Exacerbated atherosclerosis in progeria is prevented by progerin elimination in vascular smooth muscle cells but not endothelial cells.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease caused by the expression of progerin, a mutant protein that accelerates aging and precipitates death. Given that atherosclerosis complications are the main cause of death in progeria, here, we investigated whether progerin-induced atherosclerosis is prevented in HGPSrev-Cdh5-CreERT2 and HGPSrev-SM22α-Cre mice with progerin suppression in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. HGPSrev-Cdh5-CreERT2 mice were undistinguishable from HGPSrev mice with ubiquitous progerin expression, in contrast with the ameliorated progeroid phenotype of HGPSrev-SM22α-Cre mice. To study atherosclerosis, we generated atheroprone mouse models by overexpressing a PCSK9 gain-of-function mutant. While HGPSrev-Cdh5-CreERT2 and HGPSrev mice developed a similar level of excessive atherosclerosis, plaque development in HGPSrev-SM22α-Cre mice was reduced to wild-type levels. Our studies demonstrate that progerin suppression in VSMCs, but not in ECs, prevents exacerbated atherosclerosis in progeroid mice.

Coronary and carotid artery dysfunction and KV7 overexpression in a mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease caused by expression of progerin, a lamin A variant that is also expressed at low levels in non-HGPS individuals. Although HGPS patients die predominantly from myocardial infarction and stroke, the mechanisms that provoke pathological alterations in the coronary and cerebral arteries in HGPS remain ill defined. Here, we assessed vascular function in the coronary arteries (CorAs) and carotid arteries (CarAs) of progerin-expressing LmnaG609G/G609G mice (G609G), both in resting conditions and after hypoxic stimulus. Wire myography, pharmacological screening, and gene expression studies demonstrated vascular atony and stenosis, as well as other functional alterations in progeroid CorAs and CarAs and aorta. These defects were associated with loss of vascular smooth muscle cells and overexpression of the KV7 family of voltage-dependent potassium channels. Compared with wild-type controls, G609G mice showed reduced median survival upon chronic isoproterenol exposure, a baseline state of chronic cardiac hypoxia characterized by overexpression of hypoxia-inducible factor 1α and 3α genes, and increased cardiac vascularization. Our results shed light on the mechanisms underlying progerin-induced coronary and carotid artery disease and identify KV7 channels as a candidate target for the treatment of HGPS.

Lamin A/C Ablation Restricted to Vascular Smooth Muscle Cells, Cardiomyocytes, and Cardiac Fibroblasts Causes Cardiac and Vascular Dysfunction.

Mutations in the LMNA gene (encoding lamin A/C proteins) cause several human cardiac diseases, including dilated cardiomyopathies (LMNA-DCM). The main clinical risks in LMNA-DCM patients are sudden cardiac death and progressive left ventricular ejection fraction deterioration, and therefore most human and animal studies have sought to define the mechanisms through which LMNA mutations provoke cardiac alterations, with a particular focus on cardiomyocytes. To investigate if LMNA mutations also cause vascular alterations that might contribute to the etiopathogenesis of LMNA-DCM, we generated and characterized Lmnaflox/floxSM22αCre mice, which constitutively lack lamin A/C in vascular smooth muscle cells (VSMCs), cardiac fibroblasts, and cardiomyocytes. Like mice with whole body or cardiomyocyte-specific lamin A/C ablation, Lmnaflox/floxSM22αCre mice recapitulated the main hallmarks of human LMNA-DCM, including ventricular systolic dysfunction, cardiac conduction defects, cardiac fibrosis, and premature death. These alterations were associated with elevated expression of total and phosphorylated (active) Smad3 and cleaved (active) caspase 3 in the heart. Lmnaflox/floxSM22αCre mice also exhibited perivascular fibrosis in the coronary arteries and a switch of aortic VSMCs from the 'contractile' to the 'synthetic' phenotype. Ex vivo wire myography in isolated aortic rings revealed impaired maximum contraction capacity and an altered response to vasoconstrictor and vasodilator agents in Lmnaflox/floxSM22αCre mice. To our knowledge, our results provide the first evidence of phenotypic alterations in VSMCs that might contribute significantly to the pathophysiology of some forms of LMNA-DCM. Future work addressing the mechanisms underlying vascular defects in LMNA-DCM may open new therapeutic avenues for these diseases.

