Carbonic anhydrase inhibition selectively prevents amyloid β neurovascular mitochondrial toxicity

Abstract

Mounting evidence suggests that mitochondrial dysfunction plays a causal role in the etiology and progression of Alzheimer's disease (AD). We recently showed that the carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) prevents amyloid β (Aβ)-mediated onset of apoptosis in the mouse brain. In this study, we used MTZ and, for the first time, the analog CAI acetazolamide (ATZ) in neuronal and cerebral vascular cells challenged with Aβ, to clarify their protective effects and mitochondrial molecular mechanism of action. The CAIs selectively inhibited mitochondrial dysfunction pathways induced by Aβ, without affecting metabolic function. ATZ was effective at concentrations 10 times lower than MTZ. Both MTZ and ATZ prevented mitochondrial membrane depolarization and H₂ O₂ generation, with no effects on intracellular pH or ATP production. Importantly, the drugs did not primarily affect calcium homeostasis. This work suggests a new role for carbonic anhydrases (CAs) in the Aβ-induced mitochondrial toxicity associated with AD and cerebral amyloid angiopathy (CAA), and paves the way to AD clinical trials for CAIs, FDA-approved drugs with a well-known profile of brain delivery.

Keywords: Alzheimer's disease; acetazolamide; amyloid β; carbonic anhydrase inhibitors; methazolamide; mitochondria.

Figure 1

Dose–response curve of Aβ and CAIs. Apoptotic cell death was measured by Cell Death ELISA ^plus in neuronal cells (a, b) and microvascular ECs (c, d). Increasing concentrations of CAIs and 10 μM of Aβ42 (a, b) or 50 μM of Aβ40‐Q22 (c, d) were added to the cell cultures. Data in histograms are mean ± SEM of at least three independent experiments

Figure 2

Mitochondrial membrane depolarization induced by Aβ oligomers is prevented by CAIs. Graphs showing mitochondrial membrane potential (ΔΨ) measured by TMRM in SH‐SY5Y neuronal cells (left) and microvascular ECs (right). (a) The monomeric and fibrillar forms of the peptides induced a much lower mitochondrial membrane depolarization. It is interesting that the scrambled form of the peptide did not exert any effect on ΔΨ in any of the cell lines. Aβ42, on its different aggregation states, was always added at 10 μM, while the final concentration of Q22 was 50 μM. (b) ΔΨ in the neuronal cells (left panel) is reduced to about 65% of control cells. The reduction is completely prevented by MTZ and ATZ. In ECs cells (central panel), ΔΨ is reduced to 35% of the control levels by Aβ, and reverted to above 80%, after treatment with the peptide in the presence of CAIs. N‐methyl acetazolamide (100 μM), a structural analog of ATZ unable to inhibit CAs, showed no effect on ΔΨ either under control conditions or in the presence of Aβ, in SH‐SY5Y cells (right panel). Data in histograms are mean ± SEM of, at least, three independent experiments

Figure 3

Increase in mitochondrial H₂O₂ production in response to Aβ and its inhibition in the presence of CAIs. Effect of Aβ on cellular production. (a) Data show the relative amount of H₂O₂ produced by isolated mitochondria expressed as FOC of the control (Ctrl), as measured by Amplex Red in SH‐SY5Y cells (left) and in ECs (right). H₂O₂ production significantly increases when cells are treated with Aβ42 10 μM or Q22 50 μM. The release of H₂O₂ is prevented when CAIs are added together with Aβ. Both the level of H₂O₂ production and the efficiency of its inhibition are more extreme in neuronal cells, compared to ECs. General oxidative stress within the cell, measured by CellROX (b) and DCFDA (c) reagents in neuronal cells (left) and ECs (right), is not significantly affected by Aβ challenge. The addition of CAIs does not significantly change the amount of intracellular ROS present in any of the two cell types. Data in histograms are mean ± SEM of at least three independent experiments

Figure 4

Effect of Aβ on calcium homeostasis and differential impact of CAIs. (a, b) Graphs show mitochondrial calcium accumulated in the presence of Aβ (respectively, Aβ42 10 μM or Q22 50 μM) with or without MTZ (100 or 300 μM) or ATZ (10 or 100 μM) in (a) SH‐SY5Y cells and in (b) ECs. (c, d) Cytoplasmic calcium concentration in the same cell types and under the same treatments is represented. (e) Pretreatment of the cells with the drugs before challenge with Aβ does not significantly affect mitochondrial and cytoplasmic calcium concentration. Data in histograms in (a, b, c, d, and e) are mean ± SEM of at least three independent experiments

Figure 5

Intracellular pH and ATP production are not affected by CAIs. Measurement of the intracellular pH after 3 hr of treatment with the pre‐aggregated peptide is shown in (a) for SH‐SY5Y and for ECs. The same pH measurement after 16 hr of treatment with Aβ without pre‐aggregation is represented in (b) (Aβ42 10 μM or Q22 50 μM). Cellular pH is not significantly affected by Aβ and CAIs (MTZ 100 or 300 μM and ATZ 10 or 100 μM). (c) Bar histograms showing ATP production in response to Aβ peptides or to peptides in the presence of CAIs for SH‐SY5Y and ECs cell cultures. ATP production is measured by a luminometric assay (CellGlo, Promega) and is represented as A.U. Data in histograms are mean ± SEM of, at least, three independent experiments

Figure 6

Protective effect of CAIs on Aβ‐induced CytC release and caspase‐9 activation. ATZ prevents CytC release in response to Aβ in (a) SH‐SY5Y and (b) ECs cells. Images were acquired by confocal microscopy after immunocytochemistry. Chain‐like mitochondrial Cyt C (arrowheads) in healthy cells or diffused cytoplasmic CytC (arrows) in cells undergoing apoptosis is stained in green. Nuclei are marked in blue with Hoechst 33342. Nuclear condensation indicates apoptosis. The protective effect of MTZ and ATZ on caspase‐9 activation is shown in (c) for SH‐SY5Y and (d) for ECs cell cultures. Data in histograms are mean ± SEM of at least three independent experiments