Benfotiamine treatment activates the Nrf2/ARE pathway and is neuroprotective in a transgenic mouse model of tauopathy.

Impaired glucose metabolism, decreased levels of thiamine and its phosphate esters, and reduced activity of thiamine-dependent enzymes, such as pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and transketolase occur in Alzheimer's disease (AD). Thiamine deficiency exacerbates amyloid beta (Aß) deposition, tau hyperphosphorylation and oxidative stress. Benfotiamine (BFT) rescued cognitive deficits and reduced Aß burden in amyloid precursor protein (APP)/PS1 mice. In this study, we examined whether BFT confers neuroprotection against tau phosphorylation and the generation of neurofibrillary tangles (NFTs) in the P301S mouse model of tauopathy. Chronic dietary treatment with BFT increased lifespan, improved behavior, reduced glycated tau, decreased NFTs and prevented death of motor neurons. BFT administration significantly ameliorated mitochondrial dysfunction and attenuated oxidative damage and inflammation. We found that BFT and its metabolites (but not thiamine) trigger the expression of Nrf2/antioxidant response element (ARE)-dependent genes in mouse brain as well as in wild-type but not Nrf2-deficient fibroblasts. Active metabolites were more potent in activating the Nrf2 target genes than the parent molecule BFT. Docking studies showed that BFT and its metabolites (but not thiamine) bind to Keap1 with high affinity. These findings demonstrate that BFT activates the Nrf2/ARE pathway and is a promising therapeutic agent for the treatment of diseases with tau pathology, such as AD, frontotemporal dementia and progressive supranuclear palsy.

Methylene blue upregulates Nrf2/ARE genes and prevents tau-related neurotoxicity.

Methylene blue (MB, methylthioninium chloride) is a phenothiazine that crosses the blood brain barrier and acts as a redox cycler. Among its beneficial properties are its abilities to act as an antioxidant, to reduce tau protein aggregation and to improve energy metabolism. These actions are of particular interest for the treatment of neurodegenerative diseases with tau protein aggregates known as tauopathies. The present study examined the effects of MB in the P301S mouse model of tauopathy. Both 4 mg/kg MB (low dose) and 40 mg/kg MB (high dose) were administered in the diet ad libitum from 1 to 10 months of age. We assessed behavior, tau pathology, oxidative damage, inflammation and numbers of mitochondria. MB improved the behavioral abnormalities and reduced tau pathology, inflammation and oxidative damage in the P301S mice. These beneficial effects were associated with increased expression of genes regulated by NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE), which play an important role in antioxidant defenses, preventing protein aggregation, and reducing inflammation. The activation of Nrf2/ARE genes is neuroprotective in other transgenic mouse models of neurodegenerative diseases and it appears to be an important mediator of the neuroprotective effects of MB in P301S mice. Moreover, we used Nrf2 knock out fibroblasts to show that the upregulation of Nrf2/ARE genes by MB is Nrf2 dependent and not due to secondary effects of the compound. These findings provide further evidence that MB has important neuroprotective effects that may be beneficial in the treatment of human neurodegenerative diseases with tau pathology.

PGC-1α overexpression exacerbates ß-amyloid and tau deposition in a transgenic mouse model of Alzheimer's disease.

The peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) interacts with various transcription factors involved in energy metabolism and in the regulation of mitochondrial biogenesis. PGC-1α mRNA levels are reduced in a number of neurodegenerative diseases and contribute to disease pathogenesis, since increased levels ameliorate behavioral defects and neuropathology of Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. PGC-1α and its downstream targets are reduced both in postmortem brain tissue of patients with Alzheimer's disease (AD) and in transgenic mouse models of AD. Therefore, we investigated whether increased expression of PGC-1α would exert beneficial effects in the Tg19959 transgenic mouse model of AD; Tg19959 mice express the human amyloid precursor gene (APP) with 2 familial AD mutations and develop increased ß-amyloid levels, plaque deposition, and memory deficits by 2-3 mo of age. Rather than an improvement, the cross of the Tg19959 mice with mice overexpressing human PGC-1α exacerbated amyloid and tau accumulation. This was accompanied by an impairment of proteasome activity. PGC-1α overexpression induced mitochondrial abnormalities, neuronal cell death, and an exacerbation of behavioral hyperactivity in the Tg19959 mice. These findings show that PGC-1α overexpression exacerbates the neuropathological and behavioral deficits that occur in transgenic mice with mutations in APP that are associated with human AD.

Bezafibrate administration improves behavioral deficits and tau pathology in P301S mice.

