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Many cellular processes, including ribosome biogenesis, are regulated through post-transcriptional RNA modifications. Here, a genome-wide analysis of the human mitochondrial transcriptome shows that 2’- O -methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, introduced by MRM1, MRM2 and MRM3, with the modifications installed by the latter two proteins being interdependent. MRM2 controls mitochondrial respiration by regulating mitoribosome biogenesis. In its absence, mtLSU particles (visualized by cryo-EM at the resolution of 2.6 Å) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module, allowing five mtLSU biogenesis intermediates with different intersubunit interface configurations to be placed along the assembly pathway. However, mitoribosome biogenesis does not depend on the methyltransferase activity of MRM2. Disruption of the MRM2 Drosophila melanogaster orthologue leads to mitochondria-related developmental arrest. This work identifies a key checkpoint during mtLSU assembly, essential to maintain mitochondrial homeostasis. Rebelo-Guiomar et al. unveil late stage assembly intermediates of the human mitochondrial ribosome by inactivating the methyltransferase MRM2 in cells. Absence of MRM2 impairs organismal homeostasis, while its catalytic activity is dispensable for mitoribosomal biogenesis.

The ATP synthase is a mitochondrial inner membrane complex whose function is essential for cell bioenergy, being responsible for the conversion of ADP into ATP and playing a role in mitochondrial cristae morphology organization. The enzyme is composed of 18 protein subunits, 16 nuclear DNA (nDNA) encoded and two mitochondrial DNA (mtDNA) encoded, organized in two domains, FO and F1. Pathogenetic variants in genes encoding structural subunits or assembly factors are responsible for fatal human diseases. Emerging evidence also underlines the role of ATP-synthase in neurodegenerative diseases as Parkinson’s, Alzheimer’s, and motor neuron diseases such as Amyotrophic Lateral Sclerosis. Post-translational modification, epigenetic modulation of ATP gene expression and protein level, and the mechanism of mitochondrial transition pore have been deemed responsible for neuronal cell death in vivo and in vitro models for neurodegenerative diseases. In this review, we will explore ATP synthase assembly and function in physiological and pathological conditions by referring to the recent cryo-EM studies and by exploring human disease models.

BackgroundThymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy.ObjectiveTo perform a retrospective natural history study of a large cohort of patients with TK2 deficiency.MethodsThe study was conducted by 42 investigators across 31 academic medical centres.ResultsWe identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion.ConclusionsIn TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder.

Autosomal recessive mutations in the thymidine kinase 2 gene ( TK2 ) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N ( Tk2 −/− ) knock‐in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13‐day‐old Tk2 −/− mice treated with dCMP+dTMP 200 mg/kg/day each ( Tk2 −/− 200dCMP/ dTMP ) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency. Synopsis Molecular bypass therapy with orally administered deoxythymidine monophosphate and deoxycytidine monophosphate produces clinical, molecular, biochemical, and histological improvements in a mitochondrial DNA depletion syndrome Tk2 knock‐in mouse model. Deoxypyrimidine monophosphates cross biological barriers in Tk2‐deficient mice. dCMP+dTMP treatment restores mtDNA levels and ameliorates the phenotype of Tk2‐mutant mice. dCMP+dTMP have dose‐related clinical and biochemical effects in Tk2‐deficient mice. Graphical Abstract Molecular bypass therapy with orally administered deoxythymidine monophosphate and deoxycytidine monophosphate produces clinical, molecular, biochemical, and histological improvements in a mitochondrial DNA depletion syndrome Tk2 knock‐in mouse model.

Isolated methylmalonic acidemia (MMA), caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT), is often complicated by end stage renal disease that is resistant to conventional therapies, including liver transplantation. To establish a viable model of MMA renal disease, Mut was expressed in the liver of Mut ⁻/⁻ mice as a stable transgene under the control of an albumin (INS-Alb- Mut) promoter. Mut ⁻/⁻;Tg ᴵᴺS⁻ᴬˡᵇ⁻ᴹᵘᵗ mice, although completely rescued from neonatal lethality that was displayed by Mut ⁻/⁻ mice, manifested a decreased glomerular filtration rate (GFR), chronic tubulointerstitial nephritis and ultrastructural changes in the proximal tubule mitochondria associated with aberrant tubular function, as demonstrated by single-nephron GFR studies. Microarray analysis of Mut ⁻/⁻;Tg ᴵᴺS⁻ᴬˡᵇ⁻ᴹᵘᵗ kidneys identified numerous biomarkers, including lipocalin-2, which was then used to monitor the response of the GFR to antioxidant therapy in the mouse model. Renal biopsies and biomarker analysis from a large and diverse patient cohort (ClinicalTrials.gov identifier: NCT00078078) precisely replicated the findings in the animals, establishing Mut ⁻/⁻;Tg ᴵᴺS⁻ᴬˡᵇ⁻ᴹᵘᵗ mice as a unique model of MMA renal disease. Our studies suggest proximal tubular mitochondrial dysfunction is a key pathogenic mechanism of MMA-associated kidney disease, identify lipocalin-2 as a biomarker of increased oxidative stress in the renal tubule, and demonstrate that antioxidants can attenuate the renal disease of MMA.

