Regulation of Mitochondrial Function by the Actin Cytoskeleton

The regulatory role of actin cytoskeleton on mitochondrial function is a growing research field, but the underlying molecular mechanisms remain poorly understood. Specific actin-binding proteins (ABPs), such as Gelsolin, have also been shown to participate in the pathophysiology of mitochondrial OXPHOS disorders through yet to be defined mechanisms. In this mini-review, we will summarize the experimental evidence supporting the fundamental roles of actin cytoskeleton and ABPs on mitochondrial trafficking, dynamics, biogenesis, metabolism and apoptosis, with a particular focus on Gelsolin involvement in mitochondrial disorders. The functional interplay between the actin cytoskeleton, ABPs and mitochondrial membranes for the regulation of cellular homeostasis thus emerges as a new exciting field for future research and therapeutic approaches.

CXCL12/CXCR4 Axis Drives Mitochondrial Trafficking in Tumor Myeloma Microenvironment

Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) also transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. As metabolic rewiring is involved in the regulation of MSC phenotype, we first analyzed metabolic profile of healthy control (HC-) and MM-MSCs. NAD +/NADH ratio was decreased in MM-MSCs (n=8) as compared with HC-MSCs (n=4, p<0.05), meanwhile ATP/ADP ratio was not significantly different between the two groups. This led us to analyze whether MM-MSCs were much prone in transferring mitochondria than HC-MSCs. We first labeled HC- and MM-MSCs with Mitotracker Red CMXRos before co-culture with MM cells. After 24h of coculture, we quantified mitochondria transfer by flow cytometry. The obtained values were significantly higher in MM cells co-cultured with MM-MSCs (n=10) as compared to PCs co-cultured with HC-MSCs (n=5, p<0.01). In the cell-to-cell contact the gap junction-forming protein CX43 has been found critical for mitochondria uptake in lung and brain injury and it also can regulate CXCL12 secretion by MSCs. We found that MM-MSCs showed a significantly up-regulated CXCL12 expression as compared to HC-MSCs (p<0.001). Therefore, we co-cultured HS-5 cells with myeloma cell lines and observed that significantly increased CXCL12-CX43 colocalization in healthy MSCs. To evaluate the selective PC-induced activation of CXCL12 expression via CX43 in MSCs, we co-cultured HS-5 cells with MM cell lines and exposed cocultures to ioxynil octanoate (IO), a selective inhibitor of CX43-based gap junctions. We found that the up-regulation of CXCL12 induced by MM cells was reverted by exposition to the CX43 inhibitor, thereby indicating that CX43 activated by PCs regulates CXCL12 production in MSCs. Given that CX43 is involved in mitochondria trafficking, we subsequently cocultured MM cells with HS-5 in presence or not of IO. Our data showed that mitochondrial transfer was abolished by CX43 inhibitor. Given that MM PCs induced increased CX43 and CXCL12 colocalization in HS-5 cells, we supposed that CXCL12/CXCR4 signaling could regulate mitochondria trafficking throughout this axis. For this reason, we analyzed the kinetic of mitochondria uptake of several HMCLs and related their CXCR4 expression with the percentage of transferred mitochondria. Our data demonstrated that HMCLs with higher expression of CXCR4 had also higher percentage of transferred mitochondria both in time lapse and flow cytometry. The correlation between CXCR4 expression and the percentage of mitochondria uptake in HMCLs was also confirmed in primary myeloma PCs. Furthermore, plerixafor, a selective inhibitor of CXCR4, significantly reduced mitochondrial transfer from MSCs to myeloma PCs further establishing mechanistically that CXCR4/CXCL12 is directly involved in mitochondrial trafficking. Next, we investigated whether combination of plerixafor with bortezomib or carfilzomib interferes with mitochondrial transfer from MSCs to PCs. Interestingly, we found that the proteasome inhibitors promoted mitochondrial transfer while their combination with plerixafor inhibited mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM. In conclusion, we have shown that MM-MSCs are relatively low dependent on mitochondria metabolism and are inclined to transfer mitochondria to MM tumor cells. Furthermore, tumor PCs increase the expression of CX43 in MSCs leading to an increased levels of CXCL12 and stimulation of its corresponding receptor expressed on MM cells. The resulting CX43/CXCL12/CXCR4 interplay enhances mitochondrial trafficking from MSCs to myeloma PCs and can protect cancer cells against anti-myeloma agents. Understanding pro-tumorigenic phenotype of MSCs and mechanisms of adhesion and heterocellular communication favoring their interaction with cancer PCs, will allow to manipulate critical pathways, including CXCL12/CXCR4 axis, thus improving disease outcome. Disclosures Di Raimondo: Pfizer: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Jazz Pharmaceutical: Honoraria; Janssen Pharmaceuticals: Honoraria; Amgen: Honoraria.