Peroxisome proliferator-activated receptors (PPARs) are ligand-mediated transcription factors, which control both lipid and energy metabolism and inflammation pathways. PPARγ agonists are effective in the treatment of metabolic diseases and, more recently, neurodegenerative diseases, in which they show promising neuroprotective effects. We studied the effects of the pan-PPAR agonist bezafibrate on tau pathology, inflammation, lipid metabolism and behavior in transgenic mice with the P301S human tau mutation, which causes familial frontotemporal lobar degeneration. Bezafibrate treatment significantly decreased tau hyperphosphorylation using AT8 staining and the number of MC1-positive neurons. Bezafibrate treatment also diminished microglial activation and expression of both inducible nitric oxide synthase and cyclooxygenase 2. Additionally, the drug differentially affected the brain and brown fat lipidome of control and P301S mice, preventing lipid vacuoles in brown fat. These effects were associated with behavioral improvement, as evidenced by reduced hyperactivity and disinhibition in the P301S mice. Bezafibrate therefore exerts neuroprotective effects in a mouse model of tauopathy, as shown by decreased tau pathology and behavioral improvement. Since bezafibrate was given to the mice before tau pathology had developed, our data suggest that bezafibrate exerts a preventive effect on both tau pathology and its behavioral consequences. Bezafibrate is therefore a promising agent for the treatment of neurodegenerative diseases associated with tau pathology.

Behavioral improvement after chronic administration of coenzyme Q10 in P301S transgenic mice.

Coenzyme Q10 is a key component of the electron transport chain which plays an essential role in ATP production and also has antioxidant effects. Neuroprotective effects of coenzyme Q10 have been reported in both in vitro and in vivo models of neurodegenerative diseases. However, its effects have not been studied in cells or in animals with tau induced pathology. In this report, we administered coenzyme Q10 to transgenic mice with the P301S tau mutation, which causes fronto-temporal dementia in man. These mice develop tau hyperphosphorylation and neurofibrillary tangles in the brain. Coenzyme Q10 improved survival and behavioral deficits in the P301S mice. There was a modest reduction in phosphorylated tau in the cortex of P301S mice. We also examined the effects of coenzyme Q10 treatment on the electron transport chain enzymes, the mitochondrial antioxidant enzymes, and the tricarboxylic acid cycle. There was a significant increase in complex I activity and protein levels, and a reduction in lipid peroxidation. Our data show that coenzyme Q10 significantly improved behavioral deficits and survival in transgenic mice with the P301S tau mutation, upregulated key enzymes of the electron transport chain, and reduced oxidative stress.

Behavioral deficit, oxidative stress, and mitochondrial dysfunction precede tau pathology in P301S transgenic mice.

Abnormal tau accumulation can lead to the development of neurodegenerative diseases. P301S mice overexpress the human tau mutated gene, resulting in tau hyperphosphorylation and tangle formation. Mice also develop synaptic deficits and microglial activation prior to any neurodegeneration and tangles. Oxidative stress can also affect tauopathy. We studied the role of oxidative stress in relationship to behavioral abnormalities and disease progression in P301S mice at 2, 7, and 10 mo of age. At 7 mo of age, P301S mice had behavioral abnormalities, such as hyperactivity and disinhibition. At the same age, we observed increased carbonyls in P301S mitochondria (∼215 and 55% increase, males/females), and deregulation in the activity and content of mitochondrial enzymes involved in reactive oxygen species formation and energy metabolism, such as citrate synthase (∼19 and ∼5% decrease, males/females), MnSOD (∼16% decrease, males only), cytochrome C (∼19% decrease, females only), and cytochrome C oxidase (∼20% increase, females only). These changes in mitochondria proteome appeared before tau hyperphosphorylation and tangle formation, which were observed at 10 mo and were associated with GSK3ß activation. At that age, mitochondria proteome deregulation became more apparent in male P301S mitochondria. The data strongly suggest that oxidative stress and mitochondrial abnormalities appear prior to tau pathology.

Apocynin administration does not improve behavioral and neuropathological deficits in a transgenic mouse model of Alzheimer's disease.

