Bioenergetic measurements in peripheral blood mononuclear cells (PBMCs) - Influence of age, sex and circadian rhythm

dsDie Lebenserwartung ist in den letzten Jahren immer weiter angestiegen und führt in Kombination mit zurückgehenden Geburtenraten zu einer Alterung der Gesellschaft. Das Altern bringt ein erhöhtes Risiko für verschiedene Erkrankungen mit sich. Hierbei spielen vor allem die neurodegenerativen Erkrankungen eine wichtige Rolle. Die Demenzerkrankung betrifft besonders viele Personen. Schon heute sind mehr als 47 Millionen Menschen weltweit von einer Demenz betroffen und diese Zahl wird sich voraussichtlich bis 2050 sogar verdreifachen. Aus diesem Grund ist es wichtig, effektive Präventions- und Therapieansätze zu entwickeln und frühzeitige und kostengünstige Diagnosemöglichkeiten zu etablieren. Die häufigste Demenzform ist die Alzheimer Erkrankung (AD), deren häufigste Form sporadisch auftritt und deren Ursache multifaktoriell ist. Eine mitochondriale Dysfunktion spielt sowohl bei der Alterung als auch bei der AD eine entscheidende Rolle und rückt immer mehr in den Fokus der Forschung. Allerdings ist die Bestimmung der mitochondrialen Funktion im Gehirn des lebenden Menschen nur schwierig möglich. Einige Studien konnten jedoch zeigen, dass Veränderungen des Energiemetabolismus bei der Alterung nicht nur im Gehirn, sondern auch in der Peripherie auftreten. Aus diesem Grund scheinen periphere mononukleäre Blutzellen (PBMCs) ein geeignetes Kompartiment für die Erforschung der mitochondrialen Funktion zu sein. Vor diesem Hintergrund war das Hauptziel der vorliegenden Dissertation, herauszufinden, ob die Messung der mitochondrialen Funktion in PBMCs eine geeignete Methode darstellt, um Veränderungen in den Mitochondrien zu detektieren.Im ersten Teil der Arbeit wurde hierzu die Methode zunächst etabliert und mögliche Einflussfaktoren wurden identifiziert. Der Vergleich zweier Zentrifugationsröhrchen und deren Einfluss auf die Zellausbeute, die Reinheit und Zusammensetzung des Isolates sowie auf die mitochondriale Funktion zeigte keine Unterschiede. In einer zweiten Studie wurde der Einfluss des in circadianen Rhythmen ausgeschütteten, Hormons Melatonin auf die mitochondriale Funktion erforscht. Melatonin ist ein effektives Antioxidanz und kann dadurch Messungen der mitochondrialen Funktion beeinflussen. In der Studie konnte bei den männlichen Probanden eine Assoziation zwischen dem Melatonin und der mitochondrialen Aktivität identifiziert werden. Nach Abschluss der Etablierung wurde die Methode im zweiten Teil der Arbeit zur Untersuchung eines möglichen Zusammenhangs der mitochondrialen Funktion im Gehirn und in der Peripherie eingesetzt. Hierzu wurden die in den PBMCs von jungen gesunden Probanden, gemessene Citratsynthase (CS)-Aktivität sowie die ATP-Spiegel mit den im Gehirn gemessenen N-Acetylaspartat (NAA)-Konzentrationen korreliert. Die Ergebnisse der Studie zeigen eine Tendenz zu einer positiven Korrelation zwischen den mitochondrialen Parametern im Gehirn und im Blut. Im Rahmen einer anderen Studie wurde untersucht, ob sich Veränderungen der mitochondrialen Funktion in PBMCS bereits bei kognitiv gesunden alten Probanden zeigen. Hierbei konnten altersassoziierte Unterschiede beobachtet werden. Die jungen Frauen und Männer wiesen signifikant höhere ATP-Spiegel und Komplex IV-Aktivitäten auf als die weiblichen und männlichen alten Probanden. Dies deutet auf eine altersbedingte Abnahme der mitochondrialen Funktion hin. Beim Vergleich der Ergebnisse mit Patienten mit milden kognitiven Beeinträchtigungen (MCI) konnte sowohl in den jungen als auch in den alten Probanden eine signifikant höhere mitochondriale Funktion gemessen werden. Dies bestätigt erneut, dass die mitochondriale Dysfunktion bei der Entstehung der AD eine entscheidende Rolle spielt. Neben den altersassoziierten Unterschieden wurde in einer weiteren Studie der Einfluss des Geschlechts auf die mitochondriale Funktion erforscht. Hier, aber auch in den anderen durchgeführten Studien, zeigten sich geschlechtsassoziierte Unterschiede in der mitochondrialen Funktion in PBMCs. Die Komplex IV-Aktivität war in allen Studien bei den weiblichen Probanden signifikant höher als bei den Männern und auch die ATP-Spiegel lagen tendenziell in einem höheren Bereich. Im letzten Teil der Arbeit wurde die PBMC-Methode genutzt, um den Einfluss einer sechsmonatigen Intervention auf die mitochondriale Funktion in PBMCs von MCI-Patienten zu untersuchen. Es konnten keine Effekte der Intervention auf die in den PBMCs gemessenen mitochondrialen Parametern beobachtet werden. Unter Berücksichtigung dieser Einflussfaktoren und der geschlechtsassoziierten Unterschiede der mitochondrialen Funktion, ist die Messung von mitochondrialen Parametern in PBMCs eine geeignete und kostengünstige Methode, um Veränderungen der mitochondrialen Funktion frühzeitig zu detektieren. Die Methode könnte auch bei der Überwachung des Erfolges von Präventions- und Therapieansätzen, die das Mitochondrium als Ziel haben, von großem Nutzen sein

