Role of Cholesterol Metabolic Enzyme CYP46A1 and Its Metabolite 24S-Hydroxycholesterol in Ischemic Stroke

Abstract

Background: For several decades, it has been recognized that overactivation of the glutamate-gated N-methyl-D-aspartate receptors (NMDARs) and subsequent Ca²⁺ toxicity play a critical role in ischemic brain injury. 24S-hydroxycholesterol (24S-HC) is a major cholesterol metabolite in the brain, which has been identified as a potent positive allosteric modulator of NMDAR in rat hippocampal neurons. We hypothesize that 24S-HC worsens ischemic brain injury via its potentiation of the NMDAR, and reducing the production of 24S-HC by targeting its synthetic enzyme CYP46A1 provides neuroprotection.

Methods: We tested this hypothesis using electrophysiological, pharmacological, and transgenic approaches and in vitro and in vivo cerebral ischemia models.

Results: Our data show that 24S-HC potentiates NMDAR activation in primary cultured mouse cortical neurons in a concentration-dependent manner. At 10 µmol/L, it dramatically increases the steady-state currents by 51% and slightly increases the peak currents by 20%. Furthermore, 24S-HC increases NMDA and oxygen-glucose deprivation-induced cortical neuronal injury. The increased neuronal injury is largely abolished by NMDAR channel blocker MK-801, suggesting an NMDAR-dependent mechanism. Pharmacological inhibition of CYP46A1 by voriconazole or gene knockout of Cyp46a1 dramatically reduces ischemic brain injury.

Conclusions: These results identify a new mechanism and signaling cascade that critically impacts stroke outcome: CYP46A1 → 24S-HC → NMDAR → ischemic brain injury. They offer proof of principle for further development of new strategies for stroke intervention by targeting CYP46A1 or its metabolite 24S-HC.

Keywords: 24-hydroxycholesterol; brain ischemia; cholesterol; cholesterol 24-hydroxylase; stroke.

Figure 1.. Effect of 24S-HC on ionotropic glutamate receptor currents in primary cultured mouse cortical neurons.

The effect of 24S-HC on the currents of three ionotropic glutamate receptors, including NMDA, KA, and AMPA, are examined. Membrane potential was clamped at −60 mV. The currents were induced by exposing the cells to 100 μM NMDA, KA, or AMPA for 4s with or without 24S-HC. (A) Representative current traces (upper panel) show that 24S-HC (0.1, 0.3, 1, 3, and 10 μM) concentration-dependently potentiates NMDAR currents in primary cultured mouse cortical neurons. The lower panel shows an expanded view of superimposed NMDAR current traces with or without 24S-HC (0.1, 1, and 10 μM). (B) The concentration-response curves for 24S-HC on NMDAR peak (blue) and steady state (red) currents (n=5). (C-F) Representative current traces and summary data show that 24S-HC (10 μM) does not affect KA and AMPA receptor currents. The KA and AMPA receptor channel inhibitor CNQX (20 μM) was used as a positive control.

Figure 2.. 24S-HC increases NMDA toxicity in primary cultured mouse cortical neurons.

Viability and cytotoxicity were measured by FDA, PI staining, and LDH assays at 24h following 30 min NMDA (100 μM) treatment. (A) Representative phase-contrast images (left panel) and FDA (green)/PI (red) staining images of alive/dead neurons (right panel) at 24 h following NMDA treatment in the presence or absence of 24S-HC (10 μM) as indicated. (B) LDH assay shows the effect of 24S-HC on NMDA toxicity. NMDA channel blocker MK-801 (10 μM) was used as a positive control (n=8, ^##p<0.01 compared with control, *p<0.05 and **p<0.01 compared with NMDA, one-way ANOVA with Bonferroni’s multiple comparisons test). (C) LDH assay shows the effect of 24S-HC on NMDA toxicity in the presence of MK-801 (n=8, ^##p<0.01 compared with control, **p<0.01 compared with NMDA, one-way ANOVA with Bonferroni’s multiple comparisons test).

Figure 3.. 24S-HC increases OGD toxicity in primary cultured mouse cortical neurons.

Cytotoxicity was measured by LDH assay at 24h following 1.5h OGD treatment. (A) Representative phase contrast images and (B) LDH assay showing the effect of 24S-HC (1 and 10 μM) on OGD-induced neuronal injury in the presence or absence of NMDA channel blocker MK-801 (n=8, ^##p<0.01 compared with control, **p<0.01 compared with OGD, one-way ANOVA with Bonferroni’s multiple comparisons test).

Figure 4.. Inhibition of CYP46A1 reduces ischemic brain injury.

TTC staining was performed at 24h after 45 min MCAO. Vori (40 μM stock solution, 1 μl) or vehicle (0.4% ethanol in saline, 1 μl) were administrated through intracerebroventricular injection 3h before MCAO. (A-B) TTC staining shows that Vori reduces the infarct volume in male WT mice (n=5, **p<0.01, unpaired Student’s t-test).

Figure 5.. Knockout of Cyp46a1 reduces ischemic brain injury.

TTC staining and neurological deficits were evaluated 24h after 45 min MCAO. (A-B) TTC staining and summary data showing reduced infarct volume in male Cyp46a1^−/− mice compared with male WT mice (n=6, **p<0.01 compared with WT mice, unpaired Student’s t-test). (C) Neurological function evaluation showing reduced deficits (lower score) in Cyp46a1^−/− mice (n=6, **p<0.01 compared with WT mice, unpaired Student’s t-test). (D-F) TTC staining and summary data showing reduced infarct volume and neuronal functional deficits in female Cyp46a1^−/− mice compared with WT mice (n=5, **p<0.01 compared with WT mice, unpaired Student’s t-test).