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

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

Unbiased Label-Free Quantitative Proteomics of Cells Expressing Amyotrophic Lateral Sclerosis (ALS) Mutations in CCNF Reveals Activation of the Apoptosis Pathway: A Workflow to Screen Pathogenic Gene Mutations

The past decade has seen a rapid acceleration in the discovery of new genetic causes of ALS, with more than 20 putative ALS-causing genes now cited. These genes encode proteins that cover a diverse range of molecular functions, including free radical scavenging (e.g., SOD1), regulation of RNA homeostasis (e.g., TDP-43 and FUS), and protein degradation through the ubiquitin-proteasome system (e.g., ubiquilin-2 and cyclin F) and autophagy (TBK1 and sequestosome-1/p62). It is likely that the various initial triggers of disease (either genetic, environmental and/or gene-environment interaction) must converge upon a common set of molecular pathways that underlie ALS pathogenesis. Given the complexity, it is not surprising that a catalog of molecular pathways and proteostasis dysfunctions have been linked to ALS. One of the challenges in ALS research is determining, at the early stage of discovery, whether a new gene mutation is indeed disease-specific, and if it is linked to signaling pathways that trigger neuronal cell death. We have established a proof-of-concept proteogenomic workflow to assess new gene mutations, using CCNF (cyclin F) as an example, in cell culture models to screen whether potential gene candidates fit the criteria of activating apoptosis. This can provide an informative and time-efficient output that can be extended further for validation in a variety of in vitro and in vivo models and/or for mechanistic studies. As a proof-of-concept, we expressed cyclin F mutations (K97R, S195R, S509P, R574Q, S621G) in HEK293 cells for label-free quantitative proteomics that bioinformatically predicted activation of the neuronal cell death pathways, which was validated by immunoblot analysis. Proteomic analysis of induced pluripotent stem cells (iPSCs) derived from patient fibroblasts bearing the S621G mutation showed the same activation of these pathways providing compelling evidence for these candidate gene mutations to be strong candidates for further validation and mechanistic studies (such as E3 enzymatic activity assays, protein–protein and protein–substrate studies, and neuronal apoptosis and aberrant branching measurements in zebrafish). Our proteogenomics approach has great utility and provides a relatively high-throughput screening platform to explore candidate gene mutations for their propensity to cause neuronal cell death, which will guide a researcher for further experimental studies.</jats:p

Unbiased Label-Free Quantitative Proteomics of Cells Expressing Amyotrophic Lateral Sclerosis (ALS) Mutations in CCNF Reveals Activation of the Apoptosis Pathway: A Workflow to Screen Pathogenic Gene Mutations

The past decade has seen a rapid acceleration in the discovery of new genetic causes of ALS, with more than 20 putative ALS-causing genes now cited. These genes encode proteins that cover a diverse range of molecular functions, including free radical scavenging (e.g., SOD1), regulation of RNA homeostasis (e.g., TDP-43 and FUS), and protein degradation through the ubiquitin-proteasome system (e.g., ubiquilin-2 and cyclin F) and autophagy (TBK1 and sequestosome-1/p62). It is likely that the various initial triggers of disease (either genetic, environmental and/or gene-environment interaction) must converge upon a common set of molecular pathways that underlie ALS pathogenesis. Given the complexity, it is not surprising that a catalog of molecular pathways and proteostasis dysfunctions have been linked to ALS. One of the challenges in ALS research is determining, at the early stage of discovery, whether a new gene mutation is indeed disease-specific, and if it is linked to signaling pathways that trigger neuronal cell death. We have established a proof-of-concept proteogenomic workflow to assess new gene mutations, using CCNF (cyclin F) as an example, in cell culture models to screen whether potential gene candidates fit the criteria of activating apoptosis. This can provide an informative and time-efficient output that can be extended further for validation in a variety of in vitro and in vivo models and/or for mechanistic studies. As a proof-of-concept, we expressed cyclin F mutations (K97R, S195R, S509P, R574Q, S621G) in HEK293 cells for label-free quantitative proteomics that bioinformatically predicted activation of the neuronal cell death pathways, which was validated by immunoblot analysis. Proteomic analysis of induced pluripotent stem cells (iPSCs) derived from patient fibroblasts bearing the S621G mutation showed the same activation of these pathways providing compelling evidence for these candidate gene mutations to be strong candidates for further validation and mechanistic studies (such as E3 enzymatic activity assays, protein–protein and protein–substrate studies, and neuronal apoptosis and aberrant branching measurements in zebrafish). Our proteogenomics approach has great utility and provides a relatively high-throughput screening platform to explore candidate gene mutations for their propensity to cause neuronal cell death, which will guide a researcher for further experimental studies

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

International audienceIn 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field