Cerebral Mitochondrial Function and Cognitive Performance during Aging: A Longitudinal Study in NMRI Mice

Brain aging is one of the major risk factors for the development of several neurodegenerative diseases. Therefore, mitochondrial dysfunction plays an important role in processes of both, brain aging and neurodegeneration. Aged mice including NMRI mice are established model organisms to study physiological and molecular mechanisms of brain aging. However, longitudinal data evaluated in one cohort are rare but are important to understand the aging process of the brain throughout life, especially since pathological changes early in life might pave the way to neurodegeneration in advanced age. To assess the longitudinal course of brain aging, we used a cohort of female NMRI mice and measured brain mitochondrial function, cognitive performance, and molecular markers every 6 months until mice reached the age of 24 months. Furthermore, we measured citrate synthase activity and respiration of isolated brain mitochondria. Mice at the age of three months served as young controls. At six months of age, mitochondria-related genes (complex IV, creb-1, β-AMPK, and Tfam) were significantly elevated. Brain ATP levels were significantly reduced at an age of 18 months while mitochondria respiration was already reduced in middle-aged mice which is in accordance with the monitored impairments in cognitive tests. mRNA expression of genes involved in mitochondrial biogenesis (cAMP response element-binding protein 1 (creb-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), nuclear respiratory factor-1 (Nrf-1), mitochondrial transcription factor A (Tfam), growth-associated protein 43 (GAP43), and synaptophysin 1 (SYP1)) and the antioxidative defense system (catalase (Cat) and superoxide dismutase 2 (SOD2)) was measured and showed significantly decreased expression patterns in the brain starting at an age of 18 months. BDNF expression reached, a maximum after 6 months. On the basis of longitudinal data, our results demonstrate a close connection between the age-related decline of cognitive performance, energy metabolism, and mitochondrial biogenesis during the physiological brain aging process

Cerebral mitochondrial function and cognitive performance during aging: a longitudinal study in NMRI mice

Brain aging is one of the major risk factors for the development of several neurodegenerative diseases. Therefore, mitochondrial dysfunction plays an important role in processes of both, brain aging and neurodegeneration. Aged mice including NMRI mice are established model organisms to study physiological and molecular mechanisms of brain aging. However, longitudinal data evaluated in one cohort are rare but are important to understand the aging process of the brain throughout life, especially since pathological changes early in life might pave the way to neurodegeneration in advanced age. To assess the longitudinal course of brain aging, we used a cohort of female NMRI mice and measured brain mitochondrial function, cognitive performance, and molecular markers every 6 months until mice reached the age of 24 months. Furthermore, we measured citrate synthase activity and respiration of isolated brain mitochondria. Mice at the age of three months served as young controls. At six months of age, mitochondria-related genes (complex IV, creb-1, β-AMPK, and Tfam) were significantly elevated. Brain ATP levels were significantly reduced at an age of 18 months while mitochondria respiration was already reduced in middle-aged mice which is in accordance with the monitored impairments in cognitive tests. mRNA expression of genes involved in mitochondrial biogenesis (cAMP response element-binding protein 1 (creb-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), nuclear respiratory factor-1 (Nrf-1), mitochondrial transcription factor A (Tfam), growth-associated protein 43 (GAP43), and synaptophysin 1 (SYP1)) and the antioxidative defense system (catalase (Cat) and superoxide dismutase 2 (SOD2)) was measured and showed significantly decreased expression patterns in the brain starting at an age of 18 months. BDNF expression reached, a maximum after 6 months. On the basis of longitudinal data, our results demonstrate a close connection between the age-related decline of cognitive performance, energy metabolism, and mitochondrial biogenesis during the physiological brain aging process

