ReviewInsights into disease mechanisms and potential therapeutics for C9orf72-related amyotrophic lateral sclerosis/frontotemporal dementia
Introduction
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting upper and lower motor neurons (MNs) that causes progressive and relentless muscular weakness and atrophy leading to death for respiratory insufficiency. Frontotemporal dementia (FTD) is the most common form of dementia before 65 years of age and is characterized by relevant changes in social behavior and personality or language disturbance, due to degeneration of the frontal and/or temporal lobes. The term frontotemporal lobar degeneration (FTLD) denotes a neuropathological spectrum encompassing different neurodegenerative disorders, such as ALS, FTD and various combinations of them. Although the majority of these clinical syndromes are sporadic, a conspicuous part of them (ranging from 10 to 25 % in FTD and 5–10 % in ALS) is familial.
In 2011 De Jesus-Hernandez et al. and Renton et al. independently discovered that the hexanucleotide GGGGCC (G4C2) expansion in a non-coding region of Chromosome 9 Open reading frame 72 (C9orf72) gene on chromosome 9 is the most frequent genetic cause of both sporadic and familial forms of ALS and FTD in Western countries (DeJesus-Hernandez et al., 2011; Renton et al., 2011). The clinical phenotypes associated with C9orf72 hexanucleotide repeat expansion (HRE) are markedly heterogeneous (Woollacott and Mead, 2014), the number of repeats and the clinical presentation are not correlated and HRE length is highly variable in different tissues of the same patient and in parental offspring transmission.
Although the function of C9orf72 protein is not completely understood, both primary neurons from patients with C9orf72-related ALS/FTD (C9-ALS/FTD) and studies in in vitro and in vivo C9-models have improved our understanding of the etiology and pathogenesis of C9-ALS/FTD. Three possible, but not mutually exclusive pathomechanisms have emerged and are still debated in the field: loss-of-function due to C9orf72 protein haploinsufficiency, gain of toxic function from repeat-containing RNAs, also known as RNA foci, generated by bidirectional transcription of HRE, and toxic gain-of-function of dipeptide repeat proteins (DPRs) translated from sense and antisense HRE transcripts by non-canonical translation of repetitive RNAs (Balendra and Isaacs, 2018). Further, alterations in various downstream cellular pathways, such as nucleocytoplasmic transport (NCT), RNA metabolism, ubiquitin-proteasome system (UPS) and autophagy, vesicle trafficking, DNA damage, and mitochondrial metabolism, might be involved in disease pathogenesis.
The precise contribution of each mechanism into C9-ALS/FTD pathogenesis is still controversial, but they all likely play a role to some extent. The discovery of C9orf72 as the most common genetic cause of ALS/FTD has prompted the research towards an HRE-targeted therapy, opening the path for new promising pharmacologic approaches. However, though a phase I clinical trial on antisense oligonucleotides (ASOs) directed on HRE is now ongoing, an efficacious treatment for C9-ALS/FTD still lacks.
Here, we provide a literature overview on clinical, pathological and molecular aspects of C9-ALS/FTD. We report recent epidemiological data, clinical features and neuropathology underlying C9-ALS/FTD, highlighting the main similarities and differences between C9-ALS/FTD and sporadic form of ALS (sALS). We describe the gene structure and the supposed role of C9orf72 protein in cellular machinery. Through the description of neuropathological findings and in vitro and in vivo studies, we focus on the main three mechanisms involved in disease pathogenesis, investigating proofs and controversies regarding the contribution of each of them in driving neurodegeneration, and widely exploring the downstream cellular pathways. Then, we highlight the most recently investigated biochemical, structural and functional biomarkers to assess disease progression and drug efficacy in clinical trials. Finally, we provide a synopsis of the possible therapeutic approaches in C9-ALS/FTD, including ASOs specifically targeting the C9orf72 HRE, RNA interference, small molecules, genome editing techniques and DPRs-directed strategies.
We are confident that a better comprehension of the molecular mechanisms underlying C9-ALS/FTD pathogenesis would provide major and promising advances in specific disease-modifying treatments.