Quantitative Analysis of Neuronal Mitochondrial Movement Reveals Patterns Resulting from Neurotoxicity of Rotenone and 6-hydroxydopamine

Alterations in mitochondrial dynamics, including their intracellular trafficking, are common early manifestations of neuronal degeneration. However, current methodologies used to study mitochondrial trafficking events rely on parameters that are mostly altered in later stages of neurodegeneration. Our objective was to establish a reliable computational methodology to detect early alterations in neuronal mitochondrial trafficking. We propose a novel quantitative analysis of mitochondria trajectories based on innovative movement descriptors, including straightness, efficiency, anisotropy, and kurtosis. Using biological data from differentiated SH-SY5Y cells treated with the mitochondrial toxicants 6-hydroxydopamine and rotenone, we evaluated time- and dose-dependent alterations in trajectory descriptors. MitoTracker Red CMXRos-labelled mitochondria movement was analyzed by total internal reflection fluorescence microscopy followed by computational modelling to describe the process. This innovative analysis of mitochondria trajectories, based on the aforementioned trajectory descriptors, provides insights into mitochondrial movement characteristics and can be a consistent and sensitive method to detect alterations in mitochondrial trafficking occurring in the earliest time points of neurodegeneration.

Quantitative analysis of neuronal mitochondrial movement reveals patterns resulting from neurotoxicity of rotenone and 6-hydroxydopamine

Alterations in mitochondrial dynamics, including their trafficking, can present early manifestation of neuronal degeneration. However, current methodologies used to study mitochondrial trafficking events rely on parameters that are mostly altered in later stages of neurodegeneration. Our objective was to establish a reliable computational methodology to detect early alterations in neuronal mitochondrial trafficking. We propose a novel quantitative analysis of mitochondria trajectories based on innovative movement descriptors, including straightness, efficiency, anisotropy, and kurtosis. Using biological data from differentiated SH-SY5Y cells treated with mitochondrial toxicants 6-hydroxydopamine and rotenone, we evaluated time and dose-dependent alterations in trajectory descriptors. Mitochondrial movement was analyzed by total internal reflection fluorescence microscopy followed by computer modelling to describe the process. The stacks of individual images were analyzed by an open source MATLAB algorithm (www.github.com/kandelj/MitoSPT) and to characterize mitochondria trajectories, we used the Python package trajpy (https://github.com/ocbe-uio/trajpy/). Our results confirm that this computational approach is effective and accurate in order to study mitochondrial motility and trajectories in the context of healthy and diseased neurons in different stages.

Intercellular mitochondrial transfer as a means of tissue revitalization

As the crucial powerhouse for cell metabolism and tissue survival, the mitochondrion frequently undergoes morphological or positional changes when responding to various stresses and energy demands. In addition to intracellular changes, mitochondria can also be transferred intercellularly. Besides restoring stressed cells and damaged tissues due to mitochondrial dysfunction, the intercellular mitochondrial transfer also occurs under physiological conditions. In this review, the phenomenon of mitochondrial transfer is described according to its function under both physiological and pathological conditions, including tissue homeostasis, damaged tissue repair, tumor progression, and immunoregulation. Then, the mechanisms that contribute to this process are summarized, such as the trigger factors and transfer routes. Furthermore, various perspectives are explored to better understand the mysteries of cell–cell mitochondrial trafficking. In addition, potential therapeutic strategies for mitochondria-targeted application to rescue tissue damage and degeneration, as well as the inhibition of tumor progression, are discussed.