Biology subjects

Roberts, A. M.

Publications and source records attributed to Roberts, A. M..

8 recordsLinked to original sources

bioRxiv2026

Distinct signaling mechanisms and proteome phenotypes are elicited by compartment-specific genetic defects of copper homeostasis

Impairments to the complex machinery regulating copper homeostasis lead to neurodevelopmental diseases, demonstrating the importance of copper for neuronal health and maintenance. The exact mechanisms by which the brain responds to copper deficiency following disruptions to the copper transporters ATP7A and CTR1 in conditions such as Menkes disease remain unclear, though failure to supply complex IV of the respiratory chain with copper is suspected to account for substantial pathology. Here, we studied mechanisms of copper deficiency using systems biology approaches to contrast isogenic CTR1- and COX17-deficient cells, which model copper deficiency at the level of the whole cell or complex IV, respectively. Multiomics approaches revealed distinct signaling mechanisms elicited by compartment-specific genetic defects of copper homeostasis, spanning multiple organelles and biological functions. Specifically, COX17 KO cells exhibited elevated AMPK activity and blunted mTOR activity relative to CTR1-null cells. Manipulating mTOR activity elicited inverse effects on survival in CTR1-deficient cells and flies as compared to their COX17-deficient counterparts. Increased mTOR activity and downstream protein synthesis is adaptive in models of copper deficiency but deleterious in COX17-deficient cells and flies. We propose that mTOR activation represents a resilience mechanism that fails following sustained copper deficiency and impairments to mitochondrial respiration. SignificanceO_LIComparative proteomics reveals distinct molecular mechanisms downstream of compartmentalized copper deficiency C_LIO_LICOX17- and CTR1-deficient cells exhibit distinct patterns of AMPK/mTOR pathway activity C_LIO_LImTOR and downstream S6K activity is protective in cellular copper deficiency but not in compartmentalized mitochondrial copper deficiency C_LI

cell biology↗

bioRxiv2025

Endocrine therapy induces oxidative stress in ER+ breast cancer that sensitizes persister cells to ferroptosis

Despite endocrine therapy, recurrence and progression of estrogen receptor alpha (ER)-positive breast cancer remain significant clinical problems. We therefore sought to identify mechanisms underlying endocrine-tolerant persistence. Endocrine-tolerant persister ER+ breast cancer cells were oxidatively stressed during endocrine therapy. Proteomic analysis revealed upregulation of antioxidant-driving enzymes including glutathione peroxidase 4 (GPX4) in persisters. Relief of oxidative stress enhanced persister fitness. The increased oxidative state of persisters enabled lipid peroxidation and ferroptosis. Persisters had an altered lipidome with increased levels of polyunsaturated fatty acids prone to peroxidation, which was attributable in part to increased lysophosphatidylcholine acyltyransferase 3 (LPCAT3, MBOAT5) expression via loss of ER-mediated repression during endocrine therapy. Treatment with the GPX4 inhibitor RSL3 enhanced the anti-persister effects of endocrine-based therapies in xenograft-bearing mice. These findings supporting the development of therapeutic strategies to leverage the oxidative stress induced by endocrine-based therapies and drive ferroptosis as a treatment for ER+ breast cancer. Statement of SignificanceEndocrine therapy increases oxidative stress and sensitizes endocrine-tolerant persister ER+ breast cancer cells to ferroptosis, indicating that therapies targeting this metabolic dependency could help prevent disease recurrence and progression.

cancer biology↗

bioRxiv2025

Mitochondrially Transcribed dsRNA Mediates Manganese-induced Neuroinflammation

Manganese is an essential trace element required for various biological functions, but in excess is neurotoxic and leads to significant health concerns. The mechanisms underlying manganese neurotoxicity remain poorly understood. Neuropathological studies of affected brain regions reveal astrogliosis, neuronal loss, and neuroinflammation. Here, we present a novel manganese-dependent mechanism linking mitochondrial dysfunction to neuroinflammation. We found that manganese disruption of the mitochondrial transcriptome processing results in the accumulation of double stranded RNA (dsRNA). This dsRNA is released into the cytoplasm, where it activates the cytosolic sensor MDA5, triggering type I interferon responses and inflammatory cytokine production. This mechanism is evident in 100 day human cerebral organoids, where manganese-increased mitochondrial dsRNA and induced inflammatory responses in mature astrocytes. Similarly, we observed an increase in mitochondrial dsRNA content, the activation of an inflammatory transcriptome and the production of cytokines in female and male mouse brains carrying mutations in the Slc30a10 gene, a model for human hypermanganesemia with dystonia 1 disorder. These findings highlight a previously unrecognized role for mitochondrial dsRNA in manganese-induced neuroinflammation and provide insights into the molecular pathogenesis of manganism. We propose that this mitochondrial dsRNA-induced inflammatory pathway could be active in other neurological diseases caused by environmental or genetic factors. Significance StatementEnvironmental exposures and genetic defects that perturb manganese homeostasis are an underappreciated cause of neurodegeneration and neuroinflammation. We describe a new paradigm for inducible neuroinflammation, where manganese disruption of mitochondrial transcriptome processing leads to the accumulation of mitochondrial double-stranded RNA (dsRNA), which activate antiviral responses in the cytoplasm driving type I interferon dependent inflammation. This manganese-dsRNA axis is induced in cell lines in vitro and a subpopulation of mature astrocytes in exposed human cerebral organoids. Brain cortex of mice deficient in the manganese efflux transporter Slc30a10, a genetic model of chronic manganese accumulation, show dsRNA accumulation, and up-regulation of type I interferon response and astrogliosis markers, supporting a role for this pathway in neurotoxicity and parkinsonism.

