EXPLORING MITOCHONDRIA-LYSOSOMES CONTACT SITES DYNAMICS IN NEURODEGENERATION AND CANCER: NOVEL TARGETS FOR NEW BIOMEDICAL STRATEGIES
Descrizione
Mitochondrial and lysosomal functions are intricately related and critical for maintaining cellular homeostasis, as highlighted by multiple diseases linked to dysfunction of both organelles. Recent studies have revealed the existence of mitochondria–lysosomes (MT–LY) contact sites and suggested that these inter-organelle membrane contacts play an important role both in health and disease. These contact sites are important to regulate the dynamics of their network and the bidirectional exchange of ions and metabolites (e.g., Ca2+, iron and lipids) between the two organelles. In this project, we aim to deeply explore whether and how perturbations of MT-LY contact sites can impact on up/downstream related cellular functions in physiological and pathological conditions (i.e., neurodegeneration and cancer). To do this we decided to articulate the project in three different major aims: i) Characterization of MT-LY contact sites either in dopaminergic cells lines expressing Parkinson disease (PD) related proteins or in astroglial/neuronal cells treated with Alzheimer’s disease (AD) related proteins; ii) Characterization of MT-LY contact sites upon metabolism modulation in dopaminergic and glioma cells and iii) Characterization of the impact of MT-LY contact sites modulation on intracellular signaling. For each aim, several tasks, characterized by a common denominator that is the full understanding of signaling process occurring at the mitochondria lysosome interface, will be considered. It should be pointed out that the deep understanding of how these exchanges occur in physiological and pathological conditions is rather limited by the absence of adequate tools to assess contact sites in living cells. Recently, it has been developed contact sites sensors (SPLICS) based on splitGFP and bimolecular fluorescence complementation (BiFC) to directly monitor organelles contact sites remodeling by live cell imaging and confocal microscopy under different physio-pathological conditions or upon specific treatments. This approach has permitted to establish that juxtapositions at narrow (8-10 nm) and wide (40-50 nm) distances between a given pair of organelles are present, and that their differently respond to mitophagy or autophagy stimuli and to the manipulation of specific tethering factors, thus documenting the existence of at least two types of contact sites.
Finalità
The project has the potential to provide a comprehensive understanding of the regulation and functions of MT-LY communications occurring at their contact sites and of its impact on organelles function. In this project, we propose to exploit our expertise in mitochondrial metabolism, inter-organellar cross talk and signal transduction to undertake a comprehensive study to address the following questions: i) Do the PD or AD related proteins differently modulate (ER)-MT-LY crosstalk? ii) Does metabolic modulation of glycolysis, fatty acid oxidation or oxidative phosphorylation impact on (ER)-MT-LY interaction? iii) Does the (ER)-MT-LY crosstalk remodeling impact on intracellular signaling in neurodegeneration and cancer?
Risultati attesi
To answer the above questions, we will address the following aims:
Aim 1 Characterization of MT-LY contact sites in dopaminergic cells lines expressing PD related proteins (SHSY5Y and LUHMES) and in astroglial/neuronal cells treated with AD related proteins.
Task 1.1 Monitoring of the changes in ER-MT and MT-LY contact sites in response to modulation of DJ-1/PINK1/PARKIN expression (overexpression and silencing) and treatment with β-amyloid(1-42).
Task 1.2 Detection of early signs of alterations at ER-MT-LY contact sites upon mitophagy induction.
Task 1.3 Monitoring the impact of the modulation of oxidative phosphorylation (OXPHOS) on lysosome contacts with mitochondria.
Aim 2 Characterization of MT–LY contact sites upon metabolism modulation in dopaminergic and glioma cells
Task 2.1 Impact of nutrient availability on MT-LY contact sites.
Task 2.2 Impact of nutrient availability and chemotherapy on mitochondria-lysosome contacts and on TRPLM1 mediated signaling.
Aim3 Characterization of the impact of MT-LY contact sites modulation on intracellular signaling (Ca2+, ROS, RNS, transcriptional factors and relative gene expressions).
Task 3.1 Investigating TRPML1 role in the regulation of MT-LY contact sites.
Task 3.2 Impact of TRPLM1 modulation by ROS on mitochondrial Ca2+ uptake and on MT-LY tethering.
Task 3.3 Impact of AD related proteins on ROS/RNS production, mitochondrial and lysosomal gene expression, activation of Nrf2 and related genes induction.
