocampal tissue from a c9FTD case using anti-C9RANT (Rb5823) antiserum and the neuronal marker, MAP2, or the astrocytic marker, GFAP. Note that C9RANT-immunoreactive inclusions localize exclusively to neurons (A) and are not found in astrocytes (B). Scale bar = 50 μm.

Figure 4. Specificity of C9RANT pathology

p62 immunolabeling of neuronal intranuclear inclusions in c9FTD/ALS (A; cerebellum), Huntington’s disease (C; basal ganglia), spinocerebellar ataxia type 3 (E; pons), and spinal and bulbar muscular atrophy (Kennedy’s disease) (G; medulla). Anti-C9RANT-positive inclusions are specific to c9FTD/ALS (B) and absent from these other CAG repeat disorders (D–Huntington’s disease, F–spinocerebellar ataxia type 2, H–Kennedy’s disease). Additionally, C9RANT pathology is predominantly neuronal, with no inclusions in the heart (I), kidney (J), or spleen (K). The only other organ where C9RANT lesions were found was the testes, where C9RANT immunoreactive cytoplasmic and nuclear inclusions were noted in Sertoli cells (L). Scale bars in H and L=30 μm; scale bar in B and inset of L=6 μm. (See also Figure S2 and Table S1)