neuroscience↗

bioRxiv2024

Endocrine persistence in ER+ breast cancer is accompanied by metabolic vulnerability in oxidative phosphorylation

Despite adjuvant treatment with endocrine therapies, estrogen receptor-positive (ER+) breast cancers recur in a significant proportion of patients. Recurrences are attributable to clinically undetectable endocrine-tolerant persister cancer cells that retain tumor-forming potential. Therefore, strategies targeting such persister cells may prevent recurrent disease. Using CRISPR-Cas9 genome-wide knockout screening in ER+ breast cancer cells, we identified a survival mechanism involving metabolic reprogramming with reliance upon mitochondrial respiration in endocrine-tolerant persister cells. Quantitative proteomic profiling showed reduced levels of glycolytic proteins in persisters. Metabolic tracing of glucose revealed an energy-depleted state in persisters where oxidative phosphorylation was required to generate ATP. A phase II clinical trial was conducted to evaluate changes in mitochondrial markers in primary ER+/HER2-breast tumors induced by neoadjuvant endocrine therapy (NCT04568616). In an analysis of tumor specimens from 32 patients, tumors exhibiting residual cell proliferation after aromatase inhibitor-induced estrogen deprivation with letrozole showed increased mitochondrial content. Genetic profiling and barcode lineage tracing showed that endocrine-tolerant persistence occurred stochastically without genetic predisposition. Mice bearing cell line- and patient-derived xenografts were used to measure the anti-tumor effects of mitochondrial complex I inhibition in the context of endocrine therapy. Pharmacological inhibition of complex I suppressed the tumor-forming potential of persisters and synergized with the anti-estrogen fulvestrant to induce regression of patient-derived xenografts. These findings indicate that mitochondrial metabolism is essential in endocrine-tolerant persister ER+ breast cancer cells and warrant the development of treatment strategies to leverage this vulnerability in the context of endocrine-sensitive disease. Statement of SignificanceEndocrine-tolerant persister cancer cells that survive endocrine therapy can cause recurrent disease. Persister cells exhibit increased energetic dependence upon mitochondria for survival and tumor re-growth potential.

cancer biology↗

bioRxiv2024

Adaptive protein synthesis in genetic models of copper deficiency and childhood neurodegeneration

Rare inherited diseases caused by mutations in the copper transporters SLC31A1 (CTR1) or ATP7A induce copper deficiency in the brain, causing seizures and neurodegeneration in infancy through poorly understood mechanisms. Here, we used multiple model systems to characterize the molecular mechanisms by which neuronal cells respond to copper deficiency. Targeted deletion of CTR1 in neuroblastoma cells produced copper deficiency that was associated with a metabolic shift favoring glycolysis over oxidative phosphorylation. Proteomic and transcriptomic analysis of CTR1 KO cells revealed simultaneous upregulation of mTORC1 and S6K signaling and reduced PERK signaling. Patterns of gene and protein expression and pharmacogenomics show increased activation of the mTORC1-S6K pathway as a pro-survival mechanism, ultimately resulting in increased protein synthesis. Spatial transcriptomic profiling of Atp7aflx/Y :: Vil1Cre/+ mice identified upregulated protein synthesis machinery and mTORC1-S6K pathway genes in copper-deficient Purkinje neurons in the cerebellum. Genetic epistasis experiments in Drosophila demonstrated that copper deficiency dendritic phenotypes in class IV neurons are partially rescued by increased S6k expression or 4E-BP1 (Thor) RNAi, while epidermis phenotypes are exacerbated by Akt, S6k, or raptor RNAi. Overall, we demonstrate that increased mTORC1-S6K pathway activation and protein synthesis is an adaptive mechanism by which neuronal cells respond to copper deficiency. SignificanceO_LICopper deficiency is present in rare conditions such as Menkes disease and CTR1 deficiency and in more common diseases like Alzheimers. The mechanisms of resilience and ultimate susceptibility to copper deficiency and associated pathology in the brain remain unknown. C_LIO_LIWe demonstrate that in a human cell line, Drosophila, and the mouse cerebellum, copper-deficient neuronal cells exhibit increased protein synthesis through mTORC1 activation and decreased PERK (EIF2AK3) activity. C_LIO_LIUpregulation of protein synthesis facilitates resilience of neuronal cells to copper deficiency, including partial restoration of dendritic arborization. Our findings offer a new framework for understanding copper deficiency-related pathology in neurological disorders. C_LI

