Time-dependent melatonin secretion is associated with mitochondrial function in peripheral blood mononuclear cells (PBMC) of male volunteers.

Melatonin blood levels vary depending on the circadian rhythm. It also stimulates antioxidant enzymes and has positive effects on mitochondrial function. The current study investigated the effects of endogenously released melatonin on adenosine triphosphate (ATP) levels and mitochondrial respiration in peripheral blood mononuclear cells (PBMC). The current study included 20 healthy adults (mean age 25,7 ± 3.4 years). Blood was collected at 8 a.m. and 2 p.m. The activity of mitochondrial respiratory complexes and ATP levels were determined in isolated PBMC. Melatonin concentrations were determined in serum samples. Sleep behavior was assessed. In PBMCs isolated from blood samples of males, respiration of mitochondrial complex IV and ATP levels as well as serum melatonin concentration were significantly lower at 2 a.m. compared to the samples collected at 8 p.m. Mitochondrial parameters and melatonin blood levels were equal at both time points in the samples isolated from females. Although our results show that the amount of melatonin secreted may have had an influence, further investigation is needed to determine the importance of melatonin and other factors in measuring the mitochondrial function of PBMC.

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 < 0.05; p < 0.01) higher compared to males. ATP levels in the PBMCs of female participants were approximately 10% higher compared to males. Citrate synthase (CS) activity, a marker of mitochondrial content, was significantly (p < 0.05) higher in females compared to males. Sex-associated differences were also found for brain metabolites. The N-acetylaspartate (NAA) concentration was significantly higher in female participants compared to males in targeted regions. This difference was observed in white matter (WM) and an area with a high percentage (> 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.

¹H- and ¹³C-NMR spectroscopy of Thy-1-APPSL mice brain extracts indicates metabolic changes in Alzheimer's disease.

Biochemical alterations underlying the symptoms and pathomechanisms of Alzheimer's disease (AD) are not fully understood. However, alterations of glucose metabolism and mitochondrial dysfunction certainly play an important role. (1)H- and (13)C-NMR spectroscopy exhibits promising results in providing information about those alterations in vivo in patients and animals, especially regarding the mitochondrial tricarboxylic acid (TCA) cycle. Accordingly, transgenic mice expressing mutant human amyloid precursor protein (APP(SL))-serving as a model of neuropathological changes in AD-were examined with in vitro 1D (1)H- and 2D (1)H-(13)C-HSQC-NMR spectroscopy after oral administration of 1-(13)C-glucose and acquisition of brain material after 30 min. Perchloric acid extracts were measured using a 500 MHz spectrometer, providing more detailed information compared to in vivo spectra achievable nowadays. Area under curve (AUC) data of metabolite peaks were obtained and normalized in relation to the creatine signal, serving as internal reference. Besides confirming well-known metabolic alterations in AD like decreased N-acetylaspartate (NAA)/Creatine (Cr) ratio, new findings such as a decrease in phosphorylcholine (PC) are presented. Glutamate (Glu) and glutamine (Gln) concentrations were decreased while γ-aminobutyric acid (GABA) was elevated in Thy1-APP(SL) mice. (13)C-NMR spectroscopy revealed a shift in the Glx-2/Glx-4-ratio-where Glx represents a combined Glu/Gln-signal-towards Glx-2 in AD. These findings correlated well with the NAA/Cr-ratio. The Gln-4/Glu-4-ratio is altered in favor of Glu. Our findings suggest that glutamine synthetase (GS), which is predominantly present in glial cells may be impaired in the brain of Thy1-APP(SL) transgenic mice. Since GS is an ATP-dependent enzyme, mitochondrial dysfunction might contribute to reduced activity, which might also account for the increased metabolism of glutamate via the GABA shunt, a metabolic pathway to bypass intra-mitochondrial α-ketoglutarate-dehydrogenase, resulting in elevated GABA levels.

Pharmacokinetic properties of MH84, a γ-secretase modulator with PPARγ agonistic activity.