Emerging roles of interferon-stimulated gene-15 in age-related telomere attrition, the DNA damage response, and cardiovascular disease.

Population aging and age-related cardiovascular disease (CVD) are becoming increasingly prevalent worldwide, generating a huge medical and socioeconomic burden. The complex regulation of aging and CVD and the interaction between these processes are crucially dependent on cellular stress responses. Interferon-stimulated gene-15 (ISG15) encodes a ubiquitin-like protein expressed in many vertebrate cell types that can be found both free and conjugated to lysine residues of target proteins via a post-translational process termed ISGylation. Deconjugation of ISG15 (deISGylation) is catalyzed by the ubiquitin-specific peptidase 18 (USP18). The ISG15 pathway has mostly been studied in the context of viral and bacterial infections and in cancer. This minireview summarizes current knowledge on the role of ISG15 in age-related telomere shortening, genomic instability, and DNA damage accumulation, as well as in hypertension, diabetes, and obesity, major CVD risk factors prevalent in the elderly population.

Cardiovascular Progerin Suppression and Lamin A Restoration Rescue Hutchinson-Gilford Progeria Syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder characterized by premature aging and death mainly because of myocardial infarction, stroke, or heart failure. The disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells. Patients look healthy at birth, and symptoms typically emerge in the first or second year of life. Assessing the reversibility of progerin-induced damage and the relative contribution of specific cell types is critical to determining the potential benefits of late treatment and to developing new therapies. METHODS: We used CRISPR-Cas9 technology to generate LmnaHGPSrev/HGPSrev (HGPSrev) mice engineered to ubiquitously express progerin while lacking lamin A and allowing progerin suppression and lamin A restoration in a time- and cell type-specific manner on Cre recombinase activation. We characterized the phenotype of HGPSrev mice and crossed them with Cre transgenic lines to assess the effects of suppressing progerin and restoring lamin A ubiquitously at different disease stages as well as specifically in vascular smooth muscle cells and cardiomyocytes. RESULTS: Like patients with HGPS, HGPSrev mice appear healthy at birth and progressively develop HGPS symptoms, including failure to thrive, lipodystrophy, vascular smooth muscle cell loss, vascular fibrosis, electrocardiographic anomalies, and precocious death (median lifespan of 15 months versus 26 months in wild-type controls, P<0.0001). Ubiquitous progerin suppression and lamin A restoration significantly extended lifespan when induced in 6-month-old mildly symptomatic mice and even in severely ill animals aged 13 months, although the benefit was much more pronounced on early intervention (84.5% lifespan extension in mildly symptomatic mice, P<0.0001, and 6.7% in severely ill mice, P<0.01). It is remarkable that major vascular alterations were prevented and lifespan normalized in HGPSrev mice when progerin suppression and lamin A restoration were restricted to vascular smooth muscle cells and cardiomyocytes. CONCLUSIONS: HGPSrev mice constitute a new experimental model for advancing knowledge of HGPS. Our findings suggest that it is never too late to treat HGPS, although benefit is much more pronounced when progerin is targeted in mice with mild symptoms. Despite the broad expression pattern of progerin and its deleterious effects in many organs, restricting its suppression to vascular smooth muscle cells and cardiomyocytes is sufficient to prevent vascular disease and normalize lifespan.

Molecular and Cellular Mechanisms Driving Cardiovascular Disease in Hutchinson-Gilford Progeria Syndrome: Lessons Learned from Animal Models.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease that recapitulates many symptoms of physiological aging and precipitates death. Patients develop severe vascular alterations, mainly massive vascular smooth muscle cell loss, vessel stiffening, calcification, fibrosis, and generalized atherosclerosis, as well as electrical, structural, and functional anomalies in the heart. As a result, most HGPS patients die of myocardial infarction, heart failure, or stroke typically during the first or second decade of life. No cure exists for HGPS, and therefore it is of the utmost importance to define the mechanisms that control disease progression in order to develop new treatments to improve the life quality of patients and extend their lifespan. Since the discovery of the HGPS-causing mutation, several animal models have been generated to study multiple aspects of the syndrome and to analyze the contribution of different cell types to the acquisition of the HGPS-associated cardiovascular phenotype. This review discusses current knowledge about cardiovascular features in HGPS patients and animal models and the molecular and cellular mechanisms through which progerin causes cardiovascular disease.

