Olesoxime improves cerebral mitochondrial dysfunction and enhances Aß levels in preclinical models of Alzheimer's disease.

BACKGROUND: Approved drugs for Alzheimer's disease (AD) only have a symptomatic effects and do not intervene causally in the course of the disease. Olesoxime (TRO19622) has been tested in AD disease models characterized by improved amyloid precursor protein processing (AßPP) and mitochondrial dysfunction. METHODS: Three months old Thy-1-AßPPSL (tg) and wild type mice (wt) received TRO19622 (100 mg/kg b.w.) in supplemented food pellets for 15 weeks (tg TRO19622). Mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) levels were determined in dissociated brain cells (DBC). Respiration was analyzed in mitochondria isolated from brain tissue. Citrate synthase (CS) activity and beta-amyloid peptide (Aß1-40) levels were determined in brain tissue. Malondialdehyde (MDA) levels were determined as an indicator for lipid peroxidation. DBC and brain homogenates were additionally stressed with Rotenone and FeCl2, respectively. Mitochondrial respiration and Aß1-40 levels were also determined in HEK-AßPPsw-cells. RESULTS: Treatment of mice did not affect the body weight. TRO19622 was absorbed after oral treatment (plasma levels: 6,2 µg/ml). Mitochondrial respiration was significantly reduced in brains of tg-mice. Subsequently, DBC isolated from brains of tg-mice showed significantly lower MMP but not ATP levels. TRO19622 increased the activity of respiratory chain complexes and reversed complex IV (CIV) activity and MMP. Moreover, DBC isolated from brains of tg TRO19622 mice were protected from Rotenone induced inhibition of complex I activity. TRO19622 also increased the respiratory activity in HEKsw-cells. MDA basal levels were significantly higher in brain homogenates isolated from tg-mice. TRO19622 treatment had no effects on lipid peroxidation. TRO19622 increased cholesterol levels but did not change membrane fluidity of synaptosomal plasma and mitochondrial membranes isolated from brain of mice. TRO19622 significantly increased levels of Aß1-40 in both, in brains of tg TRO19622 mice and in HEKsw cells. CONCLUSIONS: TRO19622 improves mitochondrial dysfunction but enhances Aß levels in disease models of AD. Further studies must evaluate whether TRO19622 offers benefits at the mitochondrial level despite the increased formation of Aß, which could be harmful.

Mitochondrial Pharmacology of Dimebon (Latrepirdine) Calls for a New Look at its Possible Therapeutic Potential in Alzheimer's Disease.

Dimebon (latrepirdine), an old antihistaminic drug, showed divergent results in two large clinical trials in Alzheimer disease (AD), which according to our review might be related to the specific pharmacological properties of the drug and the different patient populations included in both studies. Out of the many pharmacological effects of Dimebon, improvement of impaired mitochondrial function seeems to be most relevant for the substantial effects on cognition and behaviour reported in one of the studies, as these effects are already present at the low concentrations of dimebon measured in plasma and tissues of patients and experimental animals. Since impaired mitochondrial function seems to be the major driving force for the progression of the clinical symptoms and since most of the clinical benefits of dimebon originate from an effect on the symptomatic deterioration, mitochondrial improvement can also explain the lack of efficacy of this drug in another clinical trial where symptoms of the patiets remained stable for the time of the study. Accordingly, it seems worthwhile to reevaluate the clinical data to proof that clinical response is correlated with high levels of Neuropsychiatric Symptoms as these show a good relationship to the individual speed of symptomatic decline in AD patients related to mitochondrial dysfunction.

Mitochondrial Function, Dynamics, and Permeability Transition: A Complex Love Triangle as A Possible Target for the Treatment of Brain Aging and Alzheimer's Disease.

