Biology subjects

Ravagnani, F. G.

Publications and source records attributed to Ravagnani, F. G..

3 recordsLinked to original sources

bioRxiv2026

Mitochondrial Metabolism and Calcium Handling in Parkinsons Disease hiPSC-derived Astrocytes

Parkinsons disease (PD) is the second most common neurodegenerative disorder worldwide, and mutations in the LRRK2 and PRKN genes are among the most common familial causes of the disease. In neurodegenerative diseases such as PD, disturbances in Ca{superscript 2} homeostasis and cellular bioenergetics impair the function of neurons and glial cells, contributing to disease progression. These changes are not limited to neurons; mitochondrial dysfunction and disrupted Ca2+ homeostasis in astrocytes are increasingly recognized as key contributors to PD, impairing bioenergetics, redox balance, neuroinflammatory responses, and metabolic support essential for dopaminergic neuron survival. In this study, we investigated mitochondrial calcium homeostasis, mitochondrial oxidative phosphorylation, morphology and distribution in human induced pluripotent stem cell (hiPSC)-derived astrocytes with mutations in the PD genes LRRK2 (G2019S) and PRKN (c.155delA; Ex3-4del) and wild-type controls. Intracellular calcium dynamics were assessed using Fura-2 AM. Compared with control astrocytes, LRRK2-related PD patient-derived mutant astrocytes exhibited lower intracellular calcium levels, and slower calcium extrusion following stimulation with ATP. Mitochondrial morphology was analyzed using MitoTracker Deep Red, revealing increased mitochondrial fragmentation and redistribution of mitochondria toward the cell periphery in both PD mutant cell types. Because oxidative phosphorylation is tightly regulated by mitochondrial morphology and calcium homeostasis, we next assessed oxygen consumption rates using a continuous metabolic monitoring system (Resipher) and quantified the expression of genes (RT-qPCR) and proteins (capillary electrophoresis-based western detection) involved in mitochondrial calcium transport and bioenergetics. These analyses showed that PRKN mutant astrocytes exhibit a more oxidative bioenergetic phenotype than LRRK2 mutant astrocytes, while both mutant lines displayed altered phosphorylation of mitochondrial morphology regulator DRP1 as well as decreased levels of respiratory complexes relative to control astrocytes. In summary, this study identifies astrocyte-specific mitochondrial dysfunctions and calcium dysregulation as key features of LRRK2- and PRKN-related pathology, providing new insights into how glial metabolic alterations contribute to neurodegeneration in PD.

neuroscience↗

bioRxiv2025

Transcriptomic and functional profiling reveal autophagy inhibition and persistent bioenergetic collapse following parallel photodamage to lysosomes and mitochondria

Photodynamic therapy (PDT) using 1,9-dimethyl methylene blue (DMMB) induces coordinated mitochondrial and lysosomal damage and results in strong cellular death induction. However, the underlying transcriptional regulation in response to DMMB remains elusive. We compared the transcriptome response of photoactivated DMMB (paDMMB) to the gene signature triggered by autophagy-modulating agents: rapamycin (an autophagy activator) and bafilomycin A1 (a lysosomal acidification inhibitor). Transcriptome analysis revealed a pronounced transcriptomic response to paDMMB, with 884 differentially expressed genes (DEGs), compared to 291 for bafilomycin and 154 for rapamycin. paDMMB treatment upregulated genes associated with autophagy, mitochondrial stress responses, and proteostasis, while downregulating genes involved in miRNA processing and lipid catabolism. Rapamycin treatment downregulated amino acid biosynthesis pathways, while upregulating processes associated with nutrient starvation. Conversely, bafilomycin treatment upregulated genes related to lipid metabolism, while suppressing cytoskeletal programs. Transcriptomic comparisons revealed a striking overlap (95%) between paDMMB and bafilomycin signatures. Among the several biological processes affected by paDMMB, mitochondrial-related processes were strongly enriched. To determine whether the acute transcriptome changes caused by paDMMB led to persistent functional effects, we stimulated cells with DMMB and assessed mitochondrial respiration after a recovery period. paDMMB reduced basal respiration, ATP production, proton leak, and maximal respiration. These effects were not further altered by bafilomycin co-treatment but were markedly exacerbated by rapamycin. Collectively, we show that paDMMB leads to a transcriptome rewiring, closely resembling autophagy inhibition with a sustained mitochondrial dysfunction. These findings provide a valuable resource to understand the interplay between DMMB-induced lysosomal stress, transcriptional regulation, and PDT.

cell biology↗

bioRxiv2021

Spatial proteomics reveals profound subcellular reorganization in human keratinocytes exposed to UVA light

The effects of UV light on the skin have been extensively investigated. However, systematic information about how exposure to UVA light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional assays, we mapped the subcellular reorganization of the proteome of human keratinocytes in response to UVA light. Our workflow quantified and assigned subcellular localization for over 1600 proteins, of which about 200 were found to redistribute upon UVA exposure. Reorganization of the proteome affected modulators of signaling pathways, cellular metabolism, and DNA damage response. Strikingly, mitochondria were identified as one of the main targets of UVA-induced stress. Further investigation demonstrated that UVA induces mitochondrial fragmentation, up-regulates redox-responsive proteins and attenuates respiratory rates. These observations emphasize the role of this radiation as a potent metabolic stressor in the skin.

biochemistry↗