ABSTRACT
Metabolic alterations shared between the nervous system and skin fibroblasts have emerged in amyotrophic lateral sclerosis (ALS). Recently, we found that a subgroup of sporadic ALS (sALS) fibroblasts (sALS1) is characterized by metabolic profiles distinct from other sALS cases (sALS2) and controls, suggesting that metabolic therapies could be effective in sALS. The metabolic modulators nicotinamide riboside and pterostilbene (EH301) are under clinical development for the treatment of ALS. Here, we studied the transcriptome and metabolome of sALS cells to understand the molecular bases of sALS metabotypes and the impact of EH301. Metabolomics and transcriptomics were investigated at baseline and after EH301 treatment. Moreover, weighted gene coexpression network analysis (WGCNA) was used to investigate the association of the metabolic and clinical features. We found that the sALS1 transcriptome is distinct from sALS2 and that EH301 modifies gene expression differently in sALS1, sALS2 and the controls. Furthermore, EH301 had strong protective effects against metabolic stress, an effect linked to the antiinflammatory and antioxidant pathways. WGCNA revealed that the ALS functional rating scale and metabotypes are associated with gene modules enriched for the cell cycle, immunity, autophagy and metabolic genes, which are modified by EH301. The meta-analysis of publicly available transcriptomic data from induced motor neurons by Answer ALS confirmed the functional associations of genes correlated with disease traits. A subset of genes differentially expressed in sALS fibroblasts was used in a machine learning model to predict disease progression. In conclusion, multiomic analyses highlighted the differential metabolic and transcriptomic profiles in patient-derived fibroblast sALS, which translate into differential responses to the investigational drug EH301.
Subject(s)
Amyotrophic Lateral Sclerosis , Amyotrophic Lateral Sclerosis/metabolism , Antioxidants/metabolism , Drugs, Investigational/metabolism , Drugs, Investigational/therapeutic use , Fibroblasts/metabolism , Humans , Transcriptome/geneticsABSTRACT
Brain and heart ischemia are among the leading causes of death and disability in both men and women, but there are significant sex differences in the incidence and severity of these diseases. Ca2+ dysregulation in response to ischemia/reperfusion injury (I/RI) is a well-recognized pathogenic mechanism leading to the death of affected cells. Excess intracellular Ca2+ causes mitochondrial matrix Ca2+ overload that can result in mitochondrial permeability transition (MPT), which can have severe consequences for mitochondrial function and trigger cell death. Recent findings indicate that estrogens and their related receptors are involved in the regulation of MPT, suggesting that sex differences in I/RI could be linked to estrogen-dependent modulation of mitochondrial Ca2+. Here, we review the evidence supporting sex differences in I/RI and the role of estrogen and estrogen receptors in producing these differences, the involvement of mitochondrial Ca2+ overload in disease pathogenesis, and the estrogen-dependent modulation of MPT that may contribute to sex differences.
Subject(s)
Mitochondria/metabolism , Mitochondrial Transmembrane Permeability-Driven Necrosis/physiology , Reperfusion Injury/metabolism , Sex Characteristics , Animals , Calcium/metabolism , Humans , PermeabilityABSTRACT
Recent evidence highlights a role for sex and hormonal status in regulating cellular responses to ischemic brain injury and neurodegeneration. A key pathological event in ischemic brain injury is the opening of a mitochondrial permeability transition pore (MPT) induced by excitotoxic calcium levels, which can trigger irreversible damage to mitochondria accompanied by the release of pro-apoptotic factors. However, sex differences in brain MPT modulation have not yet been explored. Here, we show that mitochondria isolated from female mouse forebrain have a lower calcium threshold for MPT than male mitochondria, and that this sex difference depends on the MPT regulator cyclophilin D (CypD). We also demonstrate that an estrogen receptor beta (ERß) antagonist inhibits MPT and knockout of ERß decreases the sensitivity of mitochondria to the CypD inhibitor, cyclosporine A. These results suggest a functional relationship between ERß and CypD in modulating brain MPT. Moreover, co-immunoprecipitation studies identify several ERß binding partners in mitochondria. Among these, we investigate the mitochondrial ATPase as a putative site of MPT regulation by ERß. We find that previously described interaction between the oligomycin sensitivity-conferring subunit of ATPase (OSCP) and CypD is decreased by ERß knockout, suggesting that ERß modulates MPT by regulating CypD interaction with OSCP. Functionally, in primary neurons and hippocampal slice cultures, modulation of ERß has protective effects against glutamate toxicity and oxygen glucose deprivation, respectively. Taken together, these results reveal a novel pathway of brain MPT regulation by ERß that could contribute to sex differences in ischemic brain injury and neurodegeneration.
