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

https://doi.org/10.1016/j.nbd.2010.08.020Get rights and content

Abstract

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.

Graphical Abstract

Research Highlights

►Intracellular amyloid β expression induces calcium influx in MC65 cells. ►Aβ neurotoxicity parallels intracellular calcium influx. ►L-type voltage-gated calcium channel blockers prevent Aβ-associated neurotoxicity. ►Isradipine appears to be the most potent calcium channel blocker. ►Isradipine is bioavailable to the brain in nanomolar concentrations.

Introduction

Alzheimer's disease (AD) is a progressive, age-related neurodegenerative disease that impairs memory and learning by affecting selective neuronal populations predominantly in the hippocampus and cerebral cortex. Accumulation of extracellular beta-amyloid (Aβ) and intracellular neurofibrillary tangles composed of hyperphosphorylated tau are the pathologic hallmarks of AD. In both early-onset familial and late-onset sporadic forms of AD, a 4-kDa Aβ peptide cleavage product of two key enzymatic reactions mediated by β and γ secretases has been considered a causal factor of AD (Selkoe, 2000, Selkoe, 2001, Selkoe, 2004, Tanzi & Bertram, 2005). The fragment sizes of Aβ vary between 39 and 43 amino acid residues, with Aβ40 being the most common species and Aβ42, a longer, hydrophobic species with a tendency to be more fibrillogenic. Recent studies demonstrate that a soluble form of oligomeric Aβ is more toxic than the monomeric form, and consequently a conscious effort has been made to reconstitute oligomers from monomers in the laboratory setting to demonstrate toxicity in neuronal cells (Li et al., 2009, Picone et al., 2009, Pitt et al., 2009, Poon et al., 2009, Sondag et al., 2009). Evidence from animals models of AD places a particular importance on the accumulation of intraneuronal Aβ oligomers and their role in early pathogenesis of AD (Billings et al., 2005, Golde & Janus, 2005).

Most of our current knowledge of Aβ oligomer toxicity appears to stem from studies that determine the effects of external application of reconstituted, synthetic oligomers. Although these studies have contributed greatly to our knowledge, models of AD that explore the pathomechanisms of intracellular Aβ oligomers have generally been limited. In vitro studies utilizing human neuroblastoma (MC65) cells appear to overcome, at least partially, the limitations of previous methods because they eliminate the need to externally apply Aβ oligomers. MC65 cells are stably transfected with the amyloid precursor protein (APP)-C99 gene and conditionally express a fusion protein composed of the amino-17 and carboxyl-99 residue-containing fragment of APP (Sopher et al., 1996, Sopher et al., 1994). Expression is controlled by a tetracycline-responsive promoter whose activity is repressed in the presence of tetracycline (Tet+). Removal of tetracycline (Tet−) leads to expression of the C-terminal APP fragment and subsequent processing of this fragment into Aβ followed by accumulation of intracellular Aβ oligomers, and precipitous cell death by 3–4 days (Sopher et al., 1996, Sopher et al., 1994). Specific inhibition of γ-secretase in these cells can rescue MC65 cells from death, further confirming that Aβ production triggers the neurotoxicity (Maezawa et al., 2006). Various antioxidants and chemical chaperones provide protection against Aβ-induced death of MC65 cells and these neuroprotective effects are associated with prevention of oligomer formation (Hong et al., 2007, Jin et al., 2002, Lin et al., 2009, Woltjer et al., 2007, Woltjer et al., 2005b).

The precise mechanism of Aβ-mediated cell death in MC65 cells or in AD, however, is still unclear. The Ca2+ hypothesis suggests that intracellular calcium increase in response to Aβ oligomer formation may lead to neuronal cell dysfunction and death in AD (Bezprozvanny and Mattson, 2008). Mechanisms that perturb normal neuronal Ca2+ homeostasis include aberrant Ca2+ influx through plasma membrane channels including the L-type voltage-gated Ca2+channels (L-VGCC) and N-methyl-d-aspartic acid (NMDA) receptors (Arispe et al., 1993, Pollard et al., 1993). Memantine, an antagonist of the NMDA- Ca2+ channel receptor, improves cognition and reduces AD-like neuropathology in mouse models of AD and has beneficial effects in AD patients (Martinez-Coria et al., 2010). Thus, treatments that interrupt aberrant Ca2+ influx may be promising therapeutic strategies for AD. L-VGCC blockers (CCBs) have demonstrated neuroprotection from Aβ in several paradigms utilizing external application of Aβ. In this study, we tested whether CCBs could inhibit neurotoxicity due to intracellular Aβ in MC65 cells. We compared the potency of four CCBs, and finally evaluated whether target brain levels could be achieved with the most promising candidate, isradipine, administered after the onset of AD pathology in a triple transgenic mouse model of AD.