Identification of common cardiometabolic alterations and deregulated pathways in mouse and pig models of aging.

Aging is the main risk factor for cardiovascular and metabolic diseases, which have become a global concern as the world population ages. These diseases and the aging process are exacerbated in Hutchinson-Gilford progeria syndrome (HGPS or progeria). Here, we evaluated the cardiometabolic disease in animal models of premature and normal aging with the aim of identifying alterations that are shared or specific to each condition. Despite differences in body composition and metabolic markers, prematurely and normally aging mice developed heart failure and similar cardiac electrical abnormalities. High-throughput proteomics of the hearts of progeric and normally aged mice revealed altered protein oxidation and glycation, as well as dysregulated pathways regulating energy metabolism, proteostasis, gene expression, and cardiac muscle contraction. These results were corroborated in the hearts of progeric pigs, underscoring the translational potential of our findings, which could help in the design of strategies to prevent or slow age-related cardiometabolic disease.

Vascular Smooth Muscle Cell-Specific Progerin Expression Provokes Contractile Impairment in a Mouse Model of Hutchinson-Gilford Progeria Syndrome that Is Ameliorated by Nitrite Treatment.

Cardiovascular disease (CVD) is the main cause of death worldwide, and aging is its leading risk factor. Aging is much accelerated in Hutchinson-Gilford progeria syndrome (HGPS), an ultra-rare genetic disorder provoked by the ubiquitous expression of a mutant protein called progerin. HGPS patients die in their teens, primarily due to cardiovascular complications. The primary causes of age-associated CVD are endothelial dysfunction and dysregulated vascular tone; however, their contribution to progerin-induced CVD remains poorly characterized. In the present study, we found that progeroid LmnaG609G/G609G mice with ubiquitous progerin expression show both endothelial dysfunction and severe contractile impairment. To assess the relative contribution of specific vascular cell types to these anomalies, we examined LmnaLCS/LCSTie2Cretg/+ and LmnaLCS/LCSSm22αCretg/+ mice, which express progerin specifically in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. Whereas vessel contraction was impaired in mice with VSMC-specific progerin expression, we observed no endothelial dysfunction in mice with progerin expression restricted to VSMCs or ECs. Vascular tone regulation in progeroid mice was ameliorated by dietary sodium nitrite supplementation. Our results identify VSMCs as the main cell type causing contractile impairment in a mouse model of HGPS that is ameliorated by nitrite treatment.

Generation and characterization of a novel knockin minipig model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder for which no cure exists. The disease is characterized by premature aging and inevitable death in adolescence due to cardiovascular complications. Most HGPS patients carry a heterozygous de novo LMNA c.1824C > T mutation, which provokes the expression of a dominant-negative mutant protein called progerin. Therapies proven effective in HGPS-like mouse models have yielded only modest benefit in HGPS clinical trials. To overcome the gap between HGPS mouse models and patients, we have generated by CRISPR-Cas9 gene editing the first large animal model for HGPS, a knockin heterozygous LMNA c.1824C > T Yucatan minipig. Like HGPS patients, HGPS minipigs endogenously co-express progerin and normal lamin A/C, and exhibit severe growth retardation, lipodystrophy, skin and bone alterations, cardiovascular disease, and die around puberty. Remarkably, the HGPS minipigs recapitulate critical cardiovascular alterations seen in patients, such as left ventricular diastolic dysfunction, altered cardiac electrical activity, and loss of vascular smooth muscle cells. Our analysis also revealed reduced myocardial perfusion due to microvascular damage and myocardial interstitial fibrosis, previously undescribed readouts potentially useful for monitoring disease progression in patients. The HGPS minipigs provide an appropriate preclinical model in which to test human-size interventional devices and optimize candidate therapies before advancing to clinical trials, thus accelerating the development of effective applications for HGPS patients.

