Age-Dependent Alterations of Cognition, Mitochondrial Function, and Beta-Amyloid Deposition in a Murine Model of Alzheimer's Disease-A Longitudinal Study.

Aging is the main risk factor for sporadic Alzheimer's disease (AD), which is characterized by the cerebral deposition of ß-amyloid peptides (Aß) and cognitive decline. Mitochondrial dysfunction is also characteristic of the disease and represents a hallmark of both, aging and neurodegeneration. We longitudinally followed Aß levels, cognition, and mitochondrial function in the same cohort of Thy1-APP751SL mice representing a murine model of AD. In the course of time, changes were most prominent at an age of 13 months including the latency time in the passive avoidance test, the activity of complexes I and IV of the mitochondrial respiration chain, and expression of genes related to mitochondrial biogenesis and synaptic plasticity including Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), CAMP responsive element binding protein 1 (CREB1), and Synaptophysin 1 (SYP1). These changes occurred in parallel with massively increasing cerebral Aß levels. Other parameters were changed in younger mice including the alteration rate in the Y-maze test and the nesting score when Aß levels were not changed yet. The results are consistent in the cohort described. However, previous, non-longitudinal studies reported divergent time points for the occurrence of the parameters studied. These findings are discussed in light of the current results.

Loss and Recovery of Glutaredoxin 5 Is Inducible by Diet in a Murine Model of Diabesity and Mediated by Free Fatty Acids In Vitro.

Free fatty acids (FFA), hyperglycemia, and inflammatory cytokines are major mediators of ß-cell toxicity in type 2 diabetes mellitus, impairing mitochondrial metabolism. Glutaredoxin 5 (Glrx5) is a mitochondrial protein involved in the assembly of iron-sulfur clusters required for complexes of the respiratory chain. We have provided evidence that islet cells are deprived of Glrx5, correlating with impaired insulin secretion during diabetes in genetically obese mice. In this study, we induced diabesity in C57BL/6J mice in vivo by feeding the mice a high-fat diet (HFD) and modelled the diabetic metabolism in MIN6 cells through exposure to FFA, glucose, or inflammatory cytokines in vitro. qRT-PCR, ELISA, immunohisto-/cytochemistry, bioluminescence, and respirometry were employed to study Glrx5, insulin secretion, and mitochondrial biomarkers. The HFD induced a depletion of islet Glrx5 concomitant with an obese phenotype, elevated FFA in serum and reactive oxygen species in islets, and impaired glucose tolerance. Exposure of MIN6 cells to FFA led to a loss of Glrx5 in vitro. The FFA-induced depletion of Glrx5 coincided with significantly altered mitochondrial biomarkers. In summary, we provide evidence that Glrx5 is regulated by FFA in type 2 diabetes mellitus and is linked to mitochondrial dysfunction and blunted insulin secretion.

Effects of different standard and special diets on cognition and brain mitochondrial function in mice.

Objectives: Human nutrition plays an important role in prevention or at least slowing down the progression of age- and diet-related diseases. Thereby, mitochondrial dysfunction represents one common underlying mechanism, which is being investigated in mouse models. However, the influence of the selected diets in preclinical studies on cognition and mitochondrial function has not yet been reported cohesively.Methods: Therefore, we present the results of three different studies that addressed this question. First, we investigated the influence of two standard control chow diets and a special diet low in antioxidants over 6 months in aged NMRI mice. Additionally, a 70% high-fat (HF) chow diet as well as a western-style diet (WSD) rich in lard and fructose were examined in C57/BL6 mice. Cognitive performance, mitochondrial function and bioenergetics in the brain were investigated. Moreover, cerebral expression of genes involved in biogenesis and antioxidant defence (citrate synthase, complex I, complex IV, SOD2, Cat1, GPx-1) were quantified.Results: The results show that a modified, low antioxidant diet increased ATP levels in the brain of aged mice, while cognitive functions remained largely unaffected. A HF diet also showed significant effects on ATP levels and gene expression levels of relevant antioxidant markers, while the WSD had marginal effects on mitochondrial function and bioenergetics in the brain.Discussion: Our results indicate that standard- and special diets have an impact on cognition and mitochondrial function in the brain. Thus, appropriate caution is warranted when selecting a suitable diet for preclinical studies in mice.

Impact of Silibinin A on Bioenergetics in PC12APPsw Cells and Mitochondrial Membrane Properties in Murine Brain Mitochondria.