Alzheimer's disease (AD) is the most common cause of dementia. Since no causative treatment is available, new therapeutic options are utmost needed. Several pirinixic acid derivatives, including MH84 (2-((4,6-bis(4-(trifluoromethyl)phenethoxy)pyrimidin-2-yl)thio)hexanoic acid), have shown promising in vitro results as γ-secretase modulators as well as PPARγ activators as potential pharmacological compounds against AD. Using a newly developed and validated sensitive LC-MS (APCI-qTOF mass analyzer) method, the pharmacokinetic and long-term accumulating properties as well as the blood-brain-barrier permeability of MH84 were evaluated in a preclinical animal study. MH84 was administered to mice by oral gavage with a dose of 12 mg/kg. Nine time points from 0.5 to 48 h with 6 animals per point were investigated. Additionally 6 animals were fed daily, for 21 days with an identical dose to determine possible long-term accumulation in plasma and brain tissue. The sample preparation was performed by a liquid-liquid extraction on Extrelut(®) columns whereas the LC separation was operated on a MulthoHigh 100 RP 18-5 µ column (125 × 4 mm) using an isocratic mobile phase of formic acid (0.1% (v/v))-methanol mixture (11:89 (v/v)) at a flow rate of 1 ml/min. The validation confirmed the new LC-MS method to be precise, accurate and reliable. After oral application, Cmax and Tmax of unmetabolized MH84 was determined to be 10.90 µg/ml and 3h in plasma. In brain tissue a constant level of 300 to maximum 320.64 ng/g was found after 1.5-6h. Daily gavage for 21 days did not lead to a long-term drug accumulation in the brain. The efficacy of the obtained MH84 levels needs to be investigated in further preclinical pharmacodynamic animal studies.

Curcumin Micelles Improve Mitochondrial Function in a Mouse Model of Alzheimer's Disease.

Preventive strategies for late-onset Alzheimer's disease (AD) should start early at a prodromal stage. Mitochondrial dysfunction has been found to play an important role in the initiation of both aging and the pathogenesis of Alzheimer's disease. Curcumin, a widely used spice and food-coloring agent, is a polyphenol derived from the rhizome of Curcuma longa. It is known to have anti-oxidant, anti-inflammatory, and anti-protein-aggregate activities which are usually considered beneficial for mitochondrial function. We assessed brain mitochondrial function and concentrations of soluble Aß40 in a mouse model of AD (Thy1-APP751SL transgenic mice) after 3-week administration of curcumin micelles. Curcumin micelles are a newly developed formulation that account for increased curcumin bioavailability. Curcumin treatment had positive effects on mitochondrial membrane potential and respiratory control ratio. Additionally, it decreased levels of soluble Aß40 in brains of Thy1-APPSL transgenic mice. Hence, curcumin micelles are a promising neutraceutical for the prevention of AD.

Neuroprotection by bilobalide in ischemia: improvement of mitochondrial function.

Bilobalide, an active constituent of Ginkgo biloba, is known to have neuroprotective properties, but its mode of action remains unclear. In this study, bilobalide significantly reduced brain damage in mice (indicated by TTC staining) when given before transient middle cerebral artery occlusion (tMCAO). As measured by microdialysis in the ischemic striatum, local perfusion with bilobalide (10 microM) reduced ischemia-induced glutamate release by 70% while glucose levels were not affected. Mitochondria isolated from ischemic brain showed a decrease of respiration compared to non-ischemic controls. Treatment with bilobalide (10 mg/kg) before tMCAO improved respiratory capacity of complex I significantly when measured ex vivo. In addition, mitochondrial swelling induced ex vivo by calcium was used to estimate opening of the mitochondrial permeability transition pore. In this assay, the changes induced by tMCAO were completely reversed when mice had received pretreatment with bilobalide. We conclude that neuroprotection by bilobalide involves a mechanism in which the drug reverses ischemia-induced changes in mitochondria, leading to a reduction of glutamate release.

Simvastatin alters membrane cholesterol distribution and beta-amyloid levels in brains of female APP751SL mice.

Statins (HMG-CoA reductase or CSE-inhibitors) strongly reduce the cellular amyloid-beta protein production by modulating the processing of amyloid precursor protein (APP) in vitro. Several in vivo studies have addressed this important issue in transgenic mouse models with inconsistent results. Recently, we showed that simvastatin alters cholesterol distribution in synaptosomal membranes (SPM) in vivo. In the present study, we tested whether these changes in cholesterol membrane distribution affect APP-processing in vivo. Female APP751SL mice were force-fed with simvastatin (50 mg/kg b.wt.) by oral gavage over a time period of 3 weeks. Our data show that chronic simvastatin treatment decreased cholesterol levels in the brain and affected cholesterol distribution within SPM. Simvastatin significantly increased the levels of insoluble Abeta1-40 and Abeta1-42 but reduced levels of soluble Abeta1-40 in the brain. The reduction of soluble Abeta1-40 levels in the brain was associated with an increase of plasma-levels of AP31.40 in simvastatin-treated animals that may indicate enhanced Abeta1-40-clearance from the brain. Although the observed alteration in transbilayer cholesterol is likely to be involved in changes of APP processing by alpha-, beta- and gamma-secretase, we cannot exclude other potential mechanisms of statins such as lipid and non-lipid related, pleiotropic effects. Our data were evaluated in reference to published studies and a possible gender effect was identified.

