Hypoxia Inducible Factor-1α binds and activates γ-secretase for Aβ production under hypoxia and cerebral hypoperfusion

Abstract

Hypoxic-ischemic injury has been linked with increased risk for developing Alzheimer's disease (AD). The underlying mechanism of this association is poorly understood. Here, we report distinct roles for hypoxia-inducible factor-1α (Hif-1α) in the regulation of BACE1 and γ-secretase activity, two proteases involved in the production of amyloid-beta (Aβ). We have demonstrated that Hif-1α upregulates both BACE1 and γ-secretase activity for Aβ production in brain hypoxia-induced either by cerebral hypoperfusion or breathing 10% O₂. Hif-1α binds to γ-secretase, which elevates the amount of active γ-secretase complex without affecting the level of individual subunits in hypoxic-ischemic mouse brains. Additionally, the expression of full length Hif-1α increases BACE1 and γ-secretase activity in primary neuronal culture, whereas a transcriptionally incompetent Hif-1α variant only activates γ-secretase. These findings indicate that Hif-1α transcriptionally upregulates BACE1 and nontranscriptionally activates γ-secretase for Aβ production in hypoxic-ischemic conditions. Consequently, Hif-1α-mediated Aβ production may be an adaptive response to hypoxic-ischemic injury, subsequently leading to increased risk for AD. Preventing the interaction of Hif-1α with γ-secretase may therefore be a promising therapeutic strategy for AD treatment.

Fig. 1. Ischemic condition leads to an accumulation of Hif-1a in neurons in BCAS mice.

A Representative western blot for Hif-1α in brains of sham and BCAS (week 1, 2,and 4) post-surgery. B Localization of Hif-1α in sham and BCAS brains was assessed by triple immunofluorescence staining. Fluorescence microscopy of Hif-1α (purple) NeuN (green), and GFAP (red) of the hippocampus is shown. Scale bar = 200 μm. C Left, quantification of Hif-1α expression in the hippocampus of sham vs. BCAS animals. Right, Hif-1α/NeuN and Hif-1α/GFAP colocalization quantification in the hippocampus of Sham vs. BCAS animals. Data are mean ± s.d. p-values were determined by two-sided Student’s t-test.

Fig. 2. γ-Secretase activity is upregulated in the ischemia mouse model.

A Sham vs. Bilateral Common Carotid Artery Stenosis (BCAS) animals were sacrificed week 1, 2,and 4 post-surgery. n = 4–6 mice per group. γ-Secretase activity was assessed using membrane fractions of the brain from 1, 2 and 4week post-surgery for Aβ₄₀ and Aβ₄₂. B γ-secretase for Notch1 cleavage at 2-weeks BCAS and sham brains. C Representative western blot for BACE1 and γ-secretase components NCT, Aph1, PS1-NTF, and PS1-CTF from Sham and BCAS mice 4 weeks post-surgery. D Quantification of western blots for BACE1. E Chemical structure of JC-8 photoprobe and western blot for PS1-NTF after γ-secretase active-site directed photolabeling using JC-8 on Sham and BCAS mice 4 weeks post-surgery. Quantification of photolabeling in Sham and BCAS, 4 weeks post-surgery. F Chemical structure of LZ26. Hif-1α expression in 4-week was confirmed in brain membrane that was used for LZ26 capture as input. Hif-1α is pulled down with LZ26 in 4-week BCAS animals and not sham control animals. Specificity of capture is demonstrated by addition of parent compound L685,458. Data are mean ± s.d. p-values were determined by two-sided Student’s t-test.

Fig. 3. Ischemia hypoxia does change the expression and activity of ADAM-10 and ADAM-17.

A The expression of ADAM-10 and −17 in BCAS and sham mouse brains (2 week) mRNA expression was assessed by qPCR. Data are mean ± s.d. B Western blot analysis of expression of pre- and mature forms of ADAM-10 and −17 in BCAS and sham mouse brains (2 week). C Quantitative analysis of ADAM10 (C) and ADAM 17 (D) Data are mean ± s.d.

Fig. 4. Hypoxia increases γ-secretase activity and BACE1 expression in mice.

A Western blot for Hif-1α in brains of wildtype 3-4 months old male C57BL/6 mice subjected to atmospheric hypoxia of 10% O₂ for 24 h. B γ-secretase activity was assessed for Aβ₄₀ and Aβ₄₂ in mice housed in hypoxia (10% O₂) for 24 h compared to normal (20% O₂) oxygen conditions. n = 4 mice for each group. C γ-secretase components, NCT, Aph1, PS1-NTF, PS1-CTF, and Pen2 and BACE1 from pooled brain membrane from hypoxia and normal oxygen mice from 2 A were assessed by Western blot. D Quantification of BACE1 expression levels normalized to normoxia controls. Data are mean ± s.d. p-values were determined by two-sided Student’s t-test.

Fig. 5. Non-transcriptional Hif-1α increases γ-secretase activity in primary neurons.

A Mouse primary cortical neurons were placed in hypoxia (5% O₂) at DIV14 for 48 hours. Media was collected and Aβ₄₀ and Aβ₄₂ was measured. B γ-secretase activity was assessed for Aβ₄₀ and Aβ₄₂ in mouse primary cortical neurons in hypoxia (5% O₂) and normal (20% O₂) oxygen conditions. C Schematic of Hif-1α domains and constructs. D Western blot analysis for Hif-1α in primary neurons at DIV 14. E Mouse cortical neurons expressing Hif-1α variants were assessed for Aβ production in the media at DIV 14. Aβ₄₀— F Mouse cortical neurons expressing Hif-1α variants were assessed for γ-secretase activity at DIV 14. G Representative western blot analysis for BACE1 expression in mouse cortical neurons expressing Hif-1α variants. H Quantification of BACE1 expression normalized to EV control. I mRNA expression was assessed by qPCR for BACE1, EV vs. Hif-1α TM. Data are mean ± s.d. p-values were determined by one-way ANOVA followed by Tukey.

Fig. 6. Dual roles of Hif-1α activate γ-secretase and increase BACE1 through distinct mechanisms.

Cerebrovascular or cardiovascular diseases cause hypoxia and an accumulation Hif-1α in neurons, enhancing BACE1 and γ-secretase through transcriptional and non-transcriptional mechanisms, respectively. Hif-1α-mediated Aβ production acts as an adaptive response to hypoxic-ischemic injury for neuronal protection and this potentially beneficial effect of Aβ may become detrimental in the setting of AD.