L'application de la métabolomique à la découverte de nouveaux biomarqueurs chez les patients atteints d'acidose lactique

L’acidose lactique du Saguenay-Lac-St-Jean, ou syndrome de Leigh de forme canadienne-française (LSFC), est une maladie mitochondriale neurodégénérative causée par des mutations du gène LRPPRC et caractérisée par des crises d’acidose menant au décès en bas âge. On ne comprend pas encore les causes exactes de ces crises, et aucun traitement n’est actuellement disponible. L’objectif de cette étude a été de comparer le profil des métabolites sanguins et urinaires chez des sujets LSFC et des témoins, avant et après un repas, par une approche métabolomique ciblée. Le projet s’inscrit dans une démarche à long terme visant l’identification de biomarqueurs prédictifs des crises, permettant d'intervenir plus rapidement afin d’éviter le décès. Les échantillons biologiques ont été prélevés chez 9 sujets atteints du LSFC et 9 témoins appariés, à jeun et 90 minutes après un repas standardisé. Les analyses incluent un bilan biochimique et hormonal, un profil des acides aminés, des acides gras, des acides organiques et des acylcarnitines. Les métabolites significativement modifiés chez les patients peuvent être classés en deux catégories : (i) le reflet d’une dysfonction mitochondriale, et plus particulièrement de l’accumulation d’équivalents réduits en amont de la chaîne respiratoire, et (ii) des indices de risque cardiométabolique, qui s’observent davantage chez les patients adultes malgré leur jeune âge. Ainsi, il serait intéressant d’inclure au traitement des stratégies visant la diminution des facteurs de risque cardiométabolique, notamment par une modification des habitudes de vie. Notre étude démontre la pertinence d’avoir recours à la métabolomique dans l’étude des désordres de la phosphorylation oxydative.L’acidose lactique du Saguenay-Lac-St-Jean, ou syndrome de Leigh de forme canadienne-française (LSFC), est une maladie mitochondriale neurodégénérative causée par des mutations du gène LRPPRC et caractérisée par des crises d’acidose menant au décès en bas âge. On ne comprend pas encore les causes exactes de ces crises, et aucun traitement n’est actuellement disponible. L’objectif de cette étude a été de comparer le profil des métabolites sanguins et urinaires chez des sujets LSFC et des témoins, avant et après un repas, par une approche métabolomique ciblée. Le projet s’inscrit dans une démarche à long terme visant l’identification de biomarqueurs prédictifs des crises, permettant d'intervenir plus rapidement afin d’éviter le décès. Les échantillons biologiques ont été prélevés chez 9 sujets atteints du LSFC et 9 témoins appariés, à jeun et 90 minutes après un repas standardisé. Les analyses incluent un bilan biochimique et hormonal, un profil des acides aminés, des acides gras, des acides organiques et des acylcarnitines. Les métabolites significativement modifiés chez les patients peuvent être classés en deux catégories : (i) le reflet d’une dysfonction mitochondriale, et plus particulièrement de l’accumulation d’équivalents réduits en amont de la chaîne respiratoire, et (ii) des indices de risque cardiométabolique, qui s’observent davantage chez les patients adultes malgré leur jeune âge. Ainsi, il serait intéressant d’inclure au traitement des stratégies visant la diminution des facteurs de risque cardiométabolique, notamment par une modification des habitudes de vie. Notre étude démontre la pertinence d’avoir recours à la métabolomique dans l’étude des désordres de la phosphorylation oxydative

Humoral responses to the measles, mumps and rubella vaccine are impaired in Leigh Syndrome French Canadian patients

Leigh Syndrome French Canadian (LSFC) is a rare autosomal recessive metabolic disorder characterized by severe lactic acidosis crises and early mortality. LSFC patients carry mutations in the Leucine Rich Pentatricopeptide Repeat Containing (LRPPRC) gene, which lead to defects in the respiratory chain complexes and mitochondrial dysfunction. Mitochondrial respiration modulates cellular metabolic activity, which impacts many cell types including the differentiation and function of immune cells. Hence, we postulated that, in addition to neurological and metabolic disorders, LSFC patients may show impaired immune activity. To gain insight into the quality of the immune response in LSFC patients, we examined the response to the measles, mumps and rubella (MMR) vaccine by measuring antibody titers to MMR in the plasma. In a cohort of eight LSFC patients, the response to the MMR vaccine was variable, with some individuals showing antibodies to all three viruses, while others had antibodies to two or fewer viruses. These results suggest that the mutations in the LRPPRC gene present in LSFC patients may affect the immune response to vaccines. Monitoring vaccine response in this fragile population should be considered to ensure full protection against pathogens

Informal caregivers of persons with dementia, their use of and needs for specific professional support: a survey of the national dementia programme