Isoquercitrin provides better bioavailability than quercetin: comparison of quercetin metabolites in body tissue and brain sections after six days administration of isoquercitrin and quercetin.

In the present study, over a period of 8 days 12 mg/kg/d quercetin aglycone and 18 mg/kg/d isoquercitrin were orally given to rats, respectively. Four hours after administration, plasma samples were taken as well as tissue samples of liver, lung, heart, kidney and the brain sections hippocampus, cerebellum, striatum, cortex and the remaining brain. A HPLC-FD method with in-line post-column complexation was employed to quantify the quercetin metabolites (QM) in plasma and tissues. Compared to the quercetin gavage the isoquercitrin gavage consistently produced higher levels of QM in tissues (double to five-fold) as well as in plasma (double to three-fold). In body tissues, the highest amounts of QM were observed in the lung. In brain tissue, the highest levels of QM were found in the cerebellum, while the striatum contained the lowest levels of QM. In conclusion, this study clearly demonstrates that orally given isoquercitrin leads to higher levels in plasma and in all investigated tissue than quercetin aglycone.

Alterations of brain membranes in schizophrenia: impact of phospholipase A(2).

Physiological enzymatic cleavage of membrane phospholipids by phospholipase A2 (PLA2) results in normal levels of phosphomonoester and phosphodiester, by which a normal dopamine neurotransmission is maintained. Data from postmortem tissue and in vivo imaging studies suggest that increased activity of intracellular calcium-independent PLA2 (iPLA2) in the brain of schizophrenic patients might accelerate the breakdown of membrane phospholipids and alter the properties of neuronal membranes, which in turn contributes to a hypodopaminergy. Alterations in PLA2 activity are probably genetically determined and represent a possible pharmacological target for Schizophrenia.

Increased brain membrane fluidity in schizophrenia.

Recent findings showing significant correlations between phospholipase A2 (PLA2) activity and structural changes in schizophrenic brains contribute to the membrane hypothesis of schizophrenia, which was hampered because a clean functional link between elevated PLA2 activity and brain structure was missing (Neuroimage, 2010; 52: 1314-1327). We measured membrane fluidity parameters and found that brain membranes isolated from the prefrontal cortex of schizophrenic patients showed significantly increased flexibility of fatty acid chains. Our findings support a possible link between elevated PLA2 activity in cortical areas of schizophrenic patients and subsequent alterations of the biophysical parameters of neuronal membranes leading to structural changes in these areas.

Lipid membranes and beta-amyloid: a harmful connection.

Gradual changes in steady-state levels of beta amyloid peptides (Abeta) in the brain are considered as initial step in the amyloid cascade hypothesis of Alzheimer's disease (AD). Abeta is a product of the secretase cleavage of the amyloid precursor protein and there is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Abeta disturbs membrane properties of artificial and isolated biological membranes and of plasma membranes in living cells. Abeta induced changes in membrane fluidity could be explained by physico-chemical interactions of the peptide with membrane components such as cholesterol, phospholipids and gangliosides. Thus, cell membranes may be the location where the neurotoxic cascade of Abeta is initiated. Perturbation of membranes, binding to lipids and alteration of cellular calcium signaling by Abeta have been reported by several studies and these topics are examined in this review.

Plant foods and brain aging: a critical appraisal.

In the 21st century, human aging will be one of the biggest challenges for most societies throughout the world. The decline in human fitness is a typical hallmark of the aging process. Aside from the cardiovascular system, the brain most often suffers significantly from the life-long impact of stressors, such as reactive oxygen and nitrogen species. Oxytosis, i.e. oxidative stress-induced cell death, has been identified to play a major role in the development and onset of chronic diseases. Foods, especially of plant origin, are rich in antioxidants and numerous in vivo data suggest that a diet rich in fruits and vegetables supports the maintenance of animal and human health. These beneficial effects also extend to the central nervous system, which, due to the presence of the blood-brain barrier, tightly controls the influx of metabolites and nutrients. In earlier studies the impact of antioxidant vitamins, such as alpha-tocopherol and ascorbic acid, on brain health has been of interest. Recently, the focus moved to assessing the potential of unsaturated fatty acids and secondary plant metabolites, particularly of polyphenols, to act as neuroprotectants. Considerable experimental evidence suggests that polyphenols and other plant-derived bioactivities affect animal and human brain function not only by directly lowering oxidative stress load but also by modulating various signal transduction pathways.

