Calcium channel blocking as a therapeutic strategy for Alzheimer's disease: the case for isradipine.

Alzheimer's disease is the most devastating neurodegenerative disorder in the elderly, yet treatment options are severely limited. The drug development effort to modify Alzheimer's disease pathology by intervention at beta amyloid production sites has been largely ineffective or inconclusive. The greatest challenge has been to identify and define downstream mechanisms reliably predictive of clinical symptoms. Beta amyloid accumulation leads to dysregulation of intracellular calcium by plasma membrane L-type calcium channels located on neuronal somatodendrites and axons in the hippocampus and cortex. Paradoxically, L-type calcium channel subtype Ca(v)1.2 also promotes synaptic plasticity and spatial memory. Increased intracellular calcium modulates amyloid precursor protein processing and affects multiple downstream pathways including increased hyperphosphorylated tau and suppression of autophagy. Isradipine is a Federal Drug Administration-approved dihydropyridine calcium channel blocker that binds selectively to Ca(v)1.2 in the hippocampus. Our studies have shown that isradipine in vitro attenuates beta amyloid oligomer toxicity by suppressing calcium influx into cytoplasm and by suppressing Ca(v)1.2 expression. We have previously shown that administration of isradipine to triple transgenic animal model for Alzheimer's disease was well-tolerated. Our results further suggest that isradipine became bioavailable, lowered tau burden, and improved autophagy function in the brain. A better understanding of brain pharmacokinetics of calcium channel blockers will be critical for designing new experiments with appropriate drug doses in any future clinical trials for Alzheimer's disease. This review highlights the importance of Ca(v)1.2 channel overexpression, the accumulation of hyperphosphorylated tau and suppression of autophagy in Alzheimer's disease and modulation of this pathway by isradipine.

A translational continuum of model systems for evaluating treatment strategies in Alzheimer's disease: isradipine as a candidate drug.

A growing body of evidence supports the 'calcium hypothesis' of Alzheimer's disease (AD), which postulates that a variety of insults might disrupt the homeostatic regulation of neuronal calcium (Ca(2+)) in the brain, resulting in the progressive symptoms that typify the disease. However, despite ongoing efforts to develop new methods for testing therapeutic compounds that might be beneficial in AD, no single bioassay permits both rapid screening and in vivo validation of candidate drugs that target specific components of the Ca(2+) regulatory machinery. To address this issue, we have integrated four distinct model systems that provide complementary information about a trial compound: the human neuroblastoma MC65 line, which provides an in vitro model of amyloid toxicity; a transgenic Drosophila model, which develops age-dependent pathologies associated with AD; the 3×TgAD transgenic mouse, which recapitulates many of the neuropathological features that typify AD; and the embryonic nervous system of Manduca, which provides a novel in vivo assay for the acute effects of amyloid peptides on neuronal motility. To demonstrate the value of this 'translational suite' of bioassays, we focused on a set of clinically approved dihydropyridines (DHPs), a class of well-defined inhibitors of L-type calcium channels that have been suggested to be neuroprotective in AD. Among the DHPs tested in this study, we found that isradipine reduced the neurotoxic consequences of ß-amyloid accumulation in all four model systems without inducing deleterious side effects. Our results provide new evidence in support of the Ca(2+) hypothesis of AD, and indicate that isradipine represents a promising drug for translation into clinical trials. In addition, these studies also demonstrate that this continuum of bioassays (representing different levels of complexity) provides an effective means of evaluating other candidate compounds that target specific components of the Ca(2+) regulatory machinery and that therefore might be beneficial in the treatment of AD.

Phytic acid as a potential treatment for alzheimer's pathology: evidence from animal and in vitro models.

Alzheimer's disease (AD) causes progressive, age-dependent cortical and hippocampal dysfunction leading to abnormal intellectual capacity and memory. We propose a novel protective treatment for AD pathology with phytic acid (inositol hexakisphosphate), a phytochemical found in food grains and a key signaling molecule in mammalian cells. We evaluated the protective and beneficial effects of phytic acid against amyloid-ß (Aß) pathology in MC65 cells and the Tg2576 mouse model. In MC65 cells, 48-72-hour treatment with phytic acid provided complete protection against amyloid precursor protein-C-terminal fragment-induced cytotoxicity by attenuating levels of increased intracellular calcium, hydrogen peroxide, superoxide, Aß oligomers, and moderately upregulated the expression of autophagy (beclin-1) protein. In a tolerance paradigm, wild type mice were treated with 2% phytic acid in drinking water for 70 days. Phytic acid was well tolerated. Ceruloplasmin activity, brain copper and iron levels, and brain superoxide dismutase and ATP levels were unaffected by the treatment. There was a significant increase in brain levels of cytochrome oxidase and a decrease in lipid peroxidation with phytic acid administration. In a treatment paradigm, 12-month old Tg2576 and wild type mice were treated with 2% phytic acid or vehicle for 6 months. Brain levels of copper, iron, and zinc were unaffected. The effects of phytic acid were modest on the expression of AßPP trafficking-associated protein AP180, autophagy-associated proteins (beclin-1, LC3B), sirtuin 1, the ratio of phosphorylated AMP-activated protein kinase (PAMPK) to AMPK, soluble Aß1-40, and insoluble Aß1-42. These results suggest that phytic acid may provide a viable treatment option for AD.

