C9ORF72 Repeat Expansion Affects the Proteome of Primary Skin Fibroblasts in ALS

Lualdi, Marta; Shafique, Adeena; Pedrini, Edoardo; Pieroni, Luisa; Greco, Viviana; Castagnola, Massimo; Cucina, Giorgia; Corrado, Lucia; Pierro, Alice Di; Marchi, Fabiola De; Camillo, Lara; Colombrita, Claudia; D’Anca, Marianna; Alberio, Tiziana; D’Alfonso, Sandra; Fasano, Mauro

doi:10.3390/ijms221910385

Cited by 6 publications

(7 citation statements)

References 52 publications

(54 reference statements)

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“…In this regard, skin fibroblasts might be a suitable tissue model to assess functional cellular and bioenergetic impairment in C9ALS/FTD patients. In this context, previous studies have successfully used this tissue to mirror the alterations observed in C9orf72 neurons [ 29 ].…”

Section: Discussionmentioning

confidence: 99%

Bioenergetic and Autophagic Characterization of Skin Fibroblasts from C9orf72 Patients

et al. 2022

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The objective of this study is to describe the alterations occurring during the neurodegenerative process in skin fibroblast cultures from C9orf72 patients. We characterized the oxidative stress, autophagy flux, small ubiquitin-related protein SUMO2/3 levels as well as the mitochondrial function in skin fibroblast cultures from C9orf72 patients. All metabolic and bioenergetic findings were further correlated with gene expression data obtained from RNA sequencing analysis. Fibroblasts from C9orf72 patients showed a 30% reduced expression of C9orf72, ~3-fold increased levels of oxidative stress and impaired mitochondrial function obtained by measuring the enzymatic activities of mitochondrial respiratory chain complexes, specifically of complex III activity. Furthermore, the results also reveal that C9orf72 patients showed an accumulation of p62 protein levels, suggesting the alteration of the autophagy process, and significantly higher protein levels of SUMO2/3 (p = 0.03). Our results provide new data reinforcing that C9orf72 cells suffer from elevated oxidative damage to biomolecules and organelles and from increased protein loads, leading to insufficient autophagy and an increase in SUMOylation processes.

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Section: Discussionmentioning

confidence: 99%

Bioenergetic and Autophagic Characterization of Skin Fibroblasts from C9orf72 Patients

et al. 2022

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“…Consistent with reports that C9ORF72 plays an important role in immune regulation (Burberry et al, 2016 ; O’Rourke et al, 2016 ; Sudria-Lopez et al, 2016 ), proteins associated with astrocytes and microglia were slightly enriched in ALS-C9 when compared to sporadic ALS (Umoh et al, 2018 ). Proteomic analysis of in vitro ALS/FTD-C9 models show that proteins involved in protein synthesis and protein degradation are predominantly affected (Hartmann et al, 2018 ; Lualdi et al, 2021 ). ALS-C9 patient-derived primary skin fibroblasts have altered expression of proteins involved in proteostasis including ribosomal proteins (RPL7 and RPL38), translation initiation and elongation factors (EIF3B, EIF4A1, EEF1A1, EEF1A2), members of the HSP90 family, proteasome 26S subunits and aminoacyl-tRNA synthetases (Lualdi et al, 2021 ).…”

Section: Als/ftd Models Of Diseasementioning

confidence: 99%

“…Proteomic analysis of in vitro ALS/FTD-C9 models show that proteins involved in protein synthesis and protein degradation are predominantly affected (Hartmann et al, 2018 ; Lualdi et al, 2021 ). ALS-C9 patient-derived primary skin fibroblasts have altered expression of proteins involved in proteostasis including ribosomal proteins (RPL7 and RPL38), translation initiation and elongation factors (EIF3B, EIF4A1, EEF1A1, EEF1A2), members of the HSP90 family, proteasome 26S subunits and aminoacyl-tRNA synthetases (Lualdi et al, 2021 ). These findings are consistent with proteomic studies from primary mouse cortical neurons overexpressing poly-(PR) 175 , one of the five possible peptides produced via RAN translation, which show significant reduction in cytosolic ribosomal proteins that corresponded with a reduction in synaptic and axonal proteins (Hartmann et al, 2018 ).…”

Section: Als/ftd Models Of Diseasementioning

confidence: 99%

“…These findings may be relevant for our understanding how C9ORF72 hexanucleotide repeat expansions can impact neuronal features. Other potentially relevant molecular pathways to neuronal morphology and synaptic function in ALS-C9 patient-derived primary skin fibroblasts include ER-phagosome crosstalk, vesicle trafficking, the Slit/Robo signaling pathway, glucose metabolism, mitochondrial transport and bioenergetics (Lualdi et al, 2021 ). Indeed, the role of C9ORF72 in mitochondria function has been previously discussed (Smith et al, 2019 ) and mitochondria defects and elevated levels of reactive oxygen species have been observed in C9ORF72 models (Dafinca et al, 2016 ; Onesto et al, 2016 ).…”

Section: Als/ftd Models Of Diseasementioning

confidence: 99%

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Synaptic dysfunction in ALS and FTD: anatomical and molecular changes provide insights into mechanisms of disease

Gelon

Dutchak

Sephton

2022

Front. Mol. Neurosci.

