Valentina Debattisti

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease.

Significance This report probes the physiological roles of mammalian mitochondrial Rho 1 (Miro1), a calcium-binding, membrane-anchored GTPase necessary for mitochondrial motility on microtubules. Using two new mouse models and primary cells, the study demonstrates a specific role for Miro1 in upper motor neuron development and retrograde transport of axonal mitochondria. Unexpectedly, Miro1 is not essential for calcium-regulated mitochondrial movement, mitochondrial-mediated calcium buffering, or maintenance of mitochondrial respiratory activity. Nevertheless, a neuron-specific Miro1 KO mouse model displays physical hallmarks of neurological disease in the brainstem and spinal cord and develops rapidly progressing upper motor neuron disease symptoms culminating in death after approximately 4 wk. These studies demonstrate that defects in mitochondrial motility and distribution alone are sufficient to cause neurological disease. Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.

Reduction of endoplasmic reticulum stress attenuates the defects caused by Drosophila mitofusin depletion

The developmental and motor defects evident in flies depleted of the mitofusin Marf can be ameliorated by treatments that reduce ER stress, confirming an active role for ER stress in the observed phenotypes.

Reliance of ER-mitochondrial calcium signaling on mitochondrial EF-hand Ca2+ binding proteins. Miros, MICUs, LETM1 and solute carriers

Endoplasmic reticulum (ER) and mitochondria are functionally distinct with regard to membrane protein biogenesis and oxidative energy production, respectively, but cooperate in several essential cell functions, including lipid biosynthesis, cell signaling and organelle dynamics. The interorganellar cooperation requires local communication that can occur at the strategically positioned and dynamic associations between ER and mitochondria. Calcium is locally transferred from ER to mitochondria at the associations and exerts regulatory effects on numerous proteins. A common Ca(2+) sensing mechanism is the EF-hand Ca(2+) binding domain, many of which can be found in proteins of the mitochondria, including Miro1&2, MICU1,2&3, LETM1 and mitochondrial solute carriers. Recently, these proteins have triggered much interest and were described in reports with diverging conclusions. The present essay focuses on their shared features and established specific functions.

Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2+ Signals is Controlled by the Stoichiometry of MICU1/2 and MCU

SUMMARY Mitochondrial Ca2+ uptake through the Ca2+ uniporter supports cell functions, including oxidative metabolism, while meeting tissue-specific calcium signaling patterns and energy needs. The molecular mechanisms underlying tissue-specific control of the uniporter are unknown. Here, we investigated a possible role for tissue-specific stoichiometry between the Ca2+-sensing regulators (MICUs) and pore unit (MCU) of the uniporter. Low MICU1:MCU protein ratio lowered the [Ca2+] threshold for Ca2+ uptake and activation of oxidative metabolism but decreased the cooperativity of uniporter activation in heart and skeletal muscle compared to liver. In MICU1-overexpressing cells, MICU1 was pulled down by MCU proportionally to MICU1 overexpression, suggesting that MICU1:MCU protein ratio directly reflected their association. Overexpressing MICU1 in the heart increased MICU1:MCU ratio, leading to liver-like mitochondrial Ca2+ uptake phenotype and cardiac contractile dysfunction. Thus, the proportion of MICU1-free and MICU1-associated MCU controls these tissue-specific uniporter phenotypes and downstream Ca2+ tuning of oxidative metabolism.