Because of the failure of all amyloid-ß directed treatment strategies for Alzheimer's disease (AD), the concept of mitochondrial dysfunction as a major pathomechanism of the cognitive decline in aging and AD has received substantial support. Accordingly, improving mitochondrial function as an alternative strategy for new drug development became of increasing interest and many different compounds have been identified which improve mitochondrial function in preclinical in vitro and in vivo experiments. However, very few if any have been investigated in clinical trials, representing a major drawback of the mitochondria directed drug development. To overcome these problems, we used a top-down approach by investigating several older antidementia drugs with clinical evidence of therapeutic efficacy. These include EGb761® (standardized ginkgo biloba extract), piracetam, and Dimebon. All improve experimentally many aspects of mitochondrial dysfunction including mitochondrial dynamics and also improve cognition and impaired neuronal plasticity, the functionally most relevant consequences of mitochondrial dysfunction. All partially inhibit opening events of the mitochondrial permeability transition pore (mPTP) which previously has mainly been discussed as a mechanism relevant for the induction of apoptosis. However, as more recent work suggests the mPTP as a master regulator of many mitochondrial functions, our data suggest the mPTP as a possible relevant drug target within the love triangle between mPTP regulation, mitochondrial dynamics, and mitochondrial function including regulation of neuronal plasticity. Drugs interfering with mPTP function will improve not only mitochondrial impairment in aging and AD but also will have beneficial effects on impaired neuronal plasticity, the pathomechanism which correlates best with functional deficits (cognition, behavior) in aging and AD.

MH84 improves mitochondrial dysfunction in a mouse model of early Alzheimer's disease.

BACKGROUND: Current approved drugs for Alzheimer's disease (AD) only attenuate symptoms, but do not cure the disease. The pirinixic acid derivate MH84 has been characterized as a dual gamma-secretase/proliferator activated receptor gamma (PPARγ) modulator in vitro. Pharmacokinetic studies in mice showed that MH84 is bioavailable after oral administration and reaches the brain. We recently demonstrated that MH84 improved mitochondrial dysfunction in a cellular model of AD. In the present study, we extended the pharmacological characterization of MH84 to 3-month-old Thy-1 AßPPSL mice (harboring the Swedish and London mutation in human amyloid precursor protein (APP)) which are characterized by enhanced AßPP processing and cerebral mitochondrial dysfunction, representing a mouse model of early AD. METHODS: Three-month-old Thy-1 AßPPSL mice received 12 mg/kg b.w. MH84 by oral gavage once a day for 21 days. Mitochondrial respiration was analyzed in isolated brain mitochondria, and mitochondrial membrane potential and ATP levels were determined in dissociated brain cells. Citrate synthase (CS) activity was determined in brain tissues and MitoTracker Green fluorescence was measured in HEK293-AßPPwt and HEK293-AßPPsw cells. Soluble Aß1-40 and Aß1-42 levels were determined using ELISA. Western blot analysis and qRT-PCR were used to measure protein and mRNA levels, respectively. RESULTS: MH84 reduced cerebral levels of the ß-secretase-related C99 peptide and of Aß40 levels. Mitochondrial dysfunction was ameliorated by restoring complex IV (cytochrome-c oxidase) respiration, mitochondrial membrane potential, and levels of ATP. Induction of PPARγ coactivator-1α (PGC-1α) mRNA and protein expression was identified as a possible mode of action that leads to increased mitochondrial mass as indicated by enhanced CS activity, OXPHOS levels, and MitoTracker Green fluorescence. CONCLUSIONS: MH84 modulates ß-secretase processing of APP and improves mitochondrial dysfunction by a PGC-1α-dependent mechanism. Thus, MH84 seems to be a new promising therapeutic agent with approved in-vivo activity for the treatment of AD.

Olesoxime suppresses calpain activation and mutant huntingtin fragmentation in the BACHD rat.

Huntington's disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntington's disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntington's disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoxime's effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntington's disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further investigations on the use of olesoxime as a therapeutic for Huntington's disease.

