Exploring mitochondrial-derived vesicles (MDVs) content, dynamics, and functions during inflammatory response

Abstract:

Mitochondria are complex organelles that house hundreds of biochemical reactions and serve as cellular signaling hubs, thus emerging as key regulators of multiple cellular processes, ranging from the production of energy to the coordination of cell death. Mitochondria communicate with the other intracellular structures by physical association, forming specific inter-organelle contacts that allow the exchange of signals and specific materials. For example, the connection between the endoplasmic reticulum (ER) and mitochondria is instrumental for lipids trafficking and calcium (Ca2+) homeostasis. Importantly, the cross-talk between organelles also relies on vesicular transport that directly involves the mitochondrial compartment. The so-called mitochondrial-derived vesicles (MDVs) incorporate protein cargoes deriving from the outer mitochondrial membrane, as well as from the internal spaces, including mitochondrial inner and matrix proteins. MDVs deliver their contents to lysosomes and peroxisomes for degradation or to multivesicular bodies that fuse with the plasma membrane, leading to the release of their internal content through extracellular vesicles (EVs). Indeed, cells secrete mitochondrial proteins and mtDNA into their environment, which has been proposed to support many functions, including serving as a quality control system, participating in long-range metabolic regulation, or stimulating the immune system. However, the composition, functions, and intracellular dynamics of MDVs, as well as their role in specific inflammatory contexts or disorders are far to be elucidated.

In this project, we propose to elucidate the trafficking of MDVs (from the generation to secretion in the extracellular milieu), their content, and functions by developing innovative methodological approaches. This strategy is based on long-term MDVs monitoring and visualization through a 3D tomographic technology and analysis of mitochondrial cargoes using an optimized protocol for the isolation of EVs. The morphological and biochemical characterization of MDVs will be carried out upon stress conditions elicited by pathogenic infections. Moreover, we will investigate the mitochondrial functions that regulate MDVs homeostasis, by focusing on mitochondrial Ca2+ signaling and ER-mitochondria association. Finally, we will shed light on the role of MDVs in the context of cystic fibrosis (CF), analyzing the contribution of MDVs to the inflammatory response. Therefore, this proposal will apply innovative technologies to provide essential clues on MDVs homeostasis, revealing novel mechanistic insights and indicating their functional contribution to CF disease.

Risultati attesi: 

The central goal of this project is to investigate how mitochondria-derived vesicles (MDVs) are formed, transported, and altered in response to various types of cellular stress, and how these
processes may contribute to inflammation, particularly in cystic fibrosis (CF) during pulmonary infection. The research is structured around three main Aims, each targeting specific aspects of MDV biology. Aim 1 focuses on understanding how MDVs behave during infections. The team will monitor MDV formation and movement within cells using advanced 3D imaging and fluorescent markers to visualize mitochondrial compartments in both live and fixed cells. They will study how bacterial pathogens such as P. aeruginosa and S. aureus stimulate MDV production and assess whether inhibiting cellular degradation pathways enhances MDV release. Another key objective is to characterize the molecular contents of MDVs released during infection. By isolating extracellular MDVs and identifying mitochondrial cargo, such as mitochondrial DNA (mtDNA), researchers aim to determine whether different infections generate distinct MDV “signatures.” These insights will shed light on how mitochondria communicate stress signals during infection and inflammation. Aim 2 examines how mitochondrial function shapes MDV formation and composition. One aspect of this work will explore the role of mitochondrial calcium (Ca2+) uptake, a critical factor for mitochondrial health and signaling. By genetically deleting or reducing the activity of the mitochondrial calcium uniporter (MCU), the team will analyze how disturbances in mitochondrial calcium levels influence MDV dynamics and cargo. Another focus is the physical interaction between mitochondria and the endoplasmic reticulum (ER), which helps regulate mitochondrial architecture and function. Disrupting ER-mitochondria contacts by targeting the tethering protein PDZD8 will provide insights into how these connections affect MDV release and composition. Finally, researchers will investigate whether alterations in mitochondrial Ca2+ handling or ER-mitochondria interactions impact inflammatory responses in immune cells exposed to MDVs. These studies aim to link mitochondrial dysfunction to immune system activation, potentially uncovering new therapeutic targets. Aim 3 brings these investigations into the clinical context of CF. Researchers will compare MDV dynamics in airway cells from healthy individuals and CF patients, particularly during infections with P. aeruginosa. They will assess whether mitochondrial defects characteristic of CF, including disrupted mitochondrial Ca2+ signaling and altered ER-mitochondria interactions, affect MDV production and cargo. Additionally, the team will study how MDVs derived from CF cells influence immune cells using an in vitro co-culture model. They will also explore whether pharmacological inhibition of the MCU complex can help restore balanced immune responses in CF models. Overall, this project seeks to unravel the intricate connections between mitochondrial stress, MDV biology, and inflammation. The findings are expected to expand fundamental knowledge of mitochondrial dynamics and may lead to the identification of novel biomarkers and therapeutic strategies for conditions, like CF, where chronic inflammation poses significant clinical challenges.