Lamin A/C augments Th1 differentiation and response against vaccinia virus and Leishmania major.

Differentiation of naive CD4+ T-cells into functionally distinct T helper (Th) subsets is critical to immunity against pathogen infection. Little is known about the role of signals emanating from the nuclear envelope for T-cell differentiation. The nuclear envelope protein lamin A/C is induced in naive CD4+ T-cells upon antigen recognition and acts as a link between the nucleus and the plasma membrane during T-cell activation. Here we demonstrate that the absence of lamin A/C in naive T-cell reduces Th1 differentiation without affecting Th2 differentiation in vitro and in vivo. Moreover, Rag1 -/- mice reconstituted with Lmna -/- CD4+CD25 - T-cells and infected with vaccinia virus show weaker Th1 responses and viral removal than mice reconstituted with wild-type T-cells. Th1 responses and pathogen clearance upon Leishmania major infection were similarly diminished in mice lacking lamin A/C in the complete immune system or selectively in T-cells. Lamin A/C mediates Th1 polarization by a mechanism involving T-bet and IFNγ production. Our results reveal a novel role for lamin A/C as key regulator of Th1 differentiation in response to viral and intracellular parasite infections.

A-type lamins and cardiovascular disease in premature aging syndromes.

Lamin A is a nuclear intermediate filament protein with important structural and regulatory roles in most differentiated mammalian cells. Excessive accumulation of its precursor prelamin A or the mutant form called 'progerin' causes premature aging syndromes. Progeroid 'laminopathies' are characterized by severe cardiovascular problems (cardiac electrical defects, vascular calcification and stiffening, atherosclerosis, myocardial infarction, and stroke) and premature death. Here, we review studies in cell and mouse models and patients that are unraveling how abnormal prelamin A and progerin accumulation accelerates cardiovascular disease and aging. This knowledge is essential for developing effective therapies to treat progeria and may help identify new mechanisms underlying normal aging.

Short Telomere Load, Telomere Length, and Subclinical Atherosclerosis: The PESA Study.

BACKGROUND: Leucocyte telomere length (LTL) shortening is associated with cardiovascular ischemic events and mortality in humans, but data on its association with subclinical atherosclerosis are scarce. Whether the incidence and severity of subclinical atherosclerosis are associated with the abundance of critically short telomeres, a major trigger of cellular senescence, remains unknown. OBJECTIVES: The authors conducted a cross-sectional exploration of the association between subclinical atherosclerosis burden and both average LTL and the abundance of short telomeres (%LTL<3 kb). METHODS: Telomere length was assessed by high-throughput quantitative fluorescence in situ hybridization in circulating leukocytes from 1,459 volunteers without established cardiovascular disease (58% men, 40 to 54 years of age) from the PESA (Progression of Early Subclinical Atherosclerosis) study. Subclinical atherosclerosis was evaluated by coronary artery calcium scan and 2-dimensional/3-dimensional ultrasound in different aortic territories. Statistical significance of differences among multiple covariates was assessed with linear regression models. Independent associations of telomere parameters with plaque presence were evaluated using general linear models. RESULTS: In men and women, age was inversely associated with LTL (Pearson's r = -0.127, p < 0.001) and directly with %LTL<3 kb (Pearson's r = 0.085; p = 0.001). Short LTL reached statistical significance as a determinant of total and femoral plaque in men, but not in women. However, this association was not sustained after adjustment for age or additional adjustment for cardiovascular risk factors. No significant independent association was found between %LTL<3 kb and plaque burden. Serum-oxidized low-density lipoprotein levels were directly associated with %LTL<3 kb in men (p = 0.008) and women (p < 0.001). CONCLUSIONS: In a cross-sectional study of a middle-aged population, average LTL and short telomere load are not significant independent determinants of subclinical atherosclerosis. Longitudinal follow-up of PESA participants will assess long-term associations between telomere length and progression of subclinical atherosclerosis.

Oil Red O and Hematoxylin and Eosin Staining for Quantification of Atherosclerosis Burden in Mouse Aorta and Aortic Root.