neuroscience↗

bioRxiv2023

Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer

PurposeClinical evidence indicates that treatment with estrogens elicits anti-cancer effects in [~]30% of patients with advanced endocrine-resistant estrogen receptor alpha (ER)-positive breast cancer. Despite the proven efficacy of estrogen therapy, its mechanism of action is unclear and this treatment remains under-utilized. Mechanistic understanding may offer strategies to enhance therapeutic efficacy. Experimental DesignWe performed genome-wide CRISPR/Cas9 screening and transcriptomic profiling in long-term estrogen-deprived (LTED) ER+ breast cancer cells to identify pathways required for therapeutic response to the estrogen 17{beta}-estradiol (E2). We validated findings in cell lines, patient-derived xenografts (PDXs), and patient samples, and developed a novel combination treatment through testing in cell lines and PDX models. ResultsCells treated with E2 exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops). Pharmacological suppression of the DNA damage response via poly(ADP-ribose) polymerase (PARP) inhibition with olaparib enhanced E2-induced DNA damage. PARP inhibition synergized with E2 to suppress growth and prevent tumor recurrence in BRCA1/2-mutant and BRCA1/2-wild-type cell line and PDX models. ConclusionsE2-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells. Inhibition of the DNA damage response using drugs such as PARP inhibitors can enhance therapeutic response to E2. These findings warrant clinical exploration of the combination of E2 with DNA damage response inhibitors in advanced ER+ breast cancer, and suggest that PARP inhibitors may synergize with therapeutics that exacerbate transcriptional stress.

cancer biology↗

bioRxiv2022

The Mitochondrial RNA Granule is Necessary for Parkinsonism-Associated Metal Toxicity

Prolonged manganese exposure causes manganism, a neurodegenerative movement disorder. The identity of adaptive and non-adaptive cellular processes targeted by manganese remains mostly unexplored. Here we study mechanisms engaged by manganese in genetic cellular models known to increase susceptibility to manganese exposure, the plasma membrane manganese efflux transporter SLC30A10 and the mitochondrial Parkinsons gene PARK2. We found that SLC30A10 and PARK2 mutations as well as manganese exposure compromised the mitochondrial RNA granule as well as mitochondrial transcript processing. These RNA granule defects led to impaired assembly and function of the mitochondrial respiratory chain. Notably, cells that survived a cytotoxic manganese challenge had impaired RNA granule function, thus suggesting that this granule phenotype was adaptive. CRISPR gene editing of subunits of the mitochondrial RNA granule, FASTKD2 or DHX30, as well as pharmacological inhibition of mitochondrial transcription-translation, were protective rather than deleterious for survival of cells acutely exposed to manganese. Similarly, adult Drosophila mutants with defects in the mitochondrial RNA granule component scully were safeguarded from manganese-induced mortality. We conclude that the downregulation of the mitochondrial RNA granule function is a protective mechanism for acute metal toxicity. Significance StatementMutations in the manganese efflux transporter SLC30A10 and the mitochondrial Parkinsons gene PARK2, cause neurodegeneration and increased susceptibility to toxic manganese exposure. Thus, molecular processes affected in both mutants could offer insight into fundamental mechanisms conferring susceptibility or resilience to environmental and genetic factors associated with neurodegeneration. Here we report that SLC30A10 and PARK2 mutations compromise an understudied structure, the mitochondrial RNA granule, which is required for processing polycistronic mitochondrial RNAs. Cells and Drosophila lacking mitochondrial RNA granule components were resistant to manganese exposure. We conclude that the downregulation of the mitochondrial RNA granule function is an adaptive mechanism for cells exposed to manganese.

cell biology↗

bioRxiv2019

Characterising the loss-of-function impact of 5' untranslated region variants in whole genome sequence data from 15,708 individuals

Upstream open reading frames (uORFs) are important tissue-specific cis-regulators of protein translation. Although isolated case reports have shown that variants that create or disrupt uORFs can cause disease, genetic sequencing approaches typically focus on protein-coding regions and ignore these variants. Here, we describe a systematic genome-wide study of variants that create and disrupt human uORFs, and explore their role in human disease using 15,708 whole genome sequences collected by the Genome Aggregation Database (gnomAD) project. We show that 14,897 variants that create new start codons upstream of the canonical coding sequence (CDS), and 2,406 variants disrupting the stop site of existing uORFs, are under strong negative selection. Furthermore, variants creating uORFs that overlap the CDS show signals of selection equivalent to coding loss-of-function variants, and uORF-perturbing variants are under strong selection when arising upstream of known disease genes and genes intolerant to loss-of-function variants. Finally, we identify specific genes where perturbation of uORFs is likely to represent an important disease mechanism, and report a novel uORF frameshift variant upstream of NF2 in families with neurofibromatosis. Our results highlight uORF-perturbing variants as an important and under-recognised functional class that can contribute to penetrant human disease, and demonstrate the power of large-scale population sequencing data to study the deleteriousness of specific classes of non-coding variants.

genomics↗