Biology subjects

Sheshadri, P.

Publications and source records attributed to Sheshadri, P..

5 recordsLinked to original sources

bioRxiv2025

Targeting Lysosomal pH Restores Mitochondrial Quality Control in GBA1-Mutant Parkinsons Disease

BackgroundHeterozygous mutations in the Glucocerebrosidase gene (GBA1), which encodes the lysosomal enzyme {beta}-glucocerebrosidase (GCase), are a genetic risk factor for Parkinsons disease (PD). The pathophysiological consequences of GBA1 mutations on dopaminergic neuronal function, especially their impact on lysosomal function, mitophagy, and mitochondrial bioenergetics, remain unclear. MethodsFibroblasts and dopaminergic neurons generated from induced pluripotent stem cells (iPSCs) derived from patients with GBA1-PD were used in the study. Live-cell imaging was performed to measure lysosomal acidification, protease activity, mitochondrial membrane potential, and mitophagy. Mitochondrial morphology and autophagic vesicles were examined using transmission electron microscopy. Oxygen consumption rate was measured by Seahorse assay. V-ATPase assembly was quantified using FLIM-FRET, and pharmacological interventions included rapamycin and acidic nanoparticles. Statistical analyses involved unpaired t-tests, one-way ANOVA, and two-way ANOVA. ResultsGCase activity, lysosomal acidification, protease activity, mitophagy and mitochondrial bioenergetic function were all impaired. Mitochondria were fragmented, with reduced membrane potential and oxygen consumption. MTORC1 was constitutively phosphorylated and FLIM-FRET measurements confirmed impaired lysosomal V-ATPase assembly, which was reversed following rapamycin treatment. Rapamycin and lysosome-targeted acidic nanoparticles rescued lysosomal pH, restored mitophagy, mitochondrial membrane potential and mitochondrial OXPHOS complex levels in GBA1 mutant dopaminergic neurons. ConclusionsWe reveal a novel mechanistic link between GBA1 mutations and mitochondrial dysfunction, as disruption of V-ATPase assembly driven by MTORC1 activation impairs lysosomal acidification. Mitophagy is therefore impaired leading to mitochondrial dysfunction, undermining dopaminergic cell function and fate. Pharmacological intervention with rapamycin or acidic nanoparticles restore lysosomal pH and rescue mitochondrial function, signposting a novel therapeutic approach for GBA1-PD.

neuroscience↗

bioRxiv2025

Pathogenic variants in autophagy-tethering factor EPG5 drive neurodegeneration through mitochondrial dysfunction and innate immune activation

The autophagy-tethering factor, ectopic P-granule 5 autophagy protein (EPG5), plays a key role in autophagosome-lysosome fusion. Impaired autophagy associated with pathogenic variants in EPG5 cause a rare devastating multisystem disorder known as Vici syndrome, which includes neurodevelopmental defects, severe progressive neurodegeneration and immunodeficiency. The pathophysiological mechanisms driving disease presentation and progression are not understood. In patient-derived fibroblasts and iPS cells differentiated to cortical neurons, we found that impaired mitophagy leads to mitochondrial bioenergetic dysfunction. Physiological Ca2+ signals resulted in paradoxical mitochondrial Ca2+ overload attributed to downregulation of MICU1/3. Ca2+ signals caused mitochondrial depolarisation, mtDNA release and activation of the cGAS-STING pathway, reversed by pharmacological inhibition of the mitochondrial permeability transition pore (mPTP) or of the STING pathway. Thus, we have identified multiple potential therapeutic targets driving disease progression associated with pathogenic EPG5 mutations, including impaired mitochondrial bioenergetics, mitochondrial Ca2+ overload, vulnerability to mPTP opening and activation of innate immune signalling.