Aim  To describe a spectrum of intracerebral large artery disease in Aicardi–Goutières syndrome (AGS) associated with mutations in the AGS5 gene SAMHD1. Method  We used clinical and radiological description and molecular analysis. Results  Five individuals (three males, two females) were identified as having biallelic mutations in SAMHD1 and a cerebral arteriopathy in association with peripheral vessel involvement resulting in chilblains and ischaemic ulceration. The cerebral vasculopathy was primarily occlusive in three patients (with terminal carotid occlusion and basal collaterals reminiscent of moyamoya syndrome) and aneurysmal in two. Three of the five patients experienced intracerebral haemorrhage, which was fatal in two individuals. Post‐mortem examination of one patient suggested that the arteriopathy was inflammatory in origin. Interpretation  Mutations in SAMHD1 are associated with a cerebral vasculopathy which is likely to have an inflammatory aetiology. A similar disease has not been observed in patients with mutations in AGS1 to AGS4, suggesting a particular role for SAMHD1 in vascular homeostasis. Our report raises important questions about the management of patients with mutations in SAMHD1.

Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) is a recently established multimodal single cell analysis technique combining the immunophenotyping capabilities of antibody labeling and cell sorting with the resolution of single-cell RNA sequencing (scRNA-seq). By simply adding a 12-bp nucleotide barcode to antibodies (cell hashing), CITE-seq can be used to sequence antibody-bound tags alongside the cellular mRNA, thus reducing costs of scRNA-seq by performing it at the same time on multiple barcoded samples in a single run. Here, we illustrate an ideal CITE-seq data analysis workflow by characterizing the transcriptome of SH-SY5Y neuroblastoma cell line, a widely used model to study neuronal function and differentiation. We obtained transcriptomes from a total of 2879 single cells, measuring an average of 1600 genes/cell. Along with standard scRNA-seq data handling procedures, such as quality checks and cell filtering procedures, we performed exploratory analyses to identify most stable genes to be possibly used as reference housekeeping genes in qPCR experiments. We also illustrate how to use some popular R packages to investigate cell heterogeneity in scRNA-seq data, namely Seurat, Monocle, and slalom. Both the CITE-seq dataset and the code used to analyze it are freely shared and fully reusable for future research.

Spinal muscular atrophy is a common motor neuron disease caused by low survival motoneuron (SMN), a key protein in the proper splicing of genes. Restoring the protein is therefore a promising therapeutic strategy. Implementation of this strategy, however, depends on defining the temporal requirements for SMN. Here, we used controlled knockdown of SMN in transgenic mice to determine the precise postnatal stage requirements for this protein. Reducing SMN in neonatal mice resulted in a classic SMA-like phenotype. Unexpectedly, depletion of SMN in adults had relatively little effect. Insensitivity to low SMN emerged abruptly at postnatal day 17, which coincided with establishment of the fully mature neuromuscular junction (NMJ). Mature animals depleted of SMN eventually exhibited evidence of selective neuromuscular pathology that was made worse by traumatic injury. The ability to regenerate the mature NMJ in aged or injured SMN-depleted mice was grossly impaired, a likely consequence of the inability to meet the surge in demand for motoneuronal SMN that was seen in controls. Our results demonstrate that relative maturity of the NMJ determines the temporal requirement for the SMN protein. These observations suggest that the use of potent but potentially deleterious SMN-enhancing agents could be tapered in human patients once the neuromuscular system matures and reintroduced as needed to enhance SMN for remodeling aged or injured NMJs.

In order to evaluate the psychiatric symptoms associated with a diagnosis of eating disorders (ED) we have administered a new psychometric instument: the Self Administrated Psychiatric Scales for Children and Adolescents (SAFA) test. SAFA was administered to a cohort of 97 patients, aged from 8.8 to 18, with an ED diagnosis. Age, body mass index (BMI) and BMI standard deviation score were analyzed. Furthermore, while looking for linkable risk factors, we evaluated other data that took an influence over the SAFA profile, like parental separation and family components' number. Compared to the range of statistical normality (based on Italian population), patients with bulimia nervosa or binge-eating disorder showed higher and pathologic values in specific subscales. When analyzing sex, males showed more pathologic values in most anxiety-related, obsessiveness-compulsiveness-related and insecurity subscales. A correlation among age, BMI and specific subscales (low self esteem, psychological aspects) emerged in participants with anorexia nervosa. In order to plan more appropriate diagnostic and therapeutic approaches in children or adolescents suffering from ED, the SAFA test can be an important instrument to evaluate psychiatric symptoms. Therefore, we propose to include this useful, simple self-administered test as a new screening tool for ED diagnosis.