Subject(s)
Adenosine Triphosphatases/genetics , Carrier Proteins/genetics , Cyclophilins/genetics , Estrogen Receptor beta/genetics , Hippocampus/metabolism , Membrane Proteins/genetics , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Prosencephalon/metabolism , Adenosine Triphosphatases/metabolism , Animals , COS Cells , Calcium/metabolism , Carrier Proteins/metabolism , Chlorocebus aethiops , Peptidyl-Prolyl Isomerase F , Cyclophilins/antagonists & inhibitors , Cyclophilins/deficiency , Cyclosporine/pharmacology , Estrogen Receptor beta/antagonists & inhibitors , Estrogen Receptor beta/deficiency , Female , Hippocampus/drug effects , Male , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/physiology , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microtomy , Mitochondria/drug effects , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , Mitochondrial Proton-Translocating ATPases , Piperidines/pharmacology , Prosencephalon/drug effects , Protein Binding , Pyrazoles/pharmacology , Sex Factors , Tissue Culture TechniquesABSTRACT
Voltage-gated ion channels are critical for neuronal integration. Some of these channels, however, are misregulated in several neurological disorders, causing both gain- and loss-of-function channelopathies in neurons. Using several transgenic mouse models of Alzheimer's disease (AD), we find that sub-threshold voltage signals strongly influenced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels progressively deteriorate over chronological aging in hippocampal CA1 pyramidal neurons. The degraded signaling via HCN channels in the transgenic mice is accompanied by an age-related global loss of their non-uniform dendritic expression. Both the aberrant signaling via HCN channels and their mislocalization could be restored using a variety of pharmacological agents that target the endoplasmic reticulum (ER). Our rescue of the HCN channelopathy helps provide molecular details into the favorable outcomes of ER-targeting drugs on the pathogenesis and synaptic/cognitive deficits in AD mouse models, and implies that they might have beneficial effects on neurological disorders linked to HCN channelopathies.
Subject(s)
Alzheimer Disease/physiopathology , CA1 Region, Hippocampal/physiology , Channelopathies/physiopathology , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/physiology , Neuronal Plasticity , Pyramidal Cells/physiology , Action Potentials , Aging , Animals , CA1 Region, Hippocampal/ultrastructure , Disease Models, Animal , Endoplasmic Reticulum/physiology , Female , Male , Mice, Transgenic , Pyramidal Cells/ultrastructureABSTRACT
OBJECTIVE: ALS is a rapidly progressive, fatal disorder caused by motor neuron degeneration, for which there is a great unmet therapeutic need. AMX0035, a combination of sodium phenylbutyrate (PB) and taurursodiol (TUDCA, TURSO), has shown promising results in early ALS clinical trials, but its mechanisms of action remain to be elucidated. Therefore, our goal was to obtain an unbiased landscape of the molecular effects of AMX0035 in ALS patient-derived cells. METHODS: We investigated the transcriptomic and metabolomic profiles of primary skin fibroblasts from sporadic ALS patients and healthy controls (n = 12/group) treated with PB, TUDCA, or PB-TUDCA combination (Combo). Data were evaluated with multiple approaches including differential gene expression and metabolite abundance, Gene Ontology and metabolic pathway analysis, weighted gene co-expression correlation analysis (WGCNA), and combined multiomics integrated analysis. RESULTS: Combo changed many more genes and metabolites than either PB or TUDCA individually. Most changes were unique to Combo and affected the expression of genes involved in nucleocytoplasmic transport, unfolded protein response, mitochondrial function, RNA metabolism, and innate immunity. WGCNA showed significant correlations between ALS gene expression modules and clinical parameters that were abolished by Combo treatment. INTERPRETATION: This study is the first to explore the molecular effects of Combo in ALS patient-derived cells. It shows that Combo has a greater and distinct impact compared with the individual compounds and provides clues to drug targets and mechanisms of action, which may underlie the benefits of this investigational drug combination.
Subject(s)
Amyotrophic Lateral Sclerosis , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Drugs, Investigational , Fibroblasts/metabolism , Humans , RNA , Taurochenodeoxycholic AcidABSTRACT
Introduction: Sex differences in brain cortical function affect cognition, behaviour and susceptibility to neural diseases, but the molecular basis of sexual dimorphism in cortical function is still largely unknown. Oestrogen and oestrogen receptors (ERs), specifically ERß, the most abundant ER in the cortex, may play a role in determining sex differences in gene expression, which could underlie functional sex differences. However, further investigation is needed to address brain region specificity of the effects of sex and ERß on gene expression. The goal of this study was to investigate sex differences in gene expression in the mouse posterior cortex, where sex differences in transcription have never been examined, and to determine how genetic ablation of ERß affects transcription. Methods: In this study, we performed unbiased transcriptomics on RNA from the posterior cortex of adult wild-type and ERß knockout mice (n = 4/sex/genotype). We used unbiased clustering to analyse whole-transcriptome changes between the groups. We also performed differential expression analysis on the data using DESeq2 to identify specific changes in gene expression. Results: We found only 27 significantly differentially expressed genes (DEGs) in wild-type (WT) males vs females, of which 17 were autosomal genes. Interestingly, in ERßKO males vs females all the autosomal DEGs were lost. Gene Ontology analysis of the subset of DEGs with sex differences only in the WT cortex revealed a significant enrichment of genes annotated with the function 'cation channel activity'. Moreover, within each sex we found only a few DEGs in ERßKO vs WT mice (8 and 5 in males and females, respectively). Conclusions: Overall, our results suggest that in the adult mouse posterior cortex there are surprisingly few sex differences in gene expression, and those that exist are mainly related to cation channel activity. Additionally, they indicate that brain region-specific functional effects of ERß may be largely post-transcriptional.