neuroscience↗

bioRxiv2024

Metabolic remodelling in hiPSC-derived myofibres carrying the m.3243A>G mutation

Mutations in mitochondrial DNA cause severe multisystem disease, frequently associated with muscle weakness. The m.3243A>G mutation is the major cause of Mitochondrial Encephalomyopathy Lactic Acidosis and Stroke Like episodes (MELAS). Experimental models that recapitulate the disease phenotype in vitro for disease modelling or drug screening are very limited. We have therefore generated hiPSC-derived muscle fibres with variable heteroplasmic mtDNA mutation load without significantly affecting muscle differentiation potential. The cells are excitable and show physiological characteristics of muscle fibres and show well organised myofibrillar structure. In cells carrying the m.3243A>G, the mitochondrial membrane potential and oxygen consumption were reduced in relation to the mutant load. We have shown through proteomic, phosphoproteomic, and metabolomic analyses that the m.3243A>G mutation variably affects the cell phenotype in relation to the mutant load. This variation is reflected by an increase in the NADH/NAD+ ratio, which in turns influences key nutrient-sensing pathways in the myofibres. This model enables detailed study of the impact of the mutation on cellular bioenergetics and on muscle physiology with the potential to provide a platform for drug screening.

cell biology↗

bioRxiv2022

Deubiquitinase USP1 influences the dedifferentiation of mouse pancreatic β-cells

Diabetes is a metabolic disease caused majorly due to loss of insulin secreting {beta}-cells. Along with apoptosis, recent reports revealed dedifferentiation to be the added reason for the reduced {beta}-cell mass. The Ubiquitin Proteasome system comprising of E3 ligase and deubiquitinases (DUBs) control several key aspects of pancreatic {beta}-cell functions. The role of deubiquitinases in orchestrating the dedifferentiation process in several cancers have been well deciphered, but its role in dedifferentiation of pancreatic {beta}-cells remains elusive. In this study, screening for key DUBs that regulate dedifferentiation, identified USP1 to be specifically involved in the process. Inhibition of USP1 either by genetic intervention or small molecule inhibitor ML323 restored epithelial phenotype of {beta}-cells, but not with inhibition of other DUBs. Conversely overexpression of USP1 was sufficient to dedifferentiate {beta}-cells, even in absence of dedifferentiation inducing cues. Mechanistic insight showed USP1 to probably mediate its effect via modulating the expression of Inhibitor of Differentiation (ID) 2. Further, in an in vivo streptozotocin (STZ) induced dedifferentiation mouse model system, treatment with ML323 rescued the hyperglycaemic state. Overall, this study assigns a novel role to USP1 in dedifferentiation of {beta}-cells and its inhibition may have a therapeutic application of reducing the {beta}-cell loss during diabetes.

cell biology↗

bioRxiv2020

Paradoxical neuronal hyperexcitability in a mouse model of mitochondrial pyruvate import deficiency

Neuronal excitation imposes a high demand of ATP in neurons. Most of the ATP derives primarily from pyruvate-mediated oxidative phosphorylation, a process that relies on import of pyruvate into mitochondria occuring exclusively via the mitochondrial pyruvate carrier (MPC). To investigate whether deficient oxidative phosphorylation impacts neuron excitability, we generated a mouse strain carrying a conditional deletion of MPC1, an essential subunit of the mitochondrial pyruvate carrier, specifically in adult glutamatergic neurons. We found that, despite decreased levels of oxidative phosphorylation in these excitatory neurons, mice were normal at rest. Paradoxically, in response to mild inhibition of GABA mediated synaptic activity, they rapidly developed severe seizures and died, whereas under similar conditions the behaviour of control mice remained unchanged. We show that neurons with a deficient MPC are intrinsically hyperexcitable as a consequence of impaired calcium homeostasis, which reduces M-type potassium channel activity. Provision of ketone bodies restores energy status, calcium homeostasis and M-channel activity and attenuates seizures in animals fed a ketogenic diet. Our results provide an explanation for the paradoxical seizures that frequently accompany a large number of neuropathologies, including cerebral ischemia and diverse mitochondriopathies, in which neurons experience an energy deficit. One Sentence SummaryDecreased OXPHOS and Ca2+-mediated neuronal hyperexcitability lead to seizure in a mouse model of mitochondrial pyruvate import deficiency.

neuroscience↗