Expression of ALS/FTD-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype

Abstract

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations.

ALS Genes in the Genomic Era and their Implications for FTD

“…Comparable with TBK1, CCNF interacts with SQSTM1, an autophagic receptor that recognizes and transfers ubiquitinated proteins for autophagic degradation [63]. In a recent study in zebrafish, disruption of axonal outgrowth by the mutant Ser621Gly CCNF was observed, suggesting a toxic gain-offunction mechanism for CCNF mutations in ALS patients [64].…”

Omics Approach to Axonal Dysfunction of Motor Neurons in Amyotrophic Lateral Sclerosis (ALS)

“…Temporary overexpression of human cyclin-F (CCNF) in zebrafish embryos increases the levels of cleaved caspase-3 and cell death in the spinal cord. The mutant CCNF zebrafish also developed an MN axonopathy, which consists of shortened primary MN axons and an increased frequency of aberrant axonal branching (Hogan et al, 2017).…”

Constitutive overexpression of the ALS-linked gene <em>CCNF</em> fusions results in cytotoxicity to preclude generation of transgenic zebrafish models

“…There is the potential for toxicity to arise when overexpression is introduced into a system and this is of importance during early development when vital systems such as the nervous system are being established. In particular, the generation of models of ALS (and likely most neurodegenerative diseases) may be fraught with difficulties as many of these genes are ubiquitously expressed during development before becoming restricted to the central nervous system [6] [13]. This suggests that the toxicity detailed here is not specific to CCNF expression during developmental stages and may be a factor in the establishment of other models.…”

“…Additionally, mutations in CCNF have recently been reported in two clinically, pathologically and genetically linked neurodegenerative diseases-amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [5]. To date, only one in vivo model has been established as a tool to investigate CCNF dysfunction-a zebrafish model that transiently overexpressed an ALS-FTD mutation in CCNF (CCNF S621G) [6]. This model demonstrated neurological defects associated with expression of CCNF S621G, including a motor axonopathy and impaired motor function.…”