Flavonoids and the aging brain.

Like in all other organs, the functional capacity of the human brain deteriorates over time. Pathological events such as oxidative stress, due to the elevated release of free radicals and reactive oxygen or nitrogen species, the subsequently enhanced oxidative modification of lipids, protein, and nucleic acids, and the modulation of apoptotic signaling pathways contribute to loss of brain function. The identification of neuroprotective food components is one strategy to facilitate healthy brain aging. Flavonoids were shown to activate key enzymes in mitochondrial respiration and to protect neuronal cells by acting as antioxidants, thus breaking the vicious cycle of oxidative stress and tissue damage. Furthermore, recent data indicate a favorable effect of flavonoids on neuro-inflammatory events. Whereas most of these effects have been shown in vitro, limited data in vivo are available, suggesting a rather low penetration of flavonoids into the brain. Nevertheless, several reports support the concept that flavonoid intake inhibits certain biochemical processes of brain aging, and might thus prevent to some extent the decline of cognitive functions with aging as well as the development or the course of neurodegenerative diseases. However, more data are needed to assess the true impact of flavonoids on brain aging.

Antioxidant properties of Mediterranean food plant extracts: geographical differences.

Locally grown, wild food plants seasonally contribute a considerable portion of the daily diet in certain Mediterranean areas and it has been suggested that the beneficial effects of the Mediterranean diet on human health partly originate from the antioxidant effect of flavonoid-rich food plants. The nutrient content of most wild plants is higher than that of cultivated ones and may vary depending on the prevailing environmental conditions. Accordingly, three local Mediterranean plant foods (i.e. Cichorium intybus, Sonchus oleraceus, Papaver rhoeas) were collected in Greece (Crete), southern Italy, and southern Spain in order to assess possible differences in their in vitro antioxidant potential. The biological assays revealed diverse intra-plant specific antioxidant effects for the tested extracts ranging from no activity to almost complete protection. Furthermore, substantial differences in the polyphenol content were found for the nutritionally used part of the same plant originating from different locations. However, no clear correlations between the polyphenol content and the extracts' antioxidant activities were found. Taken together, the data suggest that certain local Mediterranean plant foods possess promising antioxidant activity and that the observed biological effects are possibly influenced by the geographically-dependent environmental conditions prevailing during plant growth.

Murine synaptosomal lipid raft protein and lipid composition are altered by expression of human apoE 3 and 4 and by increasing age.

Apolipoprotein E (apoE) 4 and aging are risk factors for Alzheimer's disease (AD). Mice expressing human apoE4 and aged wild-type mice show a similarity in the transbilayer distribution of cholesterol in synaptic plasma membranes (SPMs) but differ markedly compared with apoE3 mice and young mice. The largest changes in cholesterol distribution were observed in the SPM exofacial leaflet where there was a doubling of cholesterol. Lipid rafts are thought to be associated with the exofacial leaflet, and we proposed that lipid raft protein and lipid composition would be associated with apoE genotype and age. Lipid rafts were isolated from synaptosomes of different age groups (2, 12, 24 months) of mice expressing human apoE3 and apoE4. Lipid raft markers, alkaline phosphatase (ALP), flotillin-1, cholesterol and sphingomyelin (SM) were examined. Lipid rafts of young apoE4 mice were more similar to older mice as compared with young apoE3 mice in reductions in alkaline phosphatase activity and flotillin-1 abundance. Lipid raft cholesterol and sphingomyelin levels were not significantly different between the young apoE3 and apoE4 mice but cholesterol levels of lipid rafts did increase with age in both genotypes. Results of the present study demonstrate that the two risk factors for Alzheimer's disease, apoE4 genotype and increasing age have similar effects on brain lipid raft protein markers and these findings support the notion that the transbilayer distribution of cholesterol is associated with lipid raft function.

Statins: drugs for Alzheimer's disease?

