Sulforaphane improves mitochondrial metabolism in fibroblasts from patients with fragile X-associated tremor and ataxia syndrome

Abstract

CGG expansions between 55 and 200 in the 5'-untranslated region of the fragile-X mental retardation gene (FMR1) increase the risk of developing the late-onset debilitating neuromuscular disease Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS). While the science behind this mutation, as a paradigm for RNA-mediated nucleotide triplet repeat expansion diseases, has progressed rapidly, no treatment has proven effective at delaying the onset or decreasing morbidity, especially at later stages of the disease. Here, we demonstrated the beneficial effect of the phytochemical sulforaphane (SFN), exerted through NRF2-dependent and independent manner, on pathways relevant to brain function, bioenergetics, unfolded protein response, proteosome, antioxidant defenses, and iron metabolism in fibroblasts from FXTAS-affected subjects at all disease stages. This study paves the way for future clinical studies with SFN in the treatment of FXTAS, substantiated by the established use of this agent in clinical trials of diseases with NRF2 dysregulation and in which age is the leading risk factor.

Keywords: Antioxidants; Bioenergetics; Brain; Fibroblasts; NRF2; Neurodegeneration; Phytochemicals; Triplet nucleotide repeat diseases; Unfolded protein response.

Conflict of interest statement

Conflict of Interest

The authors have no conflicts of interest to disclose.

Copyright © 2021. Published by Elsevier Inc.

Figures

Figure 1.. Differential proteome in primary dermal fibroblasts of FXTAS-affected carriers.

A volcano plot (A) visualized the differentially expressed proteins in primary dermal fibroblasts from FXTAS-affected carriers compared to non-carriers. Red dots with p-adj <0.1. Hierarchical clustering (visualized as a heat map; B) was performed by utilizing as input the normalized concentrations of proteins. Clustering was performed by utilizing an Euclidean distance measure with a Ward clustering algorithm according to the significance of the Student’s t test (with equal variance). Each row represents one protein and each column represents one subject. Pathway analysis (C) was performed with InnateDB (Breuer et al., 2013) by utilizing as input those proteins with a fold change of |1.5|. Only shown those with p < 0.05 (Benjamini and Hochberg adjusted p-value) against the KEGG, REACTOME, and PID NCI, INOH, PID Biocarta and NETPATH databases. Numerical values indicate enrichment. Significantly different mitochondrial outcomes are reported (D) for FXTAS-affected carriers (n = 12) compared to age- and sex-matched non-carriers (n = 5). Values of outcomes were normalized against the average of pooled controls and then expressed as log2 fold change. RCR and RCRu, respiratory control ratio under phosphorylating conditions and uncoupling conditions, respectively. BHI, bioenergetic health index calculated as described in (Chacko et al., 2014). ROS/PL: reactive oxygen species production and proton leak estimated as oligomycin-induced State 4 in the presence of glucose. Student’s p values are indicated on the plot.

Figure 2.. Transcription factor enrichment in FXTAS.

Enrichment was performed by utilizing as input only those proteins with a cut-off of 2 and −2, and then filtering by those with a combined score of 100 or higher. Analysis was performed with EnrichR (Kuleshov et al., 2016). The top 4 factors were blasted against DisGeNET (Pinero et al., 2020), a database of gene-disease associations. Each search was then filtered by diseases with CNS components and with GDA scores higher than 0.02. MYO1D was the only transcription factor with no disease associated with CNS. Sunburst chart: GDA scores of CNS disorders (labeled from 1 to 12) associated with NFE2L2, FOXO1, and REST.

Figure 3.. Gene expression of NFE2L2 and related genes.

Gene expression was performed on cDNA from sex- and age-matched 4 non-carriers and 4 carriers of the premutation. All donors were females [age and CGG repeats expressed as Mean (SD) for noncarriers and carriers: 34.5 y (4.0) and 33.5 y (2.9) p = 0.847; F value = 1.99; p = 0.5863; 31.75 repeats (2.05) and 81.25 repeats (9.45); p = 0.011; F value = 21.1; p = 0.032)]. The gene expression was normalized to that of XRCC5 as it was the most stable gene across these samples and putative housekeeping genes tested (Supplementary Figure 1). A heatmap (A) was obtained by using a Pearson’s distance measure with a Ward clustering algorithm according to the significance of the Student’s t test (with equal variance). B. Gene expression as log2FC normalized to average of pooled controls. Only statistically significant different genes (Student’s t test with equal variance) are reported. C. Correlation between NFE2L2 gene expression and that of other genes with critical roles in mitochondrial function. Genes shown had an FDR <0.05.

Figure 4.. Effect of sulforaphane on biological pathways and processes.

Differentially expressed proteins in fibroblasts from FXTAS-affected subjects treated with 5 μM SFN or vehicle for 72 h were used as seeds to evaluate affected pathways. The analysis was performed with PathVisio (Kutmon et al., 2015) and the WikiPathways database. The resulting pathways were then filtered by those with more than 2 proteins and p < 0.05. The size of the bubbles represents the number of proteins. The gene ontology “biological processes” was performed with InnateDB and utilizing a cut-off of 2 and −2 and p < 0.05.

Figure 5.. Effect of sulforaphane on cellular compartments and bioenergetics.

The gene ontology “cellular compartment” (A) was performed with InnateDB, utilizing a cut-off of 2 and −2 and p < 0.05. Bubble size represents the number of proteins. Mitochondrial outcomes (B) were evaluated in fibroblasts from FXTAS-affected carriers (n = 6) as described in the Methods, upon treatment with 5 μM SFN (or DMSO) for 72h. Outcomes are shown as log2 FC between SFN- and DMSO-treated samples. C. Linear regression of mitochondrial outcomes at 72 and 96 h for 2 of the SFN-treated FXTAS cell lines. P value was obtained by Pearson’s analysis. D. Scheme showing the NFE2L2-mediated oxidative cellular response and transcription activity, and the putative acting site of SFN. Enrichment in transcription factors was performed with InnateDB.

Figure 6.. Sulforaphane affects mainly postsynaptic translation and transmission.

Proteins differentially expressed by SFN treatment were blasted against the evidence-based, expert curated resource for synapse function and gene enrichment studies SynGO (Koopmans et al., 2019).