Age-related multifactorial diseases, such as the neurodegenerative Alzheimer's disease (AD), still remain a challenge to today's society. One mechanism associated with AD and aging in general is mitochondrial dysfunction (MD). Increasing MD is suggested to trigger other pathological processes commonly associated with neurodegenerative diseases. Silibinin A (SIL) is the main bioactive compound of the Silymarin extract from the Mediterranean plant Silybum marianum (L.) (GAERTN/Compositae). It is readily available as a herbal drug and well established in the treatment of liver diseases as a potent radical scavenger reducing lipid peroxidation and stabilize membrane properties. Recent data suggest that SIL might also act on neurological changes related to MD. PC12APPsw cells produce low levels of human Aß and thus act as a cellular model of early AD showing changed mitochondrial function. We investigated whether SIL could affect mitochondrial function by measuring ATP, MMP, as well as respiration, mitochondrial mass, cellular ROS and lactate/pyruvate concentrations. Furthermore, we investigated its effects on the mitochondrial membrane parameters of swelling and fluidity in mitochondria isolated from the brains of mice. In PC12APPsw cells, SIL exhibits strong protective effects by rescuing MMP and ATP levels from SNP-induced mitochondrial damage and improving basal ATP levels. However, SIL did not affect mitochondrial respiration and mitochondrial content. SIL significantly reduced cellular ROS and pyruvate concentrations. Incubation of murine brain mitochondria with SIL significantly reduces Ca2+ induced swelling and improves membrane fluidity. Although OXPHOS activity was unaffected at this early stage of a developing mitochondrial dysfunction, SIL showed protective effects on MMP, ATP- after SNP-insult and ROS-levels in APPsw-transfected PC12 cells. Results from experiments with isolated mitochondria imply that positive effects possibly result from an interaction of SIL with mitochondrial membranes and/or its antioxidant activity. Thus, SIL might be a promising compound to improve cellular health when changes to mitochondrial function occur.

Hesperetin Nanocrystals Improve Mitochondrial Function in a Cell Model of Early Alzheimer Disease.

Mitochondrial dysfunction represents a hallmark of both brain aging and age-related neurodegenerative disorders including Alzheimer disease (AD). AD-related mitochondrial dysfunction is characterized by an impaired electron transport chain (ETC), subsequent decreased adenosine triphoshpate (ATP) levels, and elevated generation of reactive oxygen species (ROS). The bioactive citrus flavanone hesperetin (Hst) is known to modulate inflammatory response, to function as an antioxidant, and to provide neuroprotective properties. The efficacy in improving mitochondrial dysfunction of Hst nanocrystals (HstN) with increased bioavailability has not yet been investigated. Human SH-SY5Y cells harboring neuronal amyloid precursor protein (APP695) acted as a model for the initial phase of AD. MOCK-transfected cells served as controls. The energetic metabolite ATP was determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was assessed by high-resolution respirometry using a Clarke electrode. Expression levels of mitochondrial respiratory chain complex genes were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The levels of amyloid ß-protein (Aß1-40) were measured using homogeneous time-resolved fluorescence (HTRF). ROS levels, peroxidase activity, and cytochrome c activity were determined using a fluorescence assay. Compared to pure Hst dissolved in ethanol (HstP), SH-SY5Y-APP695 cells incubated with HstN resulted in significantly reduced mitochondrial dysfunction: ATP levels and respiratory chain complex activity significantly increased. Gene expression levels of RCC I, IV, and V were significantly upregulated. In comparison, the effects of HstN on SY5Y-MOCK control cells were relatively small. Pure Hst dissolved in ethanol (HstP) had almost no effect on both cell lines. Neither HstN nor HstP led to significant changes in Aß1-40 levels. HstN and HstP were both shown to lower peroxidase activity significantly. Furthermore, HstN significantly reduced cytochrome c activity, whereas HstP had a significant effect on reducing ROS in SH-SY5Y-APP695 cells. Thus, it seems that the mechanisms involved may not be linked to altered Aß production. Nanoflavonoids such as HstN have the potential to prevent mitochondria against dysfunction. Compared to its pure form, HstN showed a greater effect in combatting mitochondrial dysfunction. Further studies should evaluate whether HstN protects against age-related mitochondrial dysfunction and thus may contribute to late-onset AD.

Investigating hesperetin nanocrystals with tailor-made sizes for the prevention and treatment of Alzheimer's disease.