L-type voltage-gated calcium channel blockade with isradipine as a therapeutic strategy for Alzheimer's disease.

There is strong evidence that intracellular calcium dysregulation plays an important pathological role in Alzheimer's disease, and specifically that beta amyloid may induce increases in intracellular calcium and lead to neuronal cell dysfunction and death. Here we investigated the feasibility of modifying Alzheimer's pathology with the L-type voltage-gated calcium channel blockers verapamil, diltiazem, isradipine and nimodipine. All four compounds protected MC65 neuroblastoma cells from amyloid beta protein precursor C-terminal fragment (APP CTF)-induced neurotoxicity. Isradipine was the most potent blocker, preventing APP CTF neurotoxicity at nanomolar concentrations. Intracellular beta amyloid expression was associated with increased expression of Cav 1.2 calcium channels and increased intracellular calcium influx from the extracellular space. Despite the cytoprotection afforded by calcium channel blockers, amyloid beta oligomer formation was not suppressed. The mechanism of cell death in MC65 cells is appeared to be caspase-3 independent. With the goal of determining if there is sufficient experimental support to move forward with animal trials of isradipine, we determined its bioavailability in the triple transgenic mouse model of AD. Subcutaneous implantation of carrier-bound isradipine (3 µg/g/day) for 60 days resulted in nanomolar concentrations in both the plasma and brain. Taken together, our in vitro results support the theory that calcium blockers exert protective effects downstream of the effects of beta amyloid. Isradipine's neuroprotective effect at concentrations that are clinically relevant and achievable in vitro and in vivo suggests that this particular calcium blocking agent may have therapeutic value in the treatment of Alzheimer's disease.

Resveratrol prevents antibody-induced apoptotic death of retinal cells through upregulation of Sirt1 and Ku70.

BACKGROUND: To determine whether resveratrol, a natural plant-derived drug, has protective effects against antibody-induced apoptosis of retinal cells in vitro and to provide insights on the mechanism of resveratrol protection. FINDINGS: E1A.NR3 retinal cells pretreated with 40 muM resveratrol were grown in the presence of anti-recoverin (Rec-1), anti-enolase (Enol-1) antibodies, and normal purified immunoglobulins. When the cells were exposed to resveratrol before treatment with Enol-1 or Rec-1 antibodies, 30-55% more cells survived compared to the resveratrol-untreated cells. Western blotting showed a reduction in proapoptotic protein Bax in the cytoplasm and mitochondria of resveratrol-treated cells. Resveratrol-pretreated cells also showed a significant decrease in intracellular calcium and an inhibition of caspase-3 activity as compared to the untreated cells. Sirt1 expression was greatly reduced in the cells grown in the presence of Rec-1 and Enol-1, but it increased about five times in the resveratrol-pretreated cells. Immunocytochemistry revealed that Sirt1 expression in the cytoplasm and nucleus was colocalized with Ku70 expression in resveratrol-treated cells, suggesting possible interaction with each other in the cell. The pattern of the Ku70 cellular localization also overlapped with the Bax cellular localization in treated and untreated cells. CONCLUSION: In vitro protection of retinal cells from apoptosis by resveratrol occurred through multiple early molecular events, such as reduction of intracellular calcium levels, down-regulation of Bax, up-regulation of Sirt1 and Ku70 activities, and inhibition of caspase-3 activity. These findings will help designing future in vivo and pre-clinical treatments for autoimmune retinopathies.

Resveratrol--a boon for treating Alzheimer's disease?

Resveratrol, a red wine polyphenol, is known to protect against cardiovascular diseases and cancers, as well as to promote antiaging effects in numerous organisms. It also modulates pathomechanisms of debilitating neurological disorders, such as strokes, ischemia, and Huntington's disease. The role of resveratrol in Alzheimer's disease is still unclear, although some recent studies on red wine bioactive compounds suggest that resveratrol modulates multiple mechanisms of Alzheimer's disease pathology. Emerging literature indicates that mechanisms of aging and Alzheimer's disease are intricately linked and that these mechanisms can be modulated by both calorie restriction regimens and calorie restriction mimetics, the prime mediator of which is the SIRT1 protein, a human homologue of yeast silent information regulator (Sir)-2, and a member of NAD+-dependent histone deacetylases. Calorie restriction regimens and calorie restriction-mimetics trigger sirtuins in a wide variety of organisms, ranging from bacteria to mouse. In a mouse model of Huntington's disease, resveratrol-induced SIRT1 was found to protect neurons against ployQ toxicity and in Wallerian degeneration slow mice, resveratrol was found to protect the degeneration of neurons from axotomy, suggesting that resveratrol may possess therapeutic value to neuronal degeneration. This paper mainly focuses on the role of resveratrol in modulating AD pathomechanisms.

Mitochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression.

Alzheimer's disease (AD) is a complex, neurodegenerative disease characterized by the impairment of cognitive function in elderly individuals. In a recent global gene expression study of APP transgenic mice, we found elevated expression of mitochondrial genes, which we hypothesize represents a compensatory response because of mitochondrial oxidative damage caused by the over-expression of mutant APP and/or amyloid beta (Abeta). We investigated this hypothesis in a series of experiments examining what forms of APP and Abeta localize to the mitochondria, and whether the presence of these species is associated with mitochondrial dysfunction and oxidative damage. Using immunoblotting, digitonin fractionation, immunofluorescence, and electron microscopy techniques, we found a relationship between mutant APP derivatives and mitochondria in brain slices from Tg2576 mice and in mouse neuroblastoma cells expressing mutant human APP. Further, to determine the functional relationship between mutant APP/Abeta and oxidative damage, we quantified Abeta levels, hydrogen peroxide production, cytochrome oxidase activity and carbonyl proteins in Tg2576 mice and age-matched wild-type (WT) littermates. Hydrogen peroxide levels were found to be significantly increased in Tg2576 mice when compared with age-matched WT littermates and directly correlated with levels of soluble Abeta in Tg2576 mice, suggesting that soluble Abeta may be responsible for the production of hydrogen peroxide in AD progression in Tg2576 mice. Cytochrome c oxidase activity was found to be decreased in Tg2576 mice when compared with age-matched WT littermates, suggesting that mutant APP and soluble Abeta impair mitochondrial metabolism in AD development and progression. An increase in hydrogen peroxide and a decrease in cytochrome oxidase activity were found in young Tg2576 mice, prior to the appearance of Abeta plaques. These findings suggest that early mitochondrially targeted therapeutic interventions may be effective in delaying AD progression in elderly individuals and in treating AD patients.

Neuronal protection by sirtuins in Alzheimer's disease.

Silent information regulator 2, a member of NAD+-dependent histone deacetylase in yeast, and its homologs in mice and humans, participate in numerous important cell functions, including cell protection and cell cycle regulation. The sirtuin family members are highly conserved evolutionarily, and are predicted to have a role in cell survival. The science of sirtuins is an emerging field and is expected to contribute significantly to the role of sirtuins in healthy aging in humans. The role of sirtuins in neuronal protection has been studied in lower organisms, such as yeast, worms, flies and rodents. Both yeast Sir2 and mammalian sirtuin proteins are up-regulated under calorie-restricted and resveratrol treatments. Increased sirtuin expression protects cells from various insults. Caloric restriction and antioxidant treatments have shown useful effects in mouse models of aging and Alzheimer's disease (AD) and in limited human AD clinical trials. The role sirtuins may play in modifying and protecting neurons in patients with neurodegenerative diseases is still unknown. However, a recent report of Huntington's disease revealed that Sirtuin protects neurons in a Huntington's disease mouse model, suggesting that sirtuins may protect neurons in patients with neurodegenerative diseases, such as AD. In this review, we discuss the possible mechanisms of sirtuins involved in neuronal protection and the potential therapeutic value of sirtuins in healthy aging and AD.

Can herbs provide a new generation of drugs for treating Alzheimer's disease?

The overall aim of this review is to discuss cellular mechanisms at work in the progression of AD and current therapeutic strategies for treating AD, with a focus on the potential efficacy of herbal treatments. Recent advances in molecular, cellular, and animal model studies have revealed that formation of the 4-kDa amyloid beta peptide is a key factor in the development and progression of AD. Several cellular changes have been identified that are related to amyloid beta plaques and neurofibrillary tangles found in the autopsied brains of AD patients and in AD animal models. Several therapeutic strategies have been developed to treat AD, including anti-inflammatory, anti-oxidant, and anti-amyloid approaches. Recently, herbal treatments have been tested in animal and cellular models of AD and in clinical trials with AD subjects. In AD animal models and cell models, herbal extracts appear to have fewer adverse effects than beneficial effects on A beta and cognitive functions. These extracts have multi-functional properties (pro-cholinergic, anti-oxidant, anti-amyloid, and anti-inflammatory), and their use in the treatment of AD patients looks promising. The chemical compositions of herbs and their potential for alleviating or reducing symptoms of AD or for affecting the disease mechanism need to be further studied.