<p>Abstract</p> <p>Background</p> <p>This paper describes both the use of and needs for informal caregivers of people with dementia, based on a questionnaire survey organized within the National Dementia Programme in the Netherlands. The National Dementia Programme is a quality collaborative of the Dutch Alzheimer's Association, the Institute of Quality of Healthcare (CBO) and the Knowledge Centre on Ageing (Vilans), instigated by the Ministry of Health, Welfare and Sport, to improve integrated care for people with dementia and their informal caregivers. The support needs of informal caregivers are important to improve caregiver well-being and delaying institutionalization of the person with dementia.</p> <p>Methods</p> <p>In the period April 2006 - January 2007, the National Dementia Programme questionnaire was completed by 984 informal caregivers. Descriptive statistics were used to analyze the use of and needs for additional professional support by informal caregivers. Chi-square tests were used to assess the relationships between characteristics of the caregivers (spouses, sons/daughters, sons/daughters in-law) and support needs on one hand and to assess the relationship between the living situation of the person with dementia (living at home or living in a nursing home or home for the elderly) and support needs on the other hand.</p> <p>Results</p> <p>Almost all informal caregivers (92.6%) received some professional support. However, two thirds (67.4%) indicated they had one or more needs for additional professional support. Informal caregivers often need additional professional advice about what to do when their relative is frightened, angry of confused. Spouses reported different needs than sons or daughters (in-law): spouses relatively often need emotional support and sons or daughters (in-law) more often need information and coordination of dementia care.</p> <p>Conclusions</p> <p>Most of the informal caregivers report that they need additional information and advice, e.g. about how to cope with behavioral problems of their relative, about the progression of the illness trajectory, emotional support and coordination of dementia care. Future support programmes, e.g. in the field of case management, should address the specific needs of informal caregivers.</p

Obstructive Sleep Apnea and Cognitive Decline: A Review of Potential Vulnerability and Protective Factors

Around 40% of dementia risk is attributable to modifiable risk factors such as physical inactivity, hypertension, diabetes and obesity. Recently, sleep disorders, including obstructive sleep apnea (OSA), have also been considered among these factors. However, despite several epidemiological studies investigating the link between OSA and cognitive decline, there is still no consensus on whether OSA increases the risk of dementia or not. Part of the heterogeneity observed in previous studies might be related to some individual characteristics that modulate the association between OSA and cognitive decline. In this narrative review, we present these individual characteristics, namely, age, sex, menopause, obesity, diabetes mellitus, hypertension, cardiovascular diseases, smoking, excessive alcohol consumption, depression, air pollution, Apolipoprotein E ε4 allele, physical activity, and cognitive reserve. To date, large cohort studies of OSA and cognitive decline tended to statistically control for the effects of these variables, but whether they interact with OSA to predict cognitive decline remains to be elucidated. Being able to better predict who is at risk of cognitive decline when they have OSA would improve clinical management and treatment decisions, particularly when patients present relatively mild OSA

Mitochondrial Vulnerability and Increased Susceptibility to Nutrient-Induced Cytotoxicity in Fibroblasts from Leigh Syndrome French Canadian Patients

<div><p>Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC), a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX) and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca²⁺-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the nature of the diets, particularly excess fat intake, as well as on the use of antioxidants in patients with LSFC and, possibly, other COX defects.</p></div

Humoral responses to the measles, mumps and rubella vaccine are impaired in Leigh Syndrome French Canadian patients.

Leigh Syndrome French Canadian (LSFC) is a rare autosomal recessive metabolic disorder characterized by severe lactic acidosis crises and early mortality. LSFC patients carry mutations in the Leucine Rich Pentatricopeptide Repeat Containing (LRPPRC) gene, which lead to defects in the respiratory chain complexes and mitochondrial dysfunction. Mitochondrial respiration modulates cellular metabolic activity, which impacts many cell types including the differentiation and function of immune cells. Hence, we postulated that, in addition to neurological and metabolic disorders, LSFC patients may show impaired immune activity. To gain insight into the quality of the immune response in LSFC patients, we examined the response to the measles, mumps and rubella (MMR) vaccine by measuring antibody titers to MMR in the plasma. In a cohort of eight LSFC patients, the response to the MMR vaccine was variable, with some individuals showing antibodies to all three viruses, while others had antibodies to two or fewer viruses. These results suggest that the mutations in the LRPPRC gene present in LSFC patients may affect the immune response to vaccines. Monitoring vaccine response in this fragile population should be considered to ensure full protection against pathogens

Image1_Human induced pluripotent stem cells (hiPSCs) derived cells reflect tissue specificity found in patients with Leigh syndrome French Canadian variant (LSFC).pdf