Poor aqueous solubility of drug substances is associated with poor bioavailability and thus hampers the effective use of many potent active pharmaceutical ingredients. Various strategies to overcome poor solubility are available, whereby drug nanocrystals represent one of the most powerful formulation strategies to enhance the kinetic solubility and dissolution rate of poorly soluble drugs. Nanocrystals are simply obtained by milling large-sized drug powders to sizes < 1 µm. The so obtained nanocrystals possess an increased dissolution rate and kinetic solubility when compared with larger-sized bulk material. The aim of this study was to produce differently sized hesperetin nanocrystals and to investigate the influence of nanocrystal size on the bioefficacy of the natural antioxidant hesperetin in two cell culture models for the prevention and treatment of Alzheimer's disease. Results showed that the testing of poorly soluble compounds is challenging and requires incredibly careful characterization. Reasons for this are possible changes of the formulations in cell culture media which can occur due to various reasons. If the changes are not considered, results obtained can be misleading and even lead to a false interpretation of the results obtained. Besides, results demonstrate the increase in dissolution rate with decreasing particle size that is especially pronounced with particle sizes < 200 nm. Data also provide clear evidence that smaller nanocrystals with higher kinetic solubility possess higher antioxidant capacity. This results in lower amounts of free radicals in the cell culture models, suggesting that hesperetin nanocrystals, that improve the poor aqueous solubility of hesperetin, are promising for the prevention and treatment of Alzheimer's disease.

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, ß-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.

Purified oleocanthal and ligstroside protect against mitochondrial dysfunction in models of early Alzheimer's disease and brain ageing.

As components of the Mediterranean diet (MedDiet) olive polyphenols may play a crucial role for the prevention of Alzheimer's disease (AD). Since mitochondrial dysfunction is involved in both, brain ageing and early AD, effects of 10 different purified phenolic secoiridoids (hydroxytyrosol, tyrosol, oleacein, oleuroside, oleuroside aglycon, oleuropein, oleocanthal, ligstroside, ligstroside aglycone and ligustaloside B) and two metabolites (the plant metabolite elenolic acid and the mammalian metabolite homovanillic acid) were tested in very low doses on mitochondrial function in SH-SY5Y-APP695 cells - a cellular model of early AD. All tested secoiridoids significantly increased basal adenosine triphosphate (ATP) levels in SY5Y-APP695 cells. Oleacein, oleuroside, oleocanthal and ligstroside showed the highest effect on ATP levels and were additionally tested on mitochondrial respiration. Only oleocanthal and ligstroside were able to enhance the capacity of respiratory chain complexes. To investigate their underlying molecular mechanisms, the expression of genes associated with mitochondrial biogenesis, respiration and antioxidative capacity (PGC-1α, SIRT1, CREB1, NRF1, TFAM, complex I, IV and V, GPx1, SOD2, CAT) were determined using qRT-PCR. Exclusively ligstroside increased mRNA expression of SIRT1, CREB1, complex I, and GPx1. Furthermore, oleocanthal but not ligstroside decreased Aß 1-40 levels in SH-SY5Y-APP695 cells. To investigate the in vivo effects of purified secoiridoids, the two most promising compounds (oleocanthal and ligstroside) were tested in a mouse model of ageing. Female NMRI mice, aged 12 months, received a diet supplemented with 50 mg/kg oleocanthal or ligstroside for 6 months (equivalent to 6.25 mg/kg b.w.). Young (3 months) and aged (18 months) mice served as controls. Ligstroside fed mice showed improved spatial working memory. Furthermore, ligstroside restored brain ATP levels in aged mice and led to a significant life extension compared to aged control animals. Our findings indicate that purified ligstroside has outstanding performance on mitochondrial bioenergetics in models of early AD and brain ageing by mechanisms that may not interfere with Aß production. Additionally, ligstroside expanded the lifespan in aged mice and enhanced cognitive function.

Differential Effects of Silibinin A on Mitochondrial Function in Neuronal PC12 and HepG2 Liver Cells.

The Mediterranean plant Silybum marianum L., commonly known as milk thistle, has been used for centuries to treat liver disorders. The flavonolignan silibinin represents a natural antioxidant and the main bioactive ingredient of silymarin (silybin), a standard extract of its seeds. Mitochondrial dysfunction and the associated generation of reactive oxygen/nitrogen species (ROS/RNS) are involved in the development of chronic liver and age-related neurodegenerative diseases. Silibinin A (SIL A) is one of two diastereomers found in silymarin and was used to evaluate the effects of silymarin on mitochondrial parameters including mitochondrial membrane potential and ATP production with and without sodium nitroprusside- (SNP-) induced nitrosative stress, oxidative phosphorylation, and citrate synthase activity in HepG2 and PC12 cells. Both cell lines were influenced by SIL A, but at different concentrations. SIL A significantly weakened nitrosative stress in both cell lines. Low concentrations not only maintained protective properties but also increased basal mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) levels. However, these effects could not be associated with oxidative phosphorylation. On the other side, high concentrations of SIL A significantly decreased MMP and ATP levels. Although SIL A did not provide a general improvement of the mitochondrial function, our findings show that SIL A protects against SNP-induced nitrosative stress at the level of mitochondria making it potentially beneficial against neurological disorders.

