C9ORF72 levels: implications for diagnosis, prognosis, and treatment of ALS and related disorders

PROJECT SUMMARY/ABSTRACT Emerging evidence suggests that chromosome 9 open reading frame 72 (C9ORF72) expression, RNA foci, and dipeptide-repeat proteins contribute to C9ORF72-related diseases; however, much remains unknown about the mechanisms causing these fatal neurodegenerative diseases. Repeat expansions in C9ORF72, which are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), are associated with considerable clinical variability. To date, three C9ORF72 transcripts have been reported (variant 1, variant 2, and variant 3), which give rise to two protein isoforms (C9-S and C9-L). On the heels of our recent discoveries and exciting preliminary data, we hypothesize that the expression of C9ORF72 transcript variants or protein isoforms might contribute to the clinical heterogeneity among expansion carriers and may serve as an urgently needed biomarker for these fatal diseases. Our evaluation of C9ORF72 RNA levels in two brain regions revealed a significant increase in transcripts containing the intronic area that precedes the repeat expansion (intron 1a) but not in the succeeding area (intron 1b), indicating at least a subset of C9ORF72 transcripts is truncated. In the cerebellum, we observed a significant association between transcripts containing the entire first intron and dipeptide-repeat proteins (i.e., poly[GP] and poly[GA]), suggesting these transcripts may serve as templates for repeat-associated non-ATG (RAN) translation. Importantly, we also noticed a significant association between C9ORF72 transcript variant 1 and survival after onset, both in the frontal cortex and cerebellum, which warrants caution for the development of new treatment strategies targeting C9ORF72 (e.g., antisense oligonucleotides [ASOs]). To address the lack of validated biomarkers to determine disease prognosis and stage, monitor target engagement and patient responses to potential therapeutic interventions, we now propose detailed expression studies. We will employ Northern blotting techniques to evaluate known C9ORF72 transcripts as well as the existence of additional transcript variants and/or abnormal species in a range of regions, such as cerebellum, frontal cortex, motor cortex, hippocampus, amygdala, basal ganglia, thalamus, medulla, and spinal cord. We will perform digital molecular barcoding on a large cohort of symptomatic and presymptomatic expansion carriers and disease controls, focusing on the expression levels of C9ORF72 transcript variant 1 in blood; however, we will also investigate other transcripts and regions (Aim 1). Finally, we will evaluate the levels of C9ORF72 protein isoforms using Western blotting techniques and sensitive immunoassays, and we will visualize protein isoforms using immunohistochemistry and RNA fluorescent in situ hybridization (FISH; Aim 2). In so doing, we will determine whether specific C9ORF72 transcripts or protein isoforms contribute to the clinical heterogeneity and may serve as a biomarker for C9ORF72-related diseases.