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Synaptic loss is a pathological feature of all neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS is a disease of the cortical and spinal motor neurons resulting in fatal paralysis due to denervation of muscles. FTD is a form of dementia that primarily affects brain regions controlling cognition, language and behavior. Once classified as two distinct diseases, ALS and FTD are now considered as part of a common disease spectrum based on overlapping clinical, pathological and genetic evidence. At the cellular level, aggregation of common proteins and overlapping gene susceptibilities are shared in both ALS and FTD. Despite the convergence of these two fields of research, the underlying disease mechanisms remain elusive. However, recent discovers from ALS and FTD patient studies and models of ALS/FTD strongly suggests that synaptic dysfunction is an early event in the disease process and a unifying hallmark of these diseases. This review provides a summary of the reported anatomical and cellular changes that occur in cortical and spinal motor neurons in ALS and FTD tissues and models of disease. We also highlight studies that identify changes in the proteome and transcriptome of ALS and FTD models and provide a conceptual overview of the processes that contribute to synaptic dysfunction in these diseases. Due to space limitations and the vast number of publications in the ALS and FTD fields, many articles have not been discussed in this review. As such, this review focuses on the three most common shared mutations in ALS and FTD, the hexanucleuotide repeat expansion within intron 1 of chromosome 9 open reading frame 72 (C9ORF72), transactive response DNA binding protein 43 (TARDBP or TDP-43) and fused in sarcoma (FUS), with the intention of highlighting common pathways that promote synaptic dysfunction in the ALS-FTD disease spectrum.

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“…Several lines of evidence point to differences in sALS skin compared to controls, such as increased collagen density (12,13), increased MMP-2 and MMP-9 (14,15), increased S100A4 (16), and an altered aging metabolism phenotype (17). Furthermore, ALS skin and fibroblasts have been shown to recapitulate pathology thought to play key roles in neuronal death, such as SOD1 misfolding (18), FUS (19) and TDP-43 (20)(21)(22)(23)(24)(25)(26)(27) pathology, mitochondrial dysfunction (28)(29)(30), abnormal stress granule formation (31,32) and RNA processing (33), as well as altered protein homeostasis in the case of C9Orf72 mutant fibroblasts (34).…”

Section: Introductionmentioning

confidence: 99%

Machine learning approaches based on fibroblast morphometry confidently identify stress but have limited ability to predict ALS

Konràd

Woo

Bredvik

et al. 2022

Preprint

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Objective: Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease with limited therapeutic options. Diagnostic and surrogate endpoint biomarkers are needed for early disease detection, clinical trial design, and personalized medicine. Methods: We tested the predictive power of a large set of primary skin fibroblast (n=443) from sporadic and familial ALS patients and healthy controls. We measured morphometric features of endoplasmic reticulum, mitochondria, and lysosomes by imaging with vital dyes. We also analysed immunofluorescence images of ALS-linked proteins, including TDP-43 and stress granule components. We studied fibroblasts under basal conditions and under metabolic (galactose medium), oxidative (arsenite), and heat stress conditions. We then employed machine learning (ML) techniques on the dataset to develop biomarkers. Results: Stress perturbations caused robust changes in the measured features, such as organellar morphology, stress granule formation, and TDP-43 mislocalization. ML approaches were able to predict the perturbation with near perfect performance (ROC-AUC > 0.99). However, when trying to predict disease state or disease groups (e.g., sporadic, or familial ALS), the performance of the ML algorithm was more modest (ROC-AUC Control vs ALS = 0.63). We also detected modest but significant scores when predicting clinical features, such as age of onset (ROC-AUC late vs early = 0.60). Conclusions: Our findings indicate that the ML morphometry we developed can accurately predict if human fibroblasts are under stress, but the differences between ALS and controls, while statistically significant, are small and pose a challenge for the development of biomarkers for clinical use by these approaches.

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C9ORF72 Repeat Expansion Affects the Proteome of Primary Skin Fibroblasts in ALS

Cited by 6 publications

References 52 publications

Bioenergetic and Autophagic Characterization of Skin Fibroblasts from C9orf72 Patients

Bioenergetic and Autophagic Characterization of Skin Fibroblasts from C9orf72 Patients

Synaptic dysfunction in ALS and FTD: anatomical and molecular changes provide insights into mechanisms of disease

Machine learning approaches based on fibroblast morphometry confidently identify stress but have limited ability to predict ALS

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