Open Access

Schamim Eckert

• To understand neurodegenerative diseases is one of the major challenges of the 21st century. This also includes Alzheimer´s disease (AD), which represents a chronic neurodegenerative disorder, with long preclinical and prodromal phases (approx. 20 years) and an average clinical duration of 8–10 years. In the early phase of this disease, patients show deterioration of memory, difficulties in finding the right words for everyday objects or mood swings. The risk of AD grows exponentially with age, doubling approximately every 5 to 6 years. AD may contribute to 60–70% of all dementia cases, being the most common cause of this disease. Dementia is one of the major causes of disability and dependency among older people worldwide. The causes of the sporadic form of AD with late onset (LOAD) are not yet known, but it seems to be a result of multiple factors. Neuropathological features are extracellular senile plaques, containing beta-amyloid peptides (Aβ) and intracellular neurofibrillary tangles, containing paired helical tau proteins, which have been associated with neuronal loss and atrophy of the cerebral cortex. Thus, misfolded proteins seem to contribute to the pathogenesis, but are not the only players in the disease process. Developing feasible therapies is difficult due to the multifactorial pathology of AD. Currently approved drugs only attenuate symptoms, but do not cure the disease. Research into AD also has had several failures in terms of developing disease-modifying therapies. Thus, new therapeutic targets in order to develop a causal therapy are desperately needed. Since AD starts many years far before the first symptoms occur, new scientific approaches focus on the early stage, which are discussed to be important in aging and the onset of AD. Today, the hypothesis of the advanced mitochondrial cascade becomes more and more the leading model for LOAD, integrating physiological aging as the main risk factor. Thus, new interventions targeting mitochondrial dysfunction are of substantial interest. Accordingly, the efficacy of Dimebon and TRO19622 to ameliorate mitochondrial dysfunction in cellular and murine models of AD were investigated. Dimebon (Latrepirdine) was, originally developed in Russia as an H1-antiallergic drug. It might specifically interfere with mechanisms relevant for the cognitive decline, especially by improving impaired mitochondrial function and/or dynamics in AD. TRO19622 (Olesoxim) has been identified in a phenotypic screening approach to promote the survival of primary motor neurons. Olesoxim is easily absorbed by cells and accumulates in mitochondria. Olesoxim’s mode of action is not fully understood, however it has been shown to modulate mitochondrial membranes and interact with the voltage-dependent anion channel (VDAC) and the translocator protein (TSPO; also known as PBR). Thereby it inhibits mitochondrial permeability transition. In this study, the effects of Aβ overproduction on mitochondrial function were investigated. The effects of Dimebon and Olesoxim were examined, using a HEK cell line stably transfected with the Swedish APP double mutation (HEKsw) and un-transfected control cells (HEKut). Mitochondrial membrane potential, ATP concentrations, and respirometry were measured. Western Blot analysis of marker proteins for fission & fusion, autophagy, mitogenesis and mPTP formation were performed. Confocal laser scanning microscopy was introduced as a novel method to visualize mitochondrial dynamics. Olesoxim was also tested in Thy-1-C57BJ/6-APPSL mice representing a murine model of AD. For the in vivo model mitochondria from brain tissue were isolated and dissociated brain cells were prepared to determine respiration, lipid peroxidation, MMP, and ATP-levels. Both, the in vitro and in vivo models were compared and discussed in relation to human post-mortem data. The research was conducted in frame of the EU-project entitled „MITOTARGET“ (Mitochondrial dysfunction in neurodegenerative diseases: towards new therapeutics) funded under FP7-Health (http://cordis.europa.eu/result/rcn/54471_en.html). HEKsw cells showed an overall reduction in the mitochondrial respiration, a significant lower MMP, and significantly reduced ATP levels compared to HEKut cells. Mitochondrial mass was equal in both cell lines. In addition most mitochondria in HEKsw cells showed truncated morphology, followed by punctuated mitochondria. Levels of the fission related protein Drp were significantly elevated in HEKsw cells whereas protein levels of fusion related OPA were strongly reduced, leading to a shift in the distribution pattern towards shorter mitochondria. Moreover, HEKsw cells showed reduced mitochondrial density. Protein levels of the translocase of the inner mitochondrial membrane (TIMM50) were strongly diminished in HEKsw cells. The OXPHOS machinery is located in the inner membrane, where the MMP is build up and ATP is generated. Reduced TIMM50 levels in HEKsw indicated a reduction of the inner mitochondrial membrane, which could explain the described deficits in OXPHOS, MMP, ATP and mitochondrial morphology and density. Concentration of both mPTP markers, the voltage-depended anion channel (VDAC) and the peripheral benzodiazepine receptor (PBR), were broadly increased in HEKsw