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.

Peptidomimetics That Inhibit and Partially Reverse the Aggregation of Aß1-42.

The peptide sequence KLVFF resembles the hydrophobic core of the Aß peptide known to form amyloid plaques in Alzheimer's disease. Starting from its retro-inverso peptide, we have synthesized three generations of peptidomimetics. Step by step natural amino acids have been replaced by aromatic building blocks accessible from the Pd-catalyzed Catellani reaction. The final compound 18 is stable against proteolytic decay and largely prevents the aggregation of Aß1-42 over extended periods of time. The activity of the new inhibitors was tested first by fluorescence correlation spectroscopy. For closer examination of compound 18, additional techniques were also applied: laser-induced liquid bead ion desorption mass spectrometry, confocal laser scanning microscopy, thioflavin T fluorescence, and gel electrophoresis. Compound 18 not only retards the aggregation of chemically synthesized Aß but also can partially dissolve the oligomeric structures. Thioflavin binding mature fibrils, however, seem to resist the inhibitor.

Rice bran extract compensates mitochondrial dysfunction in a cellular model of early Alzheimer's disease.

Mitochondrial dysfunction plays an important role in brain aging and has emerged to be an early event in Alzheimer's disease (AD), contributing to neurodegeneration and the loss of physical abilities seen in patients suffering from this disease. We examined mitochondrial dysfunction in a cell culture model of AD (PC12APPsw cells) releasing very low amyloid-ß (Aß40) levels and thus mimicking early AD stages. Our data show that these cells have impaired energy metabolism, low ATP levels, and decreased endogenous mitochondrial respiration. Furthermore, protein levels of PGC1α as well as of Mitofusin 1 were decreased. PC12APPsw cells also showed increased mitochondrial content, probably due to an attempt to compensate the impaired mitochondrial function. Recent data showed that stabilized rice bran extract (RBE) protects from mitochondrial dysfunction in vivo Pharmacol Res. (2013) 76C, 17-27. To assess the effect of RBE on mitochondrial function, we treated PC12APPsw cells for 24 h with RBE. Key components of RBE are oryzanols, tocopherols, and tocotrienols, all substances that have been found to exert beneficial effects on mitochondrial function. RBE incubation elevated ATP production and respiratory rates as well as PGC1α protein levels in PC12APPsw cells, thus improving the impaired mitochondrial function assessed in our cell culture AD model. Therefore, RBE represents to be a promising nutraceutical for the prevention of AD.

Unsymmetrically substituted 9,10-dihydro-9,10-diboraanthracenes as versatile building blocks for boron-doped π-conjugated systems.

The targeted hydrolysis of the 9,10-dihydro-9,10-diboraanthracene adduct (Me(2)S)HB(C(6)H(4))(2)BH(SMe(2)) (1) with 0.5 equiv of H(2)O leads to formation of the borinic acid anhydride [(Me(2)S)HB(C(6)H(4))(2)B](2)O (2) and thereby provides access to the field of unsymmetrically substituted 9,10-dihydro-9,10-diboraanthracenes. Compound 2 reacts with tBuC≡CH to give the corresponding vinyl derivative in an essentially quantitative conversion. Subsequent cleavage of the B-O-B bridge by LiAlH(4) with formation of hydridoborate functionalities is possible but is accompanied by partial B-C(vinyl) bond degradation. This situation changes when the related mesityl derivative [MesB(C(6)H(4))(2)B](2)O (7) is employed, which can be synthesized from BrB(C(6)H(4))(2)BBr (6) by treatment with 1 equiv of MesMgBr and subsequent hydrolysis. The reaction of 7 with LiAlH(4) in tetrahydrofuran (THF) furnishes Li[MesB(C(6)H(4))(2)BH(2)] (8); hydride elimination with Me(3)SiCl leads to formation of the THF adduct MesB(C(6)H(4))(2)BH(THF) (9·THF). Alternatively, 7 can be transformed into the bromoborane MesB(C(6)H(4))(2)BBr (10) by treatment with BBr(3). A Br/H-exchange reaction between 10 and Et(3)SiH yields the donor-free borane MesB(C(6)H(4))(2)BH (9), which forms B-H-B bridged dimers (9)(2) in the solid state. The vinyl borane MesB(C(6)H(4))(2)BC(H)=C(H)Mes (14) is accessible from MesC≡CH and either 9·THF or 9. Compared with the related compound Mes(2)BC(H)=C(H)Mes, the electronic absorption and emission spectra of 14 reveal bathochromic shifts of Δλ(abs)=17 nm and Δλ(em)=74 nm, which can be attributed to the rigid, fully delocalized π framework of the [MesB(C(6)H(4))(2)B] chromophore.