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

Abstract

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.

ALS-linked loss of Cyclin-F function affects HSP90

“…In contrast, our mass spectrometry-based quantitative ubiquitinome profiling of ALS patient-derived LCLs carrying V335M and D628V mutant CCNF uncovered a prominent decrease in signature diGly peptides for K48-and K63-linked Ub chains. Consistent with this notion, diminished E3 ligase activity was reported for the CCNF mutation S509P (26). Moreover, several diGly sites that were found decreased in CCNF KO cells were regulated in the same direction in both ALS patient-derived LCLs.…”

“…Missense mutations in CCNF contribute to the development of ALS ( 25 , 27 , 30 ). In this context, a number of studies proposed a gain of toxic function mechanism based on observations by semi-quantitative immunoblotting that levels of K48 linked polyUb increased upon expression of Cyclin-F S621G, K97R, or S195R ( 25 , 26 ). In contrast, our mass spectrometry-based quantitative ubiquitinome profiling of ALS patient-derived LCLs carrying V335M and D628V mutant CCNF uncovered a prominent decrease in signature diGly peptides for K48- and K63-linked Ub chains.…”

“…In addition, this mutation caused apoptosis activation in different cell models including patient-derived iPSCs and in zebrafish overexpressing Cyclin-F S621G. Intriguingly, these animals showed aberrant neuronal branching and reduced motor function ( 26 , 27 ). Whereas other ALS mutations in CCNF are less well characterized and distributed throughout the CCNF gene with no obvious clustering at encoded domains such as the cyclin or PEST domain ( 20 , 21 , 24 , 28 , 29 ), Cyclin-F S621G served as a paradigm to postulate a gain of toxic function mechanism.…”

Sodium valproate increases activity of the sirtuin pathway resulting in beneficial effects for spinocerebellar ataxia-3 in vivo

“…Overall, there did not appear to be obvious differences in the protein classes that were identified between the vehicle and treated samples. Therefore, we employed Ingenuity Pathway Analysis (IPA) to interrogate the proteomics datasets and make predictions of the cellular pathways and functions that changed between treatment and control groups [ 6 ].

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The Need for Biomarkers in the ALS–FTD Spectrum: A Clinical Point of View on the Role of Proteomics

“…The recent acceleration in the discovery of new genes related to ALS–FTD spectrum disorders led to the need to develop strategies to identify the molecular pathways and proteostasis dysfunctions related to them, taking advantage of cellular and animal models [ 85 ]. Regarding cellular models, different studies included mutant C9orf72, SOD1, and TDP-43 and aimed to reproduce some conditions which characterize ALS and FTD, such as protein aggregation, mitochondrial dysfunction, and cellular toxicity.…”