Mitochondrial membrane fluidity is consistently increased in different models of Huntington disease: restorative effects of olesoxime.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT). One prominent target of the mutant huntingtin protein (mhtt) is the mitochondrion, affecting its morphology, distribution, and function. Thus, mitochondria have been suggested as potential therapeutic targets for the treatment of HD. Olesoxime, a cholesterol-like compound, promotes motor neuron survival and neurite outgrowth in vitro, and its effects are presumed to occur via a direct interaction with mitochondrial membranes (MMs). We examined the properties of MMs isolated from cell and animal models of HD as well as the effects of olesoxime on MM fluidity and cholesterol levels. MMs isolated from brains of aged Hdh Q111/Q111 knock-in mice showed a significant decrease in 1,6-diphenyl-hexatriene (DPH) anisotropy, which is inversely correlated with membrane fluidity. Similar increases in MM fluidity were observed in striatal STHdh Q111/Q111 cells as well as in MMs isolated from brains of BACHD transgenic rats. Treatment of STHdh cells with olesoxime decreased the fluidity of isolated MMs. Decreased membrane fluidity was also measured in olesoxime-treated MMs isolated from brains of HD knock-in mice. In both models, treatment with olesoxime restored HD-specific changes in MMs. Accordingly, olesoxime significantly counteracted the mhtt-induced increase in MM fluidity of MMs isolated from brains of BACHD rats after 12 months of treatment in vivo, possibly by enhancing MM cholesterol levels. Thus, olesoxime may represent a novel pharmacological tool to treat mitochondrial dysfunction in HD.

Improvement of mitochondrial function and dynamics by the metabolic enhancer piracetam.

The metabolic enhancer piracetam is used in many countries to treat cognitive impairment in aging, brain injuries, as well as dementia such as AD (Alzheimer's disease). As a specific feature of piracetam, beneficial effects are usually associated with mitochondrial dysfunction. In previous studies we were able to show that piracetam enhanced ATP production, mitochondrial membrane potential as well as neurite outgrowth in cell and animal models for aging and AD. To investigate further the effects of piracetam on mitochondrial function, especially mitochondrial fission and fusion events, we decided to assess mitochondrial morphology. Human neuroblastoma cells were treated with the drug under normal conditions and under conditions imitating aging and the occurrence of ROS (reactive oxygen species) as well as in stably transfected cells with the human wild-type APP (amyloid precursor protein) gene. This AD model is characterized by expressing only 2-fold more human Aß (amyloid ß-peptide) compared with control cells and therefore representing very early stages of AD when Aß levels gradually increase over decades. Interestingly, these cells exhibit an impaired mitochondrial function and morphology under baseline conditions. Piracetam is able to restore this impairment and shifts mitochondrial morphology back to elongated forms, whereas there is no effect in control cells. After addition of a complex I inhibitor, mitochondrial morphology is distinctly shifted to punctate forms in both cell lines. Under these conditions piracetam is able to ameliorate morphology in cells suffering from the mild Aß load, as well as mitochondrial dynamics in control cells.

Rice bran extract protects from mitochondrial dysfunction in guinea pig brains.

Mitochondrial dysfunction plays a major role in the development of age-related neurodegenerative diseases and recent evidence suggests that food ingredients can improve mitochondrial function. In the current study we investigated the effects of feeding a stabilized rice bran extract (RBE) on mitochondrial function in the brain of guinea pigs. Key components of the rice bran are oryzanols, tocopherols and tocotrienols, which are supposed to have beneficial effects on mitochondrial function. Concentrations of α-tocotrienol and γ-carboxyethyl hydroxychroman (CEHC) but not γ-tocotrienol were significantly elevated in brains of RBE fed animals and thus may have provided protective properties. Overall respiration and mitochondrial coupling were significantly enhanced in isolated mitochondria, which suggests improved mitochondrial function in brains of RBE fed animals. Cells isolated from brains of RBE fed animals showed significantly higher mitochondrial membrane potential and ATP levels after sodium nitroprusside (SNP) challenge indicating resistance against mitochondrial dysfunction. Experimental evidence indicated increased mitochondrial mass in guinea pig brains, e.g. enhanced citrate synthase activity, increased cardiolipin as well as respiratory chain complex I and II and TIMM levels. In addition levels of Drp1 and fis1 were also increased in brains of guinea pigs fed RBE, indicating enhanced fission events. Thus, RBE represents a potential nutraceutical for the prevention of mitochondrial dysfunction and oxidative stress in brain aging and neurodegenerative diseases.

Mitochondria: mitochondrial membranes in brain ageing and neurodegeneration.