Effects of Long-Term Treatment with a Blend of Highly Purified Olive Secoiridoids on Cognition and Brain ATP Levels in Aged NMRI Mice

Aging represents a major risk factor for developing neurodegenerative diseases such as Alzheimer’s disease (AD). As components of the Mediterranean diet, olive polyphenols may play a crucial role in the prevention of AD. Since mitochondrial dysfunction acts as a final pathway in both brain aging and AD, respectively, the effects of a mixture of highly purified olive secoiridoids were tested on cognition and ATP levels in a commonly used mouse model for brain aging. Over 6 months, female NMRI mice (12 months of age) were fed with a blend containing highly purified olive secoiridoids (POS) including oleuropein, hydroxytyrosol and oleurosid standardized for 50 mg oleuropein/kg diet (equivalent to 13.75 mg POS/kg b.w.) or the study diet without POS as control. Mice aged 3 months served as young controls. Behavioral tests showed deficits in cognition in aged mice. Levels of ATP and mRNA levels of NADH-reductase, cytochrome-c-oxidase, and citrate synthase were significantly reduced in the brains of aged mice indicating mitochondrial dysfunction. Moreover, gene expression of Sirt1, CREB, Gap43, and GPx-1 was significantly reduced in the brain tissue of aged mice. POS-fed mice showed improved spatial working memory. Furthermore, POS restored brain ATP levels in aged mice which were significantly increased. Our results show that a diet rich in purified olive polyphenols has positive long-term effects on cognition and energy metabolism in the brain of aged mice

A Walnut Diet in Combination with Enriched Environment Improves Cognitive Function and Affects Lipid Metabolites in Brain and Liver of Aged NMRI Mice

This in vivo study aimed to test if a diet enriched with 6% walnuts alone or in combination with physical activity supports healthy ageing by changing the oxylipin profile in brain and liver, improving motor function, cognition, and cerebral mitochondrial function. Female NMRI mice were fed a 6% walnut diet starting at an age of 12 months for 24 weeks. One group was additionally maintained in an enriched environment, one group without intervention served as control. After three months, one additional control group of young mice (3 weeks old) was introduced. Motor and cognitive functions were measured using Open Field, Y-Maze, Rotarod and Passive Avoidance tests. Lipid metabolite profiles were determined using RP-LC-ESI(-)-MS/MS in brain and liver tissues of mice. Cerebral mitochondrial function was characterized by the determination of ATP levels, mitochondrial membrane potential and mitochondrial respiration. Expression of genes involved with mito- and neurogenesis, inflammation, and synaptic plasticity were determined using qRT-PCR. A 6% walnut-enriched diet alone improved spatial memory in a Y-Maze alternation test (p < 0.05) in mice. Additional physical enrichment enhanced the significance, although the overall benefit was virtually identical. Instead, physical enrichment improved motor performance in a Rotarod experiment (p* < 0.05) which was unaffected by walnuts alone. Bioactive oxylipins like hydroxy-polyunsaturated fatty acids (OH-PUFA) derived from linoleic acid (LA) were significantly increased in brain (p** < 0.01) and liver (p*** < 0.0001) compared to control mice, while OH-PUFA of α-linolenic acid (ALA) could only be detected in the brains of mice fed with walnuts. In the brain, walnuts combined with physical activity reduced arachidonic acid (ARA)-based oxylipin levels (p < 0.05). Effects of walnut lipids were not linked to mitochondrial function, as ATP production, mitochondrial membrane potential and mitochondrial respiration were unaffected. Furthermore, common markers for synaptic plasticity and neuronal growth, key genes in the regulation of cytoprotective response to oxidative stress and neuronal growth were unaffected. Taken together, walnuts change the oxylipin profile in liver and brain, which could have beneficial effects for healthy ageing, an effect that can be further enhanced with an active lifestyle. Further studies may focus on specific nutrient lipids that potentially provide preventive effects in the brain

Sex-associated differences in mitochondrial function in human peripheral blood mononuclear cells (PBMCs) and brain

