Biology subjects

Shutt, T.

Publications and source records attributed to Shutt, T..

7 recordsLinked to original sources

bioRxiv2024

Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation

Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2 kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that pharmacologic activation of the ISR reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

cell biology↗

bioRxiv2023

Measuring mtDNA turnover, synthesis, and supercoiling via selective bromodeoxyuridine incorporation

An often-overlooked aspect of mitochondrial biology is the mitochondrial DNA (mtDNA). The multi-copy mtDNA is highly dynamic, with changes in supercoiling, synthesis rates, and turnover rates that are tightly associated with mitochondrial and cellular functions. To better understand the state of mtDNA, here we describe a protocol that selectively incorporates bromodeoxyuridine into mtDNA for subsequent measurement via an adapted Southern blot followed by immunoblotting (a.k.a. Southwestern blot). This basic protocol can be applied with slight modifications for the measurement of mtDNA synthesis, turnover or supercoiling to understand mtDNA changes.

molecular biology↗

bioRxiv2022

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT

The recognition that cytosolic mtDNA activates cGAS-STING innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early-onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association between TOP1MT and autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed in TOP1MT knockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels, and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.

cell biology↗

bioRxiv2020

Impaired phosphatidylethanolamine metabolism activates a reversible stress response that detects and resolves mutant mitochondrial precursors

Phosphatidylethanolamine made in mitochondria has long been recognized as an important precursor for phosphatidylcholine production that occurs in the endoplasmic reticulum (ER). Recently, the strict mitochondrial localization of the enzyme that makes PE in the mitochondrion, phosphatidylserine decarboxylase 1 (Psd1), was questioned. Since a dual localization of Psd1 to the ER would have far-reaching implications, we initiated our study to independently re-assess the subcellular distribution of Psd1. Our results support the unavoidable conclusion that the vast majority, if not all, of functional Psd1 resides in the mitochondrion. Through our efforts, we discovered that mutant forms of Psd1 that impair a self-processing step needed for it to become functional are dually localized to the ER when expressed in a PE-limiting environment. We conclude that severely impaired cellular PE metabolism provokes an ER-assisted adaptive response that is capable of identifying and resolving nonfunctional mitochondrial precursors.

cell biology↗

bioRxiv2020

A novel synthetic peptide provides neuroprotection and rescues rat cortical neurons from anesthetic-induced cell death and modulation of growth and synaptic assembly

Anesthetics, although necessary for many modern procedures, exert neurotoxic effects in various experimental models; the underlying mechanisms, however, remain unknown. In the absence of this information, any in vivo mitigation strategy would be challenging. To help address these gaps, we sought to determine whether preserving mitochondrial network integrity with a non-toxic, short-life synthetic peptide, P110, could protect cortical neurons against both inhalational and intravenous anesthetic-induced neurotoxicity. This study provides the first direct comparative account of three key general anesthetics (desflurane, propofol, and ketamine) under identical conditions and demonstrates their impact on cellular viability, neurite outgrowth, and synaptic network assembly on neonatal rat cortical neurons. Further, we discovered that the inhibition of Fis1 receptor-mediated fission reverses anesthetic-induced aberrations in an agent-specific manner. Our data thus underscore the importance of a reductionist approach, offering unique opportunities to explore non-toxic, mitochondria-based therapeutic tools for mitigating anesthetic-induced harm to the developing brain.

neuroscience↗

bioRxiv2019

Reversible Mitochondrial Fragmentation in iPSC-Derived Cardiomyocytes from Children with DCMA, a Mitochondrial Cardiomyopathy

BackgroundThe dilated cardiomyopathy with ataxia syndrome (DCMA) is an understudied autosomal recessive disease caused by loss-of-function mutations in the poorly characterized gene DNAJC19. Clinically, DCMA is commonly associated with heart failure and early death in affected children through an unknown mechanism. DCMA has been linked to Barth syndrome, a rare but well-studied disorder caused by deficient maturation of cardiolipin (CL), a key mitochondrial membrane phospholipid.\n\nMethodsPeripheral blood mononuclear cells from two children with DCMA and severe cardiac dysfunction were reprogrammed into induced pluripotent stem cells (iPSCs). Patient and control iPSCs were differentiated into beating cardiomyocytes (iPSC-CMs) using a metabolic selection strategy and mitochondrial structure and CL content before and after incubation with the mitochondrially-targeted peptide SS-31 were quantified.\n\nResultsPatient iPSCs carry the causative DNAJC19 mutation (rs137854888) found in the Hutterite population and the iPSC-CMs demonstrated highly fragmented and abnormally-shaped mitochondria associated with an imbalanced isoform ratio of the mitochondrial protein OPA1, an important regulator of mitochondrial fusion. These abnormalities were reversible by incubation with SS-31 for 24 hours. Differentiation of iPSCs into iPSC-CMs increased the number of CL species observed but consistent, significant differences in CL content were not seen between patients and control.\n\nConclusionsWe describe a unique and novel cellular model that provides insight into the mitochondrial abnormalities present in DCMA and identifies SS-31 as a potential therapeutic for this devastating disease.

cell biology↗

bioRxiv2019

SS-31 Reverses Mitochondrial Fragmentation in Fibroblasts from Patients with DCMA, a Mitochondrial Cardiomyopathy

ObjectivesWe used patient dermal fibroblasts to characterize the mitochondrial abnormalities associated with the dilated cardiomyopathy with ataxia syndrome (DCMA) and to study the effect of the mitochondrially-targeted peptide SS-31 as a potential novel therapeutic.\n\nBackgroundDCMA is an understudied autosomal recessive disorder thought to be related to Barth syndrome but caused by mutations in DNAJC19, a protein of unknown function localized to the mitochondria. The clinical disease is characterized by 3-methylglutaconic aciduria, dilated cardiomyopathy, abnormal neurological development and other heterogeneous features. Until recently no effective therapies had been identified and affected patients frequently died in early childhood from intractable heart failure.\n\nMethodsDermal fibroblasts from four pediatric patients with DCMA were used to establish parameters of mitochondrial dysfunction. Mitochondrial structure, reactive oxygen species (ROS) production, cardiolipin composition and gene expression were evaluated.\n\nResultsImmunocytochemistry with semi-automated quantification of mitochondrial structural metrics and transmission electron microscopy demonstrated mitochondria to be highly fragmented in DCMA fibroblasts compared to healthy control cells. Live-cell imaging demonstrated significantly increased ROS production in patient cells. These structural and functional abnormalities were reversed by treating DCMA fibroblasts with SS-31, a synthetic peptide that localizes to the inner mitochondrial membrane. Levels of cardiolipin were not significantly different between control and DCMA cells and were unaffected by SS-31 treatment.\n\nConclusionsOur results demonstrate the abnormal mitochondrial structure and function in fibroblasts from patients with DCMA and suggest that SS-31 may represent a potential therapy for this devastating disease.

cell biology↗