In addition to mitochondria, NADPH oxidase (NOX) is a source of oxidative stress, which can induce oxidative damage in Alzheimer's disease (AD). For this reason, several groups have investigated the effect of its inhibition. In AD mice, NADPH oxidase 2 (NOX2) deficiency improved behavior and cerebrovascular function, and reduced oxidative stress. In our study, we administered the NOX inhibitor apocynin to Tg19959 mice, and found that it did not improve cognitive and synaptic deficits, and did not decrease amyloid deposition, microgliosis and hyperphosphorylated tau. However, apocynin reduced carbonyl levels in the cerebral cortex but not the hippocampus, which may have not been sufficient to ameliorate symptoms. Also, the reduction of NOX-mediated oxidative stress may not be sufficient to prevent AD, since other sources of reactive oxygen species such as mitochondria may be more important.

Triterpenoids CDDO-ethyl amide and CDDO-trifluoroethyl amide improve the behavioral phenotype and brain pathology in a transgenic mouse model of Huntington's disease.

Oxidative stress is a prominent feature of Huntington's disease (HD) due to mitochondrial dysfunction and the ensuing overproduction of reactive oxygen species (ROS). This phenomenon ultimately contributes to cognitive and motor impairment, as well as brain pathology, especially in the striatum. Targeting the transcription of the endogenous antioxidant machinery could be a promising therapeutic approach. The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway is an important pathway involved in antioxidant and anti-inflammatory responses. Synthetic triterpenoids, which are derived from 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic acid (CDDO) activate the Nrf2/ARE pathway and reduce oxidative stress in animal models of neurodegenerative diseases. We investigated the effects of CDDO-ethyl amide (CDDO-EA) and CDDO-trifluoroethyl amide (CDDO-TFEA) in N171-82Q mice, a transgenic mouse model of HD. CDDO-EA or CDDO-TFEA were administered in the diet at various concentrations, starting at 30days of age. CDDO-EA and CDDO-TFEA upregulated Nrf2/ARE induced genes in the brain and peripheral tissues, reduced oxidative stress, improved motor impairment and increased longevity. They also rescued striatal atrophy in the brain and vacuolation in the brown adipose tissue. Therefore compounds targeting the Nrf2/ARE pathway show great promise for the treatment of HD.

Mice deficient in dihydrolipoyl succinyl transferase show increased vulnerability to mitochondrial toxins.

The activity of a key mitochondrial tricarboxylic acid cycle enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in many neurodegenerative diseases. KGDHC consists of three subunits. The dihydrolipoyl succinyl transferase (DLST) component is unique to KGDHC. DLST(+/-) mice showed reduced mRNA and protein levels and decreased brain mitochondrial KGDHC activity. Neurotoxic effects of mitochondrial toxins were exacerbated in DLST(+/-) mice. MPTP produced a significantly greater reduction of striatal dopamine and tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta of DLST(+/-) mice. DLST deficiency enhanced the severity of lipid peroxidation in the substantia nigra after MPTP treatment. Striatal lesions induced by either malonate or 3-nitropropionic acid (3-NP) were significantly larger in DLST(+/-) mice than in wildtype controls. DLST deficiency enhanced the 3-NP inhibition of mitochondria enzymes, and 3-NP induced protein and DNA oxidations. These observations support the hypothesis that reductions in KGDHC may impair the adaptability of the brain and contribute to the pathogenesis of neurodegenerative diseases.

Reduction of oxidative stress, amyloid deposition, and memory deficit by manganese superoxide dismutase overexpression in a transgenic mouse model of Alzheimer's disease.

In Alzheimer's disease (AD), oxidative stress is present early and contributes to disease pathogenesis. We previously reported that in Tg19959 transgenic AD mice, partial deficiency of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) exacerbated amyloid pathology. We therefore asked whether MnSOD overexpression would prove beneficial against AD pathogenesis, by studying the offspring of Tg19959 mice crossed with MnSOD-overexpressing mice. At 4 mo of age, there was a 2- to 3-fold increase in MnSOD protein levels in Tg19959-MnSOD mice compared to Tg19959 littermates. Tg19959-MnSOD mice also had a 50% increase in catalase protein levels, a 50% decrease in levels of oxidized protein, and a 33% reduction in cortical plaque burden compared to Tg19959 littermates. Spatial memory was impaired and synaptophysin levels were decreased in Tg19959 mice compared to wild-type littermates, but memory and synaptophysin levels were restored to wild-type levels in Tg19959-MnSOD littermates. These benefits occurred without changes in sodium dodecyl sulfate-soluble or formic acid-soluble Abeta pools or Abeta oligomers in Tg19959-MnSOD mice compared to Tg19959 littermates. These data demonstrate that facilitation of the mitochondrial antioxidant response improves resistance to Abeta, slows plaque formation or increases plaque degradation, and markedly attenuates the phenotype in a transgenic AD mouse model.