Section snippets

Materials

Cell culture media, l-glutamine, and trypsin/EDTA were obtained from Invitrogen (Carlsbad, CA); fetal bovine serum was from Atlanta Biologicals (Lawrenceville, GA); and calcium channel blockers (verapamil, diltiazem, nimodipine, and isradipine) and thapsigargin (TPG) were from Sigma (St. Louis, MO). 6E10 monoclonal antibody was obtained from Covance Laboratories (Princeton, NJ), L-type Ca++ CP α1C antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and caspase-3

Calcium channel blockers (CCBs) protect MC65 cells from death

Cell survival in Tet− treatment was significantly (P  0.05) lower than Tet+ at 24, 48, and 72 h (Fig. 1A and B). In agreement with our previous findings, cell death increased progressively in response to tetracycline withdrawal and became severe after 72 h, as a direct result of increased expression of intracellular Aβ oligomer species (Woltjer et al., 2007, Woltjer et al., 2005b). The survival response to different CCB treatments was variable. Survival in verapamil was significantly (P < 0.05)

Discussion

Our study results demonstrate: (1) neuroprotection against Aβ toxicity by CCBs in a cell culture model of intracellular Aβ oligomer production, (2) efficacy of isradipine at nanomolar concentrations, and (3) generation of nanomolar brain concentrations of systemically administered isradipine in the 3xTg mouse model of Alzheimer's disease. Our results in MC65 cells for the first time clearly show that after endogenous generation of Aβ oligomers, these cells up-regulate the expression of L-VGCCs.

Conclusion

Intracellular Ca2+ dysregulation in AD is emerging as a unifying mechanism of disease pathology. While the current study is focused on L-VGCC blockers, it is important to emphasize that Ca2+ trafficking is much more complex than described here, involving numerous organelles and mediators. This study shows that Aβ oligomers are strongly associated with increased intracellular Ca2+ and CCBs, especially isradipine, can prevent such an influx at nanomolar concentrations and protect MC65 cells.

Acknowledgments

This work was supported by NIH/NEI 5R21 EY 018708-02 grant to TSA, financial support to TSA by NIH T32 AT002688-05 grant (P.I. Dr. Barry Oken), Department of Veterans Affairs Merit Review Grant to JFQ, and NIH P30AG008017 as well as the OHSU Dean's Award to RLW and JFQ. We thank Dr. Dennis Koop, Director of the OHSU Bioanalytical/ Pharmacokinetics Core Facility, for help with LC-MS/MS analysis.

References (44)

  • C.L. Lin

    Epigallocatechin gallate (EGCG) suppresses beta-amyloid-induced neurotoxicity through inhibiting c-Abl/FE65 nuclear translocation and GSK3 beta activation

    Neurobiol. Aging

    (2009)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method

    Methods

    (2001)
  • H. Martinez-Coria

    Memantine improves cognition and reduces Alzheimer's-like neuropathology in transgenic mice

    Am. J. Pathol.

    (2010)
  • Y. Mochizuki et al.

    Application of coomassie brilliant blue staining to cultured hepatocytes

    Cell Biol. Int. Rep.

    (1987)
  • J.H. Park

    Quantification of isradipine in human plasma using LC-MS/MS for pharmacokinetic and bioequivalence study

    J Chromatogr B Analyt Technol Biomed Life Sci.

    (2009)
  • P. Picone

    Abeta oligomers and fibrillar aggregates induce different apoptotic pathways in LAN5 neuroblastoma cell cultures

    Biophys. J.

    (2009)
  • J. Pitt

    Alzheimer's-associated Abeta oligomers show altered structure, immunoreactivity and synaptotoxicity with low doses of oleocanthal

    Toxicol. Appl. Pharmacol.

    (2009)
  • J.F. Quinn

    Chronic dietary alpha-lipoic acid reduces deficits in hippocampal memory of aged Tg2576 mice

    Neurobiol. Aging

    (2007)
  • B.L. Sopher

    Cytotoxicity mediated by conditional expression of a carboxyl-terminal derivative of the beta-amyloid precursor protein

    Brain Res. Mol. Brain Res.

    (1994)
  • R.E. Tanzi et al.

    Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective

    Cell

    (2005)
  • G.D. Tollefson

    Short-term effects of the calcium channel blocker nimodipine (Bay-e-9736) in the management of primary degenerative dementia

    Biol. Psychiatry

    (1990)
  • R.L. Woltjer

    Effects of chemical chaperones on oxidative stress and detergent-insoluble species formation following conditional expression of amyloid precursor protein carboxy-terminal fragment

    Neurobiol. Dis.

    (2007)
  • Cited by (123)

    View all citing articles on Scopus
    View full text