Elucidating the Role of Cerebellar Synaptic Dysfunction in C9orf72-ALS/FTD - a Systematic Review and Meta-Analysis

Abstract

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) with synaptic dysfunction identified as an early pathological hallmark. Although TDP-43 pathology and overt neurodegeneration are largely absent from the cerebellum, the pathological hallmarks of RNA foci and dipeptide repeat protein (DPR) inclusions are most abundant. Here, we present a systematic literature search in the databases of PubMed, Scopus, Embase, Web of Science and Science Direct up until March 5, 2021, which yielded 19,515 publications. Following the exclusion criteria, 72 articles were included having referred to C9orf72, synapses and the cerebellum. Meta-analyses were conducted on studies which reported experimental and control groups with means and standard deviations extracted from figures using the online tool PlotDigitizer. This revealed dendritic defects (P = 0.03), reduced C9orf72 in human patients (P = 0.005) and DPR-related neuronal loss (P = 0.0006) but no neuromuscular junction abnormalities (P = 0.29) or cerebellar neuronal loss (P = 0.23). Our results suggest that dendritic arborisation defects, synaptic gene dysregulation and altered synaptic neurotransmission may drive cerebellar synaptic dysfunction in C9-ALS/FTD. In this review, we discuss how the chronological appearance of the different pathological hallmarks alters synaptic integrity which may have profound implications for disease progression. We conclude that a reduction in C9orf72 protein levels combined with the accumulation of RNA foci and DPRs act synergistically to drive C9 synaptopathy in the cerebellum of C9-ALS/FTD patients.

Keywords: ALS; C9orf72; Cerebellum; FTD; Synaptic dysfunction.

Fig. 1

Putative mechanisms underlying synaptic dysfunction in C9orf72-ALS/FTD. A schematic detailing the role of the hexanucleotide expansion, (G₄C₂)_n, of the C9orf72 gene in driving synaptic, axonal and dendritic dysfunction. This operates through the three main pathogenic mechanisms implicated in C9-ALS/FTD which are haploinsufficiency of the C9orf72 protein and the accumulation of RNA foci and dipeptide repeats (DPRs). Abbreviations: p53, tumour protein p53; RAN, repeat-associated non-AUG; Ca²⁺, calcium ions; mRNP, messenger ribonucleoprotein; RNA, ribonucleic acid

Fig. 2

Inclusion of articles by Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram

Fig. 3

Frequency graphs illustrating the characteristics of selected studies. (A) Shows the number of studies that report synaptic dysfunction. (B) Illustrates the number of studies which report ALS/FTD pathology in C9-patients. (C) Shows the number of different animal models used in in vivo experiments. (D) Shows the number of different in vitro models used in the selected studies. (E) illustrates the pathomechanism type assessed in different animal models. (F) Illustrates the number of DPR constructs introduced to different animal models. Abbreviations: DPRs, dipeptide repeat proteins; NMJ, neuromuscular junction; cDNA, circular DNA

Fig. 4

Meta-analysis using a random effects model of selected studies relating to synaptic deficits. (A) Shows the meta-analysis for dendritic defects assessing reductions in arborisations such as crossings and branchpoints (P = 0.03). (B) Shows the meta-analysis for neuromuscular junction (NMJs) abnormalities assessing synaptic bouton counts and fractured NMJs (P = 0.29). (C) Shows the meta-analysis for reductions of C9orf72 protein in human patients using frontal cortex and cerebellar samples (P = 0.005). All studies were highly heterogenous (I² ≥ 84%; P ≤ 0.0004). The figure was generated using the RevMan 5.4 software. Abbreviations: SD, standard deviation; CI, confidence interval

Fig. 5

Meta-analysis using a random effects model of selected studies relating to neuronal loss in C9orf72. (A) Shows the meta-analysis of neuronal loss in the cerebellum as a result of G₄C₂ repeats and DPR models (P = 0.23). (B) Shows the meta-analysis of DPR-related neuronal loss using animal models transfected with DPR constructs (P = 0.0006). Studies were highly heterogenous (I² $\ge$ 78%; P ≤ 0.001). The figure was generated using RevMan 5.4. Abbreviations: SD, standard deviation; CI, confidence interval

Fig. 6

Splice variants of human C9orf72 mRNA. An overview of the exons included in the long and short isoforms of C9orf72 and their subcellular localisation. Abbreviations: aa, amino acids; kDa, kilodalton; RNA, ribonucleic acid; V1/2/3, variant 1/2/3