Background: Alzheimer’s disease (AD) is the most common form of dementia, and it affects more women than men. Mitochondrial dysfunction (MD) plays a key role in AD, and it is detectable at an early stage of the degenerative process in peripheral tissues, such as peripheral mononuclear blood cells (PBMCs). However, whether these changes are also reflected in cerebral energy metabolism and whether sex-specific differences in mitochondrial function occur are not clear. Therefore, we estimated the correlation between mitochondrial function in PBMCs and brain energy metabolites and examined sex-specific differences in healthy participants to elucidate these issues. Methods: The current pilot study included 9 male and 15 female healthy adults (mean age 30.8 ± 7.1 years). Respiration and activity of mitochondrial respiratory complexes were measured using a Clarke-electrode (Oxygraph-2k system), and adenosine triphosphate (ATP) levels were determined using a bioluminescence-based assay in isolated PBMCs. Citrate synthase activity as a mitochondrial marker was measured using a photometric assay. Concentrations of brain energy metabolites were quantified in the same individuals using 1H-magnetic resonance spectroscopy (MRS). Results: We detected sex-associated differences in mitochondrial function. Mitochondrial complexes I, I+II, and IV and uncoupled respiration and electron transport system (ETS) capacity in PBMCs isolated from blood samples of females were significantly (p  50%) of gray matter (GM) (p < 0.05; p < 0.01). The effect sizes indicated a strong influence of sex on these parameters. Sex-associated differences were found in PBMCs and brain, but the determined parameters were not significantly correlated. Conclusions: Our study revealed sex-associated differences in mitochondrial function in healthy participants. The underlying mechanisms must be elucidated in more detail, but our study suggests that mitochondrial function in PBMCs is a feasible surrogate marker to detect differences in mitochondrial function and energy metabolism in humans and it underscores the necessity of sex-specific approaches in therapies that target mitochondrial dysfunction

Real-world treatment patterns and healthcare resource utilization among migraine patients: A German claims database analysis

Despite migraine being one of the most common neurological diseases, affected patients are often not effectively treated. This analysis describes the burden of migraine in Germany and assesses real-world treatment patterns and healthcare resource utilization (HCRU) of preventive-treated migraine patients from the perspective of the Statutory Health Insurance. A retrospective analysis was conducted using InGef Research Database claims data from 2018-2019. Migraine patients were stratified into cohorts by acute and preventive treatment status. Patients on preventive treatment were further stratified according to type of prophylaxis received. Disease burden in preventively treated migraine patients was reported via treatment patterns, pathways, and comorbidities. HCRU was assessed through outpatient provider visits, hospitalizations, and sick leave. 160,164 adult migraine patients were identified, of which 55,378 (34.6%) were prescribed preventive treatment with conventional (n = 25,984, 46.9%), calcitonin gene-related peptide monoclonal antibody (CGRP mAb) (n = 613, 1.1%), or off-label therapies (n = 28,781, 52.0%). 936 (1.7%) patients received Botulinum Neurotoxin Type A (BoNTA). CGRP mAb-treated patients had a high rate of triptan prescriptions (2018: 95.5%; 2019: 88.9%), migraine-related hospitalizations (2018: 33.0%; 2019: 21.0%), and sick leave (2018: 26.8%; 2019: 22.5%). A high proportion of CGRP mAb- and BoNTA-treated patients was diagnosed with abdominal and pelvic pain (34.3% and 36.2%) and low back pain (34.1% and 35.3%). These patients also showed a high prevalence of depressive episodes (49.1% and 50.1%) and chronic pain disorders (37.5% and 32.9%). This study focused on descriptive analyses which do not allow for assessment of causality when comparing treatment groups. Disease burden was high in patients receiving CGRP mAbs suggesting that patients treated preventively with CGRP mAbs shortly after product launch in Germany were severely affected chronic migraine patients. The same may be true for patients receiving BoNTA who also showed an increased disease burden.</p

Hepatitis C virus: Current steps toward elimination in Germany and barriers to reaching the 2030 goal

Hepatitis C virus (HCV) affects over 70 million people globally, with an estimated 399 000 HCV‐related deaths in 2016. The World Health Organization (WHO) has set a goal to eliminate HCV by 2030. Despite the availability of direct‐acting antivirals—highly effective and well‐tolerated therapies for HCV—many patients infected with HCV in Germany have not initiated treatment, including a majority of those who are aware of their positive diagnosis. Barriers to screening, diagnosis, and treatment are major factors taking many countries off track for HCV elimination by 2030. Identifying country‐specific barriers and challenges, particularly in at‐risk populations such as people who inject drugs or men who have sex with men, has the potential to create tailored programs and strategies to increase access to screening or treatment and engage at‐risk populations. This review aims to report the current steps toward HCV elimination in Germany, the country‐specific barriers and challenges that will potentially prevent reaching the 2030 HCV elimination goal and describe good practice examples to overcome these barriers