cells. Thy1-APPSL transgenic mice were characterized as in vivo model of AD. Those mice are modified to express the human form of APP, containing both, the Swedish (KM670/671NL) and the London (V717L) double mutations under the murine Thy1 promotor. Beginning at the age of 3 months, Thy1-APPSL mice develop elevated Aβ levels and mitochondrial dysfunction. Mitochondria isolated from brains of Thy-1-C57BJ/6-APPSL mice showed significant impaired respiration, resulting in a reduced MMP. However, ATP levels in dissociated brain cells did not differ compared to controls. Protein levels of FIS were unchanged, whereas Drp levels were significantly increased. Levels of the mitochondrial fusion marker optic atrophie-1 (Opa) protein were significantly reduced. Peroxisome proliferation-activated receptor gamma coactivator 1-alpha (PGC1) is a transcription factor, which represents a master regulator of mitochondrial biogenesis. PGC1 expression was significantly elevated in brains of Thy-1-C57BJ/6-APPSL mice. However, mitochondrial mass seemed to be equal in both mouse lines. Both LC3-Isoforms, the cytosolic and the autophagosomal form, were not changed in brains of Thy-1-C57BJ/6-APPSL mice, which indicates equal mitophagic activity. In brain homogenates, isolated from Thy-1-C57BJ/6-APPSL mice, both mPTP marker, VDAC and PBR, were considerably increased, which is in accordance with the findings in HEKsw cells. In conclusion, both, the cellular (HEKsw) and the animal model of AD (Thy1-APPSL) broadly match pathophysiological features, which have been found in post-mortem samples from AD patients. Thus, HEKsw cells and Thy1-APPSL mice seem to be suitable models to study new treatments against AD. Incubation of HEKsw cells with Dimebon resulted in a remarkable increase in respiratory activity and restored the MMP after impairing the cells with rotenon. Dimebon had no effects on ATP levels in both cell lines, neither after challenging cells with rotenon, nor under basal conditions. By adding Dimebon, citrate synthase (CS) activity in HEKsw cells was increased and mitochondrial morphology was shifted to a tubular shape. Dimebon further enhanced protein levels of Drp and resulted in the compensation of reduced OPA levels. Moreover, Dimebon restored the increased expression levels of the mPTP markers VDAC and PBR. Aβ1-40 levels were significantly decreased in HEKsw cells. However, changes in Aβ1-40 levels seemed to be too small, to solely explain the much larger effects of Dimebon on impaired mitochondrial function. In conclusion, Dimebon treatment restored diverse defects in Aβ overexpressing cells: Aβ levels were reduced, autophagy marker were increased, mitophagy as repair and renewal mechanism was elevated, mitochondrial mass and density were increased, OXPHOS capacity was restored, mitochondrial dynamics were balanced, mitochondrial shape showed a normal distribution, expression levels of the mPTP constituents were reduced, TIMM50 levels augmented to control levels and stress induced MMP and ROS levels were reduced. All these effects were observed after incubation of cells with a rather low concentration of 100 nmol/L. Based on these findings and in addition to already existing literature, Dimebon presents a potential therapeutic option for diseases with accompanied mitochondrial dysfunction. Although, clinical findings published so far are inconsistent. Olesoxim induced a general increase in respiratory activity and enhanced the electron transport (ETS) capacity in HEKsw cells. In addition it normalized the OXPHOS activity almost to control levels. However, incubation using different Olesoxim concentrations led to a dose independent decline in the MMP and decreased ATP levels. Adding Olesoxim caused a dose-dependent change in the length of mitochondria strongly shifting the pattern towards longer mitochondria. In HEKsw cells a reduced mitochondrial density was observed which was reversed by Olesoxim dose-dependently. Olesoxim completely compensated the severely reduced expression levels of TIMM50, but had no effects on TOMM22 levels. An unexpected finding was that 10 µM Olesoxim significantly increased Aβ1-40 levels. Effects of Olesoxim were also tested in vivo. Treatment of Thy-1-C57BJ/6-APPSL mice with Olesoxim restored the impaired MMP in dissociated brain cells, but had no effects on ATP-levels. Olesoxim increased the respiratory activity in isolated brain mitochondria and restored impaired respiration complex activities almost to control levels, without having an effect on CS activity. However, treatment with Olesoxim caused an increase of PGC1 protein levels in brains of Thy-1-C57BJ/6-APPSL mice,beyond basal levels of littermate controls. The mPTP marker proteins voltage-depended anion channel (VDAC) and peripheral benzodiazepine receptor (PBR) were significantly reduced. As well as in the cell models, treatment of Thy-1-C57 BJ/6-APPSL mice with Olesoxim significantly enhanced total human, soluble human and soluble mouse Aβ1-40 levels. Further investigation needs the observation that Olesoxim caused partly negative effects in controls. For instance, Olesoxim reduced the OXPHOS capacity and enhanced protein levels of VADAC and PBR in brains of C57BJ/6 littermate control mice, which could limit the applicability of Olesoxim in further preclinical studies.