Section snippets
Epidemiology and clinical features
HRE in C9orf72 is the most common genetic cause of ALS and FTD all over the world and in particular in the Caucasian population, while it is very infrequent in the Asiatic population. Large population studies conducted by Majounie and colleagues in 2012 have shown that the prevalence of G4C2 repeat expansion in C9orf72 accounts for 5–7% of sporadic ALS and FTD forms in white Europeans, Americans, and Australians (Majounie et al., 2012b). The pathogenic expansion is more frequent in white (7%)
C9orf72 gene and the G4C2 repeat expansion
The C9orf72 gene is placed on the short arm of chromosome 9 (9p21), contains twelve exons and generates three transcription variants by using alternative transcription start and termination sites (Fig. 1). The HRE is located in a non-coding portion of the gene, in the first intron of variants 1 and 3, and within the promoter region of the variant 2. Despite the intronic location of the G4C2 repeats in variants 1 and 3, they are likely retained in the mature messenger RNA (mRNA) and successfully
Protein function
C9orf72-related transcript variants encode for two different protein isoforms. Transcript variants 2 and 3 are translated into the full-length C9orf72 protein (481 amino acids), and variant 1 yields a predicted short protein (222 amino acids) (Fig. 1) (Woollacott and Mead, 2014). It is not clear whether the predicted short isoform C9orf72 is present in nature. Xiao et al. have shown a decreased short isoform expression in nuclear membranes of C9-ALS/FTD cases compared with controls, by using an
Neuropathology
Most ALS and approximately half of FTD cases show TAR-DNA binding protein 43 (TDP-43) inclusions in the nucleus and cytoplasm of neurons and oligodendroglia (Neumann et al., 2006), and are usually acknowledged as FTLD-TDP. These aggregates display abnormal TDP-43 phosphorylation and ubiquitination and truncated C-terminal fragments of TDP-43 (Arai et al., 2006; Neumann et al., 2006). Neuropathology and genetic alterations are correlated and C9-ALS/FTD cases are associated with type A and type B
Mechanisms of neurodegeneration
Three different but not mutually exclusive mechanisms contribute to C9orf72-mediated neurodegeneration: loss of function of C9orf72 protein due to haploinsufficiency, toxic gain of function from sense and antisense RNA foci, which are bidirectionally transcribed from HRE into repetitive RNAs, and toxic gain of function from DPRs, derived from repetitive RNA non-canonical translation. All these processes play a role in C9-ALS/FTD pathogenesis to some extent, and they are likely responsible for
Downstream mechanisms
In addition to C9orf72 loss- and gain-of-function mechanism, various studies have focused on downstream cellular alterations resulting from the HRE pathogenic cascade, including altered nucleocytoplasmic transport and RNA metabolism, disrupted proteostasis as well as other cellular functions. Researchers have used multiple human and non-human model systems for this purpose (Fig. 3).
Biochemical biomarkers
Biomarkers in ALS may help to make an earlier diagnosis, to enroll patients in clinical trials and to monitor the efficacy of therapeutic interventions. Biochemical biomarkers include DPRs, neurofilaments (Nfs) and other proteins that are related to the pathogenesis of C9-ALS/FTD and can be measured in biofluids or biological tissues.
CSF poly-GP levels discriminate symptomatic and pre-symptomatic carriers from non-carriers, thus they may be useful as pharmacodynamic biomarkers (Floeter and
Therapeutics
To date, no cure exists for C9-ALS/FTD. Different studies using ASO technology, small molecules, CRISPR-Cas9 as well as RNA interference (RNAi) strategies have targeted various pathogenic pathways, including C9orf72 RNA and DNA, DPRs, TDP-43 and downstream mechanisms (Table 2).
Closing remarks
Since the identification of C9orf72 repeat expansion, many studies have focused on unraveling the underlying pathogenic mechanisms in C9-ALS/FTD. At the same time, several disease models in vitro and in vivo have provided insights into the loss/gain of function-related neurotoxicity, as well as the downstream cellular function abnormalities. Despite thorough investigations of both biomarkers and new therapies have proceeded enthusiastically, disease-modifying therapies for C9-ALS/FTD still
Author statement
All authors have approved the final draft of the article.
Funding
This study was funded by Italian Ministry Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico Ricerca Corrente 2020 to NB and GPC.
CRediT authorship contribution statement
Delia Gagliardi: Conceptualization, Investigation, Writing - original draft. Gianluca Costamagna: Conceptualization, Investigation, Writing - original draft. Michela Taiana: Investigation, Writing - review & editing. Luca Andreoli: Visualization. Fabio Biella: Investigation, Visualization. Margherita Bersani: Visualization. Nereo Bresolin: Supervision. Giacomo Pietro Comi: Supervision. Stefania Corti: Conceptualization, Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors reported no declarations of interest.
Acknowledgments
We gratefully thank the Associazione Centro Dino Ferrari for its support. Regione Lombardia TRANS-ALS and RF-2013-02355764/Italian Ministry of Health to GPC is gratefully acknowledged. Italian Ministry of health "RF-2018-12366357 grant to SC is gratefully acknowledged.
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These authors contributed equally to this work.