Loss of Sirt1 Promotes Prostatic Intraepithelial Neoplasia, Reduces Mitophagy, and Delays Park2 Translocation to Mitochondria

Prostatic intraepithelial neoplasia is a precursor to prostate cancer. Herein, deletion of the NAD(+)-dependent histone deacetylase Sirt1 induced histological features of prostatic intraepithelial neoplasia at 7 months of age; these features were associated with increased cell proliferation and enhanced mitophagy. In human prostate cancer, lower Sirt1 expression in the luminal epithelium was associated with poor prognosis. Genetic deletion of Sirt1 increased mitochondrial superoxide dismutase 2 (Sod2) acetylation of lysine residue 68, thereby enhancing reactive oxygen species (ROS) production and reducing SOD2 activity. The PARK2 gene, which has several features of a tumor suppressor, encodes an E3 ubiquitin ligase that participates in removal of damaged mitochondria via mitophagy. Increased ROS in Sirt1(-/-) cells enhanced the recruitment of Park2 to the mitochondria, inducing mitophagy. Sirt1 restoration inhibited PARK2 translocation and ROS production requiring the Sirt1 catalytic domain. Thus, the NAD(+)-dependent inhibition of SOD2 activity and ROS by SIRT1 provides a gatekeeper function to reduce PARK2-mediated mitophagy and aberrant cell survival.

ROS Control Mitochondrial Motility through p38 and the Motor Adaptor Miro/Trak

Mitochondrial distribution and motility are recognized as central to many cellular functions, but their regulation by signaling mechanisms remains to be elucidated. Here, we report that reactive oxygen species (ROS), either derived from an extracellular source or intracellularly generated, control mitochondrial distribution and function by dose-dependently, specifically, and reversibly decreasing mitochondrial motility in both rat hippocampal primary cultured neurons and cell lines. ROS decrease motility independently of cytoplasmic [Ca2+], mitochondrial membrane potential, or permeability transition pore opening, known effectors of oxidative stress. However, multiple lines of genetic and pharmacological evidence support that a ROS-activated mitogen-activated protein kinase (MAPK), p38α, is required for the motility inhibition. Furthermore, anchoring mitochondria directly to kinesins without involvement of the physiological adaptors between the organelles and the motor protein prevents the H2O2-induced decrease in mitochondrial motility. Thus, ROS engage p38α and the motor adaptor complex to exert changes in mitochondrial motility, which likely has both physiological and pathophysiological relevance.

Increased mitochondrial nanotunneling activity, induced by calcium imbalance, affects intermitochondrial matrix exchanges.

Significance Nanotunnels are long, thin mitochondrial extensions that have been implied in direct long-distance (1 to >5 µm) communication between mitochondria of cardiac myocytes. The engineered RyR2A4860G+/− mutation, resulting in loss of function of the sarcoplasmic reticulum calcium release channel and arrhythmia, induces a striking increase in the frequency of long-distance intermitochondrial communication via nanotunnels without involvement of obvious mitochondrial migration. We use this model for exploring the significance of mitochondrial nanotunneling in myocardium and the contribution of microtubules to the formation of these unusual organelle extensions using EM tomography and live confocal imaging. This study constitutes an approach to arrhythmia investigations that focuses on a new target: the mitochondria. Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca2+ imbalance by depressing RyR2 channel activity during excitation–contraction coupling, resulting in random bursts of Ca2+ release probably due to Ca2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2A4860G+/− cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower.

Regulation of ER-mitochondria contacts by Parkin via Mfn2

&NA; Parkin, an E3 ubiquitin ligase and a Parkinsons disease (PD) related gene, translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy. Mitochondrial pro‐fusion protein Mitofusins (Mfn1 and Mfn2) were found to be a target for Parkin mediated ubiquitination. Mfns are transmembrane GTPase embedded in the outer membrane of mitochondria, which are required on adjacent mitochondria to mediate fusion. In mammals, Mfn2 also forms complexes that are capable of tethering mitochondria to endoplasmic reticulum (ER), a structural feature essential for mitochondrial energy metabolism, calcium (Ca2+) transfer between the organelles and Ca2+ dependent cell death. Despite its fundamental physiological role, the molecular mechanisms that control ER‐mitochondria cross talk are obscure. Ubiquitination has recently emerged as a powerful tool to modulate protein function, via regulation of protein subcellular localization and protein ability to interact with other proteins. Ubiquitination is also a reversible mechanism, which can be actively controlled by opposing ubiquitination‐deubiquitination events. In this work we found that in Parkin deficient cells and parkin mutant human fibroblasts, the tether between ER and mitochondria is decreased. We identified the site of Parkin dependent ubiquitination and showed that the non‐ubiquitinatable Mfn2 mutant fails to restore ER‐mitochondria physical and functional interaction. Finally, we took advantage of an established in vivo model of PD to demonstrate that manipulation of ER‐mitochondria tethering by expressing an ER‐mitochondria synthetic linker is sufficient to rescue the locomotor deficit associated to an in vivo Drosophila model of PD. Graphical abstract Figure. No caption available.