Leigh syndrome French Canadian type (LSFC) is a recessive neurodegenerative disease characterized by tissue-specific deficiency in cytochrome c oxidase (COX), the fourth complex in the oxidative phosphorylation system. LSFC is caused by mutations in the leucine rich pentatricopeptide repeat containing gene (LRPPRC). Most LSFC patients in Quebec are homozygous for an A354V substitution that causes a decrease in the expression of the LRPPRC protein. While LRPPRC is ubiquitously expressed and is involved in multiple cellular functions, tissue-specific expression of LRPPRC and COX activity is correlated with clinical features. In this proof-of-principle study, we developed human induced pluripotent stem cell (hiPSC)-based models from fibroblasts taken from a patient with LSFC, homozygous for the LRPPRC*354V allele, and from a control, homozygous for the LRPPRC*A354 allele. Specifically, for both of these fibroblast lines we generated hiPSC, hiPSC-derived cardiomyocytes (hiPSC-CMs) and hepatocyte-like cell (hiPSC-HLCs) lines, as well as the three germ layers. We observed that LRPPRC protein expression is reduced in all cell lines/layers derived from LSFC patient compared to control cells, with a reduction ranging from ∼70% in hiPSC-CMs to undetectable levels in hiPSC-HLC, reflecting tissue heterogeneity observed in patient tissues. We next performed exploratory analyses of these cell lines and observed that COX protein expression was reduced in all cell lines derived from LSFC patient compared to control cells. We also observed that mutant LRPPRC was associated with altered expression of key markers of endoplasmic reticulum stress response in hiPSC-HLCs but not in other cell types that were tested. While this demonstrates feasibility of the approach to experimentally study genotype-based differences that have tissue-specific impacts, this study will need to be extended to a larger number of patients and controls to not only validate the current observations but also to delve more deeply in the pathogenic mechanisms of LSFC.</p

Lipidomics unveils lipid dyshomeostasis and low circulating plasmalogens as biomarkers in a monogenic mitochondrial disorder

Mitochondrial dysfunction characterizes many rare and common age-associated diseases. The biochemical consequences, underlying clinical manifestations, and potential therapeutic targets, remain to be better understood. We tested the hypothesis that lipid dyshomeostasis in mitochondrial disorders goes beyond mitochondrial fatty acid β-oxidation, particularly in liver. This was achieved using comprehensive untargeted and targeted lipidomics in a case-control cohort of patients with Leigh syndrome French-Canadian variant (LSFC), a mitochondrial disease caused by mutations in LRPPRC, and in mice harboring liver-specific inactivation of Lrpprc (H-Lrpprc–/–). We discovered a plasma lipid signature discriminating LSFC patients from controls encompassing lower levels of plasmalogens and conjugated bile acids, which suggest perturbations in peroxisomal lipid metabolism. This premise was reinforced in H-Lrpprc–/– mice, which compared with littermates recapitulated a similar, albeit stronger peroxisomal metabolic signature in plasma and liver including elevated levels of very-long-chain acylcarnitines. These mice also presented higher transcript levels for hepatic markers of peroxisome proliferation in addition to lipid remodeling reminiscent of nonalcoholic fatty liver diseases. Our study underscores the value of lipidomics to unveil unexpected mechanisms underlying lipid dyshomeostasis ensuing from mitochondrial dysfunction herein implying peroxisomes and liver, which likely contribute to the pathophysiology of LSFC, but also other rare and common mitochondrial diseases

Control and Leigh Syndrome French Canadian (LSFC) skin fibroblast cell lines.

<p>Control and Leigh Syndrome French Canadian (LSFC) skin fibroblast cell lines.</p

Effect of the LRPPRC A354V mutation on LRPPRC content, mitochondrial content and cellular ATP levels.

<p><b>(A)</b> Immunoblot and densitometric analysis of LRPPRC content in whole cell lysates from control and LSFC fibroblasts (n = 4). Representative immunofluorescence images of control <b>(B;C)</b> and LSFC fibroblasts <b>(D;E)</b> labeled with anti-LRPPRC (green) and anti-pyruvate dehydrogenase (red) antibodies. Overlay images (yellow), and line scan analysis show the cellular distribution of LRPPRC in the mitochondrial compartment. <b>(F)</b> Cytochrome c oxidase (COX) enzyme activity in whole cell lysates from control and LSFC fibroblasts was normalized to that of the mitochondrial marker citrate synthase (CS) to take into account possible differences in mitochondrial content (n = 4). <b>(G)</b> Immunoblot and densitometric analysis of VDAC, a mitochondrial marker protein, in whole cell lysates from control and LSFC fibroblasts (n = 3). <b>(H)</b> Citrate synthase activity in whole cell lysates from control and LSFC fibroblasts (n = 4). <b>(I)</b> Cellular ATP content in control and LSFC fibroblasts (n = 4). Data are expressed as means ± S.E. Experiments were performed in one control (EBS-4) and one LSFC cell line (AL-006). Difference between control and LSFC cells was assessed with a paired t-test. ** Significantly different from the control group <i>p</i> ≤ 0.01. Statistical power for LRPPRC and COX was 97%.</p