Methods for staining tissues with Oil Red O and hematoxylin-eosin are classical histological techniques that are widely used to quantify atherosclerotic burden in mouse tissues because of their ease of use, reliability, and the large amount of information they provide. These stains can provide quantitative data about the impact of a genetic or environmental factor on atherosclerotic burden and on the initiation, progression, or regression of the disease, and can also be used to evaluate the efficacy of drugs designed to prevent or treat atherosclerosis. This chapter provides protocols for quantifying atherosclerotic burden in mouse aorta and aortic root, including methods for dissection, Oil Red O staining, hematoxylin-eosin staining, and image analysis.

Tissue-specific oxidative stress and loss of mitochondria in CoQ-deficient Pdss2 mutant mice.

Primary human CoQ(10) deficiencies are clinically heterogeneous diseases caused by mutations in PDSS2 and other genes required for CoQ(10) biosynthesis. Our in vitro studies of PDSS2 mutant fibroblasts, with <20% CoQ(10) of control cells, revealed reduced activity of CoQ(10)-dependent complex II+III and ATP synthesis, without amplification of reactive oxygen species (ROS), markers of oxidative damage, or antioxidant defenses. In contrast, COQ2 and ADCK3 mutant fibroblasts, with 30-50% CoQ(10) of controls, showed milder bioenergetic defects but significantly increased ROS and oxidation of lipids and proteins. We hypothesized that absence of oxidative stress markers and cell death in PDSS2 mutant fibroblasts were due to the extreme severity of CoQ(10) deficiency. Here, we have investigated in vivo effects of Pdss2 deficiency in affected and unaffected organs of CBA/Pdss2(kd/kd) mice at presymptomatic, phenotypic-onset, and end-stages of the disease. Although Pdss2 mutant mice manifest widespread CoQ(9) deficiency and mitochondrial respiratory chain abnormalities, only affected organs show increased ROS production, oxidative stress, mitochondrial DNA depletion, and reduced citrate synthase activity, an index of mitochondrial mass. Our data indicate that kidney-specific loss of mitochondria triggered by oxidative stress may be the cause of renal failure in Pdss2(kd/kd) mice.

Decreased hippocampal expression of calbindin D28K and cognitive impairment in MELAS.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a maternally inherited mitochondrial syndrome characterized by seizures, migrainous headaches, lactic acidosis, vomiting, and recurrent stroke-like episodes. Patients often suffer from cognitive dysfunction of unclear pathogenesis. In this study, we explored a possible link between cognitive dysfunction and hippocampal expression of calbindin D(28KD) (CB), a high affinity calcium-binding protein, in four MELAS patients, using post mortem hippocampal tissues. We found significantly reduced CB levels in all patients by immunohistochemistry, Western blot, and quantitative real-time PCR. Reduction in CB expression has been associated with aging and with neurodegenerative disorders, including Alzheimer's disease. We postulate that the reduced CB expression may play a role in the cognitive abnormalities associated with MELAS.

Measurement of mitochondrial dNTP pools.

Because deoxyribonucleoside triphosphates (dNTPs) are the critical substrates for DNA replication and repair, dNTP pools have been studied in context of multiple basic biochemical processes. Over the last 12 years, interest in dNTPs, and specifically the mitochondrial dNTP pools, has expanded to biomedical science because several mitochondrial diseases have been found to be caused by dysfunctions of several enzymes involved in dNTP catabolism or anabolism. Techniques to reliably measure mitochondrial dNTPs should be sensitive and specific to avoid interference caused by the abundant ribonucleotides. Here, we describe detailed protocols to measure mitochondrial dNTPs from two specific samples, cultured skin fibroblasts and mouse liver. The methods can be easily adapted to other types of samples. The protocol follows a polymerase-based method, which is the most widely used approach to measure dNTP pools. Our description is based on the latest update of the technique, which minimizes the potential interference from ribonucleotides.

Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity.

The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1(-) cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase γ, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.

Onset and organ specificity of Tk2 deficiency depends on Tk1 down-regulation and transcriptional compensation.