Stato dell’arte
Many physiological processes are regulated by the functional and physical interaction between cellular organelles. Active and bidirectional exchange of molecules, lipids and ions has been proposed to occur through contact between organelles that come in proximity at membrane contact sites (MCS). The length, the duration and the number of MCS could impact on cell wellness in terms of survival, metabolism, sensitivity to cell death or proliferation. MCS between mitochondria (MT) and endoplasmic reticulum (ER) membranes are well characterized but contact sites between MT and lysosomes (LY), nucleus, peroxisomes and plasma membrane have recently received great attention. ER-MT contacts are essential to preserve normal bioenergetics and defective ER–MT communication results in mitochondrial damage, Ca2+ dis-homeostasis, ER stress and autophagy. Many of these alterations occur both in neurodegenerative diseases and in cancer. We have shown that PD-related proteins such as alpha synuclein, DJ-1, parkin, and PINK1 contribute to the modulation of ER-MT tethering. Other studies have shown that cancer cells are dependent from Ca2+ flux by ER-MT contacts and are more susceptible to death once this communication is disturbed. Recently, MCS between MT and LY have also been identified in multiple cell types under healthy conditions. They occur at an average distance of ~10 nm and dynamically form distinctly from lysosomal degradation routes such as mitophagy or mitochondrial-derived vesicles. MT–LY contacts regulate the dynamics of both mitochondria and lysosomes as well as bidirectional exchange of Ca2+, cholesterol, and iron. Tight regulation by tethering proteins underpins the dynamics of MT–LY contacts. The small GTPase Rab7 and its activating protein TBC1D15 are the master regulators. In addition, the recent finding that mitofusin 2 (MFN2), which plays a major role in ER-MT tethering and ganglioside induced differentiation-associated protein 1 (GDAP1), an atypical glutathione S-transferase of the outer mitochondrial membrane, are involved in the MT–LY tethering by interacting with the lysosome-associated membrane protein-1 (LAMP-1), suggests the existence of a crosstalk between different MCS and highlights the relevance of mitochondrial dynamics in MCS formation. Both GDAP1 and MFN2 have been associated to mitochondrial ROS accumulation suggesting that MCS participate in ROS signalling. Impairments in MT-LY contacts have been related to the onset of neurodegenerative diseases including PD and AD. Furthermore, the activation of the transient receptor potential mucolipin 1 (TRPML1), a lysosomal/late endosomal cation channel involved in lysosomes biogenesis, exocytosis and positioning plays a pivotal role in regulating Ca2+ dynamics at the MT-LY contacts by promoting Ca2+ transfer into mitochondria through the mitochondrial calcium uniporter (MCU) as well transcription factor EB (TFEB) mediated-autophagy activation via calcineurin. TFEB activation has been found to ameliorate neurotoxicity and rescue neurodegeneration in PD and AD animal models. Intriguingly, increasing mitochondrial ROS stimulates TRPML1-induced Ca2+ release. Dysfunctions in TRP channels are involved in cancer growth, metastasis and chemoresistance. It has been demonstrated that in glioma cells TRPML1 activation triggers intracellular Ca2+ influx that leads to apoptosis and TRPML1-dependent autophagy is activated by oxidative stress associated with mitochondria damage. Moreover, the loss of TRPML1 expression correlates with poor prognosis in glioblastoma patients. Modulation of oxidative stress is essential for metabolic adaptation and chemoresistance of glioma cells, since they are able to use intermediate level of ROS and RNS for growth and invasion and to suppress them when they prejudice cell survival. During oxidative stress, the activation of transcription factor Nrf2 promotes the transcription of phase II detoxifying and antioxidant genes and System Xc− subunit xCT, a cystine/glutamate antiporter that is crucial to glutathione production and protection from oxidative stress in glioblastoma [41]. Moreover, the control of System Xc− expression, via lysosomal degradation, regulates glioma cell viability under glucose-limited conditions. Several PD proteins have been shown to promote the oncogenic progression in glioma. Among them, DJ-1, by decreasing ROS, is involved in the maintenance of stemness; Parkin controls proliferation and PINK1 suppresses ROS and tumor growth. In this scenario, the deep investigation of the MT–LY crosstalk will be critical to dissect the pathways that underline its modulation and cellular dis-homeostasis caused by defects in the transfer of information by key signaling mediators, i.e., Ca2+ ions and ROS and RNS.
Riferimento: PRIN 2022 – Codice progetto: 20223ABZ82 – CUP: F53D23005220006
Investimento totale del progetto: 225.930,00 € (finanziamento MUR: 187.473,00 €)
Partner/proponente: Prof.ssa Marisa BRINI – Università degli Studi di PADOVA, Prof.ssa Consuelo AMANTINI – Università degli Studi di CAMERINO
Coordinatore dell’UdR Università degli Studi Roma Tre: Prof. Marco Colasanti