Mitochondria are membrane bound organelles that provide cellular energy in form of ATP. In addition to ATP synthesis mitochondria are key regulators of calcium homeostasis, free radical production, steroid synthesis and apoptosis, each of these factors could also be associated with essential mechanisms involved in neurodegenerative diseases. Recent studies revealed that changes in mitochondria membrane fluidity might have a direct impact on membrane-based processes such as fission-associated morphogenic changes, opening of the mitochondrial permeability transition pore or oxidative phosphorylation at the complexes of the electron transport chain. We investigated synaptosomal plasma and mitochondrial membranes isolated from brains of mouse models for ageing, Alzheimer's disease, Huntington's disease and Amyotrophic lateral sclerosis. Membrane properties are disease specifically altered, identifying mitochondrial membranes as targets for possible therapeutic strategies in neurodegenerative diseases. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Mitochondrial dysfunction--a pharmacological target in Alzheimer's disease.

Increasing evidences suggest that mitochondrial dysfunction plays an important role in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). Alterations of mitochondrial efficiency and function are mainly related to alterations in mitochondrial content, amount of respiratory enzymes, or changes in enzyme activities leading to oxidative stress, mitochondrial permeability transition pore opening, and enhanced apoptosis. More recently, structural changes of the network are related to bioenergetic function, and its consequences are a matter of intensive research. Several mitochondria-targeting compounds with potential efficacy in AD including dimebon, methylene blue, piracetam, simvastatin, Ginkgo biloba, curcumin, and omega-3 polyunsaturated fatty acids have been identified. The majority of preclinical data indicate beneficial effects, whereas most controlled clinical trials did not meet the expectations. Since mitochondrial dysfunction represents an early event in disease progression, one reason for the disappointing clinical results could be that pharmacological interventions might came too late. Thus, more studies are needed that focus on therapeutic strategies starting before severe disease progress.

Dimebon ameliorates amyloid-ß induced impairments of mitochondrial form and function.

Due to their role in producing energy, as major sources of free radicals, and as critical regulators of apoptosis, mitochondria play a dominant role in the central nervous system (CNS). Mitochondrial dysfunction represents one major pathomechanism of Alzheimer's disease (AD), including impaired function of mitochondrial respiratory chain complexes and deficits of mitochondrial dynamics, such as impaired balance between fission and fusion mechanisms and reduced mitochondrial trafficking. Major consequences are enhanced depletion of mitochondria in axons and dendrites, synaptic dysfunction, and finally neuronal loss. Interfering with impaired mitochondrial dynamics has been proposed as novel strategy for antidementia drugs. Dimebon has been shown to improve cognition in animal models and seems to be beneficial in AD patients. Regardless of the final proof of Dimebon's clinical efficacy, it might specifically interfere with mechanisms relevant for the cognitive decline, especially by improving impaired mitochondrial function and/or dynamics in AD. Herein, we tested the effects of Dimebon on mitochondrial function and dynamics in a cellular model, overexpressing neurotoxic Aß peptides, one of the hallmarks of AD. Dimebon exerted pronounced effects on mitochondrial morphology, respiratory chain complex activities, and enlarged mitochondrial mass. In summary, form and function of mitochondria are altered in the Aß overexpressing cell model and precisely those changes are restored by nanomolar Dimebon treatment. Our findings support the idea that Dimebon improves mitochondrial function and that these "disease specific" effects might be relevant for interpretation and planning of future clinical trials.

Mitochondrion-derived reactive oxygen species lead to enhanced amyloid beta formation.

AIMS: Intracellular amyloid beta (Aß) oligomers and extracellular Aß plaques are key players in the progression of sporadic Alzheimer's disease (AD). Still, the molecular signals triggering Aß production are largely unclear. We asked whether mitochondrion-derived reactive oxygen species (ROS) are sufficient to increase Aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. RESULTS: Complex I and III dysfunction was induced in a cell model using the respiratory inhibitors rotenone and antimycin, resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Aß. Presence of an antioxidant rescued mitochondrial function and reduced formation of Aß, demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Aß was partly reduced by an antioxidant, indicating that Aß formation was also ROS dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aß levels in vivo. INNOVATION: We show for the first time that mitochondrion-derived ROS are sufficient to trigger Aß production in vitro and in vivo. CONCLUSION: Several lines of evidence show that mitochondrion-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aß itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD.