MTFP1 controls mitochondrial fusion to regulate inner membrane quality control and maintain mtDNA levels.

Mitochondrial dynamics play a critical role in cell fate decisions and in controlling mtDNA levels and distribution. However, the molecular mechanisms linking mitochondrial membrane remodeling and quality control to mtDNA copy number (CN) regulation remain elusive. Here, we demonstrate that the inner mitochondrial membrane (IMM) protein mitochondrial fission process 1 (MTFP1) negatively regulates IMM fusion. Moreover, manipulation of mitochondrial fusion through the regulation of MTFP1 levels results in mtDNA CN modulation. Mechanistically, we found that MTFP1 inhibits mitochondrial fusion to isolate and exclude damaged IMM subdomains from the rest of the network. Subsequently, peripheral fission ensures their segregation into small MTFP1-enriched mitochondria (SMEM) that are targeted for degradation in an autophagic-dependent manner. Remarkably, MTFP1-dependent IMM quality control is essential for basal nucleoid recycling and therefore to maintain adequate mtDNA levels within the cell.

High-throughput single-cell analysis reveals progressive mitochondrial DNA mosaicism throughout life.

Heteroplasmic mitochondrial DNA (mtDNA) mutations are a major cause of inherited disease and contribute to common late-onset human disorders. The late onset and clinical progression of mtDNA-associated disease is thought to be due to changing heteroplasmy levels, but it is not known how and when this occurs. Performing high-throughput single-cell genotyping in two mouse models of human mtDNA disease, we saw unanticipated cell-to-cell differences in mtDNA heteroplasmy levels that emerged prenatally and progressively increased throughout life. Proliferating spleen cells and nondividing brain cells had a similar single-cell heteroplasmy variance, implicating mtDNA or organelle turnover as the major force determining cell heteroplasmy levels. The two different mtDNA mutations segregated at different rates with no evidence of selection, consistent with different rates of random genetic drift in vivo, leading to the accumulation of cells with a very high mutation burden at different rates. This provides an explanation for differences in severity seen in human diseases caused by similar mtDNA mutations.

Cell lineage-specific mitochondrial resilience during mammalian organogenesis.

Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments.

The translocator protein (TSPO) is prodromal to mitophagy loss in neurotoxicity.

Dysfunctional mitochondria characterise Parkinson's Disease (PD). Uncovering etiological molecules, which harm the homeostasis of mitochondria in response to pathological cues, is therefore pivotal to inform early diagnosis and therapy in the condition, especially in its idiopathic forms. This study proposes the 18 kDa Translocator Protein (TSPO) to be one of those. Both in vitro and in vivo data show that neurotoxins, which phenotypically mimic PD, increase TSPO to enhance cellular redox-stress, susceptibility to dopamine-induced cell death, and repression of ubiquitin-dependent mitophagy. TSPO amplifies the extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signalling, forming positive feedback, which represses the transcription factor EB (TFEB) and the controlled production of lysosomes. Finally, genetic variances in the transcriptome confirm that TSPO is required to alter the autophagy-lysosomal pathway during neurotoxicity.

Novel defect in phosphatidylinositol 4-kinase type 2-alpha (PI4K2A) at the membrane-enzyme interface is associated with metabolic cutis laxa.

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4-kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4-phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl-chain pools of PI4P and PI(4,5)P2 were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4-kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P2 and PI(3,4,5)P3 . Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.

Reversible Keap1 inhibitors are preferential pharmacological tools to modulate cellular mitophagy.

Mitophagy orchestrates the autophagic degradation of dysfunctional mitochondria preventing their pathological accumulation and contributing to cellular homeostasis. We previously identified a novel chemical tool (hereafter referred to as PMI), which drives mitochondria into autophagy without collapsing their membrane potential (ΔΨm). PMI is an inhibitor of the protein-protein interaction (PPI) between the transcription factor Nrf2 and its negative regulator, Keap1 and is able to up-regulate the expression of autophagy-associated proteins, including p62/SQSTM1. Here we show that PMI promotes mitochondrial respiration, leading to a superoxide-dependent activation of mitophagy. Structurally distinct Keap1-Nrf2 PPI inhibitors promote mitochondrial turnover, while covalent Keap1 modifiers, including sulforaphane (SFN) and dimethyl fumarate (DMF), are unable to induce a similar response. Additionally, we demonstrate that SFN reverses the effects of PMI in co-treated cells by reducing the accumulation of p62 in mitochondria and subsequently limiting their autophagic degradation. This study highlights the unique features of Keap1-Nrf2 PPI inhibitors as inducers of mitophagy and their potential as pharmacological agents for the treatment of pathological conditions characterized by impaired mitochondrial quality control.

A role for TSPO in mitochondrial Ca2+ homeostasis and redox stress signaling.

The 18 kDa translocator protein TSPO localizes on the outer mitochondrial membrane (OMM). Systematically overexpressed at sites of neuroinflammation it is adopted as a biomarker of brain conditions. TSPO inhibits the autophagic removal of mitochondria by limiting PARK2-mediated mitochondrial ubiquitination via a peri-organelle accumulation of reactive oxygen species (ROS). Here we describe that TSPO deregulates mitochondrial Ca2+ signaling leading to a parallel increase in the cytosolic Ca2+ pools that activate the Ca2+-dependent NADPH oxidase (NOX) thereby increasing ROS. The inhibition of mitochondrial Ca2+ uptake by TSPO is a consequence of the phosphorylation of the voltage-dependent anion channel (VDAC1) by the protein kinase A (PKA), which is recruited to the mitochondria, in complex with the Acyl-CoA binding domain containing 3 (ACBD3). Notably, the neurotransmitter glutamate, which contributes neuronal toxicity in age-dependent conditions, triggers this TSPO-dependent mechanism of cell signaling leading to cellular demise. TSPO is therefore proposed as a novel OMM-based pathway to control intracellular Ca2+ dynamics and redox transients in neuronal cytotoxicity.

Controlled and Impaired Mitochondrial Quality in Neurons: Molecular Physiology and Prospective Pharmacology.

Tuned mitochondrial physiology is fundamental for qualitative cellular function. This is particularly relevant for neurons, whose pathology is frequently associated with mitochondrial deficiencies. Defects in mitochondria are indeed key features in most neurodegenerative diseases such as Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease (HD) and Amyotrophic Lateral Sclerosis (ALS). When mitochondrial coupling impairs, so does cell metabolism, trafficking and the signaling depending on the homeostasis of the mitochondrial network. Moreover, the quality control of mitochondria - via the process of mitochondrial autophagy - results biased in neurodegeneration stemming major interest on the molecular determinants of this process among neuroscientists. In this review, we highlight the most notable and acknowledged deficiencies of mitochondrial function and their relationship with diseases occurring in neurons and their transmission. The physiological aspects of mitochondrial biology in relation to bio-energy, dynamics and quality control will be discussed with the finality to form a comprehensive picture of the mitochondrial contribution to the pathophysiology of neurodegenerative syndromes. In this way we aim to set the scene to conceive novel strategies to better diagnose and target these debilitative conditions.