Characterization of Mitochondrial Calcium Uptake in Skeletal Muscle

Mitochondrial Ca2+ uptake provides fundamental input to the control of oxidative metabolism and contributes to cell survival regulating signaling mechanisms. Early studies of mitochondria isolated from various tissues have demonstrated that the Ca2+ uptake is driven by the membrane potential (ΔΨm), is mediated by an electrogenic uniport, which is completely inhibited by ruthenium red (RuRed) and was attributed to the “Ca2+ uniporter” (mtCU). mtCU has been recently identified as a protein complex constituted by the pore-forming MCU, its dominant-negative form, MCUb, a scaffold, EMRE, and the Ca2+-sensitive regulators, MICU1, MICU2 and MICU3 which determine both the threshold and cooperative activation of the mtCU by Ca2+. Although mtCU is ubiquitously present in the whole body, mitochondria isolated from several tissues displayed a range of Ca2+ current activities through the inner membrane: very low in heart, moderate in liver and kidney, and high in skeletal muscle (SM). This suggests an important role of mitochondrial calcium uptake specifically in SM function. Indeed, calcium homeostasis is crucial for both excitation-contraction (EC) coupling and relaxation. However, mtCU composition and Ca2+ transport activity in SM remains to be characterized. The goal of this project was to fill this void. We evaluated the abundance of each mtCU forming protein and found that both MCU and MICU1 showed a high expression level in SM compared to other tissues. Furthermore, SM mitochondria took up Ca2+ at surprisingly low concentrations and showed a high maximal activity (assessed by both fluorometric measurement of mitochondrial clearance of added Ca2+ and 45Ca2+ sequestration), supporting the hypothesis of a relevant contribution of mitochondria to clearance of sarcoplasmic Ca2+ in SM.

Reactive Oxygen Species (ROS) Suppress Mitochondrial Motility

Mitochondrial distribution and transport play pivotal roles for many cellular functions, including cell differentiation, cell division to ensure proper inheritance, apoptosis, ATP supply at the local sites of demand, Ca2+ buffering for intracellular Ca2+ homeostasis.We previously showed that mitochondrial motility (mito-motility) is regulated by the cytosolic Ca2+ concentration ([Ca2+]c), providing the basis for a homeostatic circuit in which the organelles decrease their movements along microtubules to locally buffer high [Ca2+]c and contribute to ATP supply. Mitochondria are also a major site for production and scavenging of ROS that serve as both a messenger and regulator of calcium signaling and are particularly relevant for the control of mitochondrial function. Here we tested the hypothesis that ROS target mito-motility to control mitochondrial function. H9c2 myoblast cells were transfected with a mitochondrial matrix targeted YFP and then loaded with fura2, to monitor the mito-motility simultaneously with [Ca2+]c. H2O2 (100 µM) caused a decrease in mito-motility (64±8 %) and an elevation in [Ca2+]c (from 55±8 to 91±8 nM) at the same time. When the cells were incubated in a Ca2+-free medium and were pretreated with thapsigargin to prevent Ca2+ entry and intracellular Ca2+ mobilization, respectively, H2O2 continued to inhibit the mito-motility dose-dependently without any changes in [Ca2+]c. These results indicate that H2O2 can cause suppression of mito-motility through a Ca2+ independent mechanism we are currently analyzing.