Evidences from cell culture experiments and animal studies suggest a strong link between cholesterol and Alzheimer's disease (AD). This relationship is supported by retrospective epidemiological studies demonstrating that statin treatment reduced the prevalence of AD in patients suffering from hypercholesterolaemia. The alternative processing of the amyloid-precursor protein (APP) in the brain of AD patients leads to the production of the neurotoxic amyloid-beta protein (Abeta), a causative factor for AD pathology. In vitro, this mechanism is modulated by alterations in cellular cholesterol levels. Moreover, lowering cholesterol in animal experiments reduced the production of Abeta in most but not all studies. These findings led to prospective clinical trials of cholesterol-lowering statins in AD patients, even if many studies do not support elevated cholesterol levels in serum and brain as a risk factor for Alzheimer's disease. Most of these studies were negative. Thus, up to date there is insufficient evidence to suggest the use of statins for treatment in patients with AD.

Brain cholesterol, statins and Alzheimer's Disease.

Growing evidence suggests that cellular cholesterol homeostasis is causally involved in different steps leading to pathological events in the brain of Alzheimer's Disease (AD) patients. It was previously demonstrated that the processing of the amyloid beta-peptide precursor protein (APP) is modulated by pronounced alterations in cellular cholesterol levels using statins or cholesterol extracting agents. However, a cholesterol-rich diet was found to enhance amyloid beta-peptide (Abeta) burden in the brain of transgenic mice without clearly affecting total brain cholesterol levels. Recent retrospective epidemiological studies have reported that the use of statins potentially suppresses the development of AD. Although some HMG-CoA reductase inhibitors seem to influence the central cholesterol pool in vivo, the above epidemiological findings are probably not linked to statin-induced changes in brain membrane cholesterol levels per se since not all statins active in preventing AD enter the central nervous system (CNS). Recently, we reported that different statins, regardless of their brain availability, induce alterations in cellular cholesterol distribution in the brain. Such pleiotropic, cholesterol-synthesis independent statin effects might be indirect and are possibly mediated at the blood-brain barrier (BBB) via nitric oxide (NO) or apolipoprotein E (ApoE).

Brain-membrane cholesterol in Alzheimer's disease.

Cholesterol represents an important determinant of the physical state of biological membranes. Growing evidence indicate that changes in brain cholesterol and variations in neuronal membrane structure are involved in the development of Alzheimer's disease (AD). Cholesterol modulates the cleavage of the amyloid precursor protein and thus affect cellular production of beta-amyloid peptides (Ab). On the other hand, cholesterol seems to be protective against the neurotoxic and membrane disordering properties of Ab. Present review summarizes reports focusing on brain membrane changes in AD and the effects of Ab on these structures. Since it has been shown that these Ab effects are cholesterol dependent, recent findings are presented indicating that the modulation of membrane cholesterol refers to different cholesterol pools within the membranes. Further, consequences thereof for possible pharmacological strategies are discussed.

Effects of aging and beta-amyloid on the properties of brain synaptic and mitochondrial membranes.

The effects of aging and of different amyloid beta-peptides (A beta) on the properties of purified synaptosomal plasma and mitochondrial membranes were studied using different fluorescent dyes. Aging led to opposite membrane alterations in both mouse brain fractions. Cholesterol levels were significantly enhanced in synaptosomal plasma membranes (SPM) from aged mice only. Flexibility of membrane fatty acids was decreased in synaptosomal plasma and mitochondrial membranes, mobility of pyrene was enhanced, but in SPM only. With regard to acyl chain flexibility in aged brain membranes, both membrane preparations were less sensitive to A beta. By contrast, effects of A beta on the mobility of pyrene were not reduced for aged synaptic membranes, but even seemed to be enhanced in the case of aged mitochondrial membranes. The data presented significantly enhance our understanding of the mechanism of the A beta's disordering effects on synaptosomal membranes that are also detectable for mitochondrial membranes and show for the first time that A beta effects are modified by brain aging. This is of special interest since membrane alterations and in particular modifications of membrane cholesterol were recently linked to Alzheimer's Disease.

Membrane-disordering effects of beta-amyloid peptides.

beta-Amyloid (Abeta) protein is the major constituent of senile plaques and cerebrovascular deposits characteristic of Alzheimer's disease (AD). The causal relationship between Abeta and AD-specific lesions like neurodegeneration and atrophy is still not known. The present article summarizes our studies indicating that rather low concentrations of Abeta significantly alter the fluidity of cell membranes and subcellular fractions from different tissues and different species including humans, as a possible initial step of its biological effects. Using different fluorescent probes our data show clearly that Abeta peptides specifically disturb the acyl-chain layer of cell membranes in a very distinct fashion. By contrast, membrane properties at the level of the polar heads of the phospholipid bilayer at the interface with membrane proteins are much less affected.