Deficiency of thymidine kinase 2 (TK2) is a frequent cause of isolated myopathy or encephalomyopathy in children with mitochondrial DNA (mtDNA) depletion. To determine the bases of disease onset, organ specificity and severity of TK2 deficiency, we have carefully characterized Tk2 H126N knockin mice (Tk2-/-). Although normal until postnatal day 8, Tk2-/- mice rapidly develop fatal encephalomyopathy between postnatal days 10 and 13. We have observed that wild-type Tk2 activity is constant in the second week of life, while Tk1 activity decreases significantly between postnatal days 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2-/- mice and correlates with the onset of mtDNA depletion in the brain and the heart. Resistance to pathology in Tk2 mutant organs depends on compensatory mechanisms to the reduced mtDNA level. Our analyses at postnatal day 13 have revealed that Tk2-/- heart significantly increases mitochondrial transcript levels relative to the mtDNA content. This transcriptional compensation allows the heart to maintain normal levels of mtDNA-encoded proteins. The up-regulation in mitochondrial transcripts is not due to increased expression of the master mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and nuclear respiratory factors 1 and 2, or to enhanced expression of the mitochondrial transcription factors A, B1 or B2. Instead, Tk2-/- heart compensates for mtDNA depletion by down-regulating the expression of the mitochondrial transcriptional terminator transcription factor 3 (MTERF3). Understanding the molecular mechanisms that allow Tk2 mutant organs to be spared may help design therapies for Tk2 deficiency.

Thymidine kinase 2 deficiency-induced mitochondrial DNA depletion causes abnormal development of adipose tissues and adipokine levels in mice.

Mammal adipose tissues require mitochondrial activity for proper development and differentiation. The components of the mitochondrial respiratory chain/oxidative phosphorylation system (OXPHOS) are encoded by both mitochondrial and nuclear genomes. The maintenance of mitochondrial DNA (mtDNA) is a key element for a functional mitochondrial oxidative activity in mammalian cells. To ascertain the role of mtDNA levels in adipose tissue, we have analyzed the alterations in white (WAT) and brown (BAT) adipose tissues in thymidine kinase 2 (Tk2) H126N knockin mice, a model of TK2 deficiency-induced mtDNA depletion. We observed respectively severe and moderate mtDNA depletion in TK2-deficient BAT and WAT, showing both tissues moderate hypotrophy and reduced fat accumulation. Electron microscopy revealed altered mitochondrial morphology in brown but not in white adipocytes from TK2-deficient mice. Although significant reduction in mtDNA-encoded transcripts was observed both in WAT and BAT, protein levels from distinct OXPHOS complexes were significantly reduced only in TK2-deficient BAT. Accordingly, the activity of cytochrome c oxidase was significantly lowered only in BAT from TK2-deficient mice. The analysis of transcripts encoding up to fourteen components of specific adipose tissue functions revealed that, in both TK2-deficient WAT and BAT, there was a consistent reduction of thermogenesis related gene expression and a severe reduction in leptin mRNA. Reduced levels of resistin mRNA were found in BAT from TK2-deficient mice. Analysis of serum indicated a dramatic reduction in circulating levels of leptin and resistin. In summary, our present study establishes that mtDNA depletion leads to a moderate impairment in mitochondrial respiratory function, especially in BAT, causes substantial alterations in WAT and BAT development, and has a profound impact in the endocrine properties of adipose tissues.

Reactive oxygen species, oxidative stress, and cell death correlate with level of CoQ10 deficiency.

Coenzyme Q(10) (CoQ(10)) is essential for electron transport in the mitochondrial respiratory chain and antioxidant defense. The relative importance of respiratory chain defects, ROS production, and apoptosis in the pathogenesis of CoQ(10) deficiency is unknown. We determined previously that severe CoQ(10) deficiency in cultured skin fibroblasts harboring COQ2 and PDSS2 mutations produces divergent alterations of bioenergetics and oxidative stress. Here, to better understand the pathogenesis of CoQ(10) deficiency, we have characterized the effects of varying severities of CoQ(10) deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ(10) biosynthesis. Levels of CoQ(10) seem to correlate with ROS production; 10-15% and >60% residual CoQ(10) are not associated with significant ROS production, whereas 30-50% residual CoQ(10) is accompanied by increased ROS production and cell death. Our results confirm that varying degrees of CoQ(10) deficiency cause variable defects of ATP synthesis and oxidative stress. These findings may lead to more rational therapeutic strategies for CoQ(10) deficiency.