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

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.

Figure 1

Effects of L-type voltage-gated Ca²⁺ channel blockers (CCBs) on survival and intracellular calcium levels in MC65 cells. Cells grown for 24 to 72 hours in the presence (Tet+) or absence (Tet−) of tetracycline and without CCBs served as positive and negative controls, respectively. (A) Effects of CCBs on cell survival. Cells were grown in Tet+, Tet−, and Tet− condition for 24, 48, and 72 h under the following CCB treatments: verapamil (25 µM), diltiazem (25 µM), nimodipine (10 µM), and isradipine (0.05 µM). Cell survival was assessed using the MTS assay after each treatment period. (B) MC65 cells were stained with Coomassie Blue at 24, 48, and 72 h after exposing to Tet+ and Tet− conditions. Live cells attached to the bottom of the slide are shown in the image. (C) Effects of CCBs on intracellular Ca²⁺ levels. Cells were cultured and treated as in (A). Intracellular Ca²⁺ levels in live cells were assessed as relative fluorescence units (RFU/Live cells) after each treatment period. (D) Effects of different concentrations of isradipine (0.001–5 µM) on survival levels in MC65 cells grown for 72 hours. Cell survival was assessed using the MTS assay. (E) Effects of different concentrations of isradipine (0.001–5 µM) on intracellular Ca²⁺ levels in MC65 cells grown for 72 hours. Intracellular Ca²⁺ in live cells was assessed as relative fluorescence units (RFU/Live cells). (F) Effects of thapsigargin (TPG; 2 µM) treatment on intracellular Ca²⁺ levels in MC65 cells cultured under Tet+ and Tet− conditions for 24 and 72 h. Each treatment was replicated on average 6 times in each of 3–5 experiments, and the error bars represent an average percentage of cell survival for the experiments. Pair-wise comparison with Bonferroni post-hoc tests were made on percent survival between Tet+ and other treatments. Statistical significance levels: * p≤0.05, ** p≤0.01, *** p≤0.001.

Figure 2

Expression of Ca²⁺ channels in MC65 cells and the downstream effects of increased Ca²⁺. (A) mRNA expression of Ca²⁺ channel Cav1.2 was determined by qRT-PCR. (B) Cav1.2 protein expression was determined by western blot (representative blot shown for three extracts from each time point) and quantified by densitometry. MC65 cells were grown for 24, 48, or 72 hours under Tet+ and Tet− treatments. (C) Calpain activity was assessed in the cells grown for 24 and 48 hours under Tet+ and Tet− treatments. (D) Expression of caspase-3, an executioner protein diagnostic of mitochondria-mediated apoptosis, was determined by western blot. The cells were grown for 48 h under Tet+ and Tet− in the presence of vehicle (Veh) or the following CCBs (µM): diltiazem (Dil, 25), verapamil (Ver, 25), nimodipine (Nim, 10), and isradipine (Isr, 0.05). The blot was probed with anti-caspase-3 polyclonal antibody. (E) Densitometry analysis was performed on a 35 kDa pro-caspase-3 and the bands originating from cleaved fragments of 19 and 17 kDa. The bars represent average for each band relative to Tet+ band and adjusted for the loading control β-tubulin. Error bars are ± SEM.

Figure 3

Effects of isradipine on the expression of Aβ oligomers and Aβ fibril formation. (A) Expression of Aβ oligomers in MC65 cells was determined by western blot. The cells were grown under Tet+ and Tet− in the presence of vehicle (Veh) or isradipine (Isr, 0.05µM). The proteins were fractionated on 16% tricine gels and transferred to nitrocellulose membranes. The membrane was probed with 6E10 monoclonal antibody to Aβ oligomers. Tet+ and Tet−, respectively, indicate the presence and absence of tetracycline in the medium, and the cells were treated with CCBs for 72 h. APP= amyloid precursor protein. Aβ oligomers appear in the range of 16 and 44 kDa. C99 = C-terminal 99 amino acid-fragment, and P8 = an 8-kDa Aβ domain containing fragment. (B) Densitometry analysis of 16–32 kDa range Aβ oligomers. The bars represent average for each band relative to Tet+ Veh band and adjusted for the loading control β-tubulin. Error bars are ± SEM. (C) Thioflavin T (ThT) fluorescence assay to determine the effect of vehicle and isradipine (0.05 µM) on Aβ fibril formation.

Figure 4

The bioavailability of isradipine in the plasma and brain homogenates of 3xTg-AD mice treated subcutaneously with isradipine (N=4) or vehicle pellets (N=3) for 60 days. (A) Vehicle plasma, (B) Treated plasma, (C) Vehicle brain, (D) Treated brain. The selective reaction monitoring (SRM) transitions shown as mass-to-charge ratios (m/z) are for isradipine 372-312 and internal standard (amlodipine) 409-238 for each set. The retention time for isradipine was 5.5 min and amlodipine 5.0.

Figure 5

A proposed mechanism of cell death due to increased intracellular Ca²⁺ in MC65 cells. A C-terminal 99-amino acid-fragment (C99) is sequentially processed by β-secretase and γ-secretase enzymes to generate Aβ monomers, which in turn oligomerize rapidly inside the cell. Aβ oligomers increase intracellular Ca²⁺ levels and ultimately cause cell death. CCBs used in this study attenuated the intracellular Ca²⁺ rise without influencing Aβ formation or oligomerization. In stark contrast to the effects of CCBs, previously published studies have shown that antioxidants lead to inhibition of both Aβ oligomerization and subsequent cell death.