Abstract
SUMMARYA 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 (Ca2+) 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 Ca2+ 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 β-amyloid accumulation in all four model systems without inducing deleterious side effects. Our results provide new evidence in support of the Ca2+ 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 Ca2+ regulatory machinery and that therefore might be beneficial in the treatment of AD.
Highlights
As originally proposed by Khachaturian and colleagues (Khachaturian, 1987), the ‘Ca2+ hypothesis of Alzheimer’s disease (AD)’ was based on observations that age-dependent increases in cellular Ca2+ are exacerbated in AD, which in turn might perturb multiple aspects of neuronal metabolism and activity (Peterson et al, 1985; Gibson and Peterson, 1987; Toescu et al, 2004)
We found that isradipine was neuroprotective in all four of our model systems, providing an important ‘proof of principle’ that this combination of progressively more complex bioassays can be used to identify neuroprotective drugs that mitigate the deleterious effects of neurotoxic amyloid
To assess whether the accumulation of A in the MC65 cells modulated their expression of L-type calcium channels (LTCCs), we used quantitative realtime PCR to measure the expression levels of Cav1.2 ( 1c) and Cav1.3 ( 1d), the predominant LTCC -subunits that are RESULTS DHPs are protective in an in vitro assay of A toxicity (MC65 cells) MC65 cells are derived from a human neuroblastoma cell line (SKN-MC) that has been stably transfected with an APP-derived construct, consisting of the N-terminal 17 residues of APP fused to the C-terminal fragment of APP generated by -secretase cleavage (C99) (Gossen and Bujard, 1992; Sopher et al, 1994)
Summary
As originally proposed by Khachaturian and colleagues (Khachaturian, 1987), the ‘Ca2+ hypothesis of Alzheimer’s disease (AD)’ was based on observations that age-dependent increases in cellular Ca2+ are exacerbated in AD, which in turn might perturb multiple aspects of neuronal metabolism and activity (Peterson et al, 1985; Gibson and Peterson, 1987; Toescu et al, 2004) Both genetic and environmental insults that disrupt Ca2+ homeostasis in the brain can accelerate the accumulation of neurotoxic amyloid (A ) peptides and hyperphosphorylated tau, and pathological aggregates of these proteins can themselves alter neuronal Ca2+ levels via a variety of signaling pathways (reviewed in Green and LaFerla, 2008; Yu et al, 2009; Berridge, 2010). A number of dihydropyridines (DHPs) targeting specific LTCC subtypes are currently used to treat hypertension and related conditions, making them attractive candidate drugs that could be ‘repurposed’ to treat the mild-tomoderate stages of AD
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