Amyloid-β inhibits No-cGMP signaling in a CD36- and CD47-dependent manner

Abstract

Amyloid-β interacts with two cell surface receptors, CD36 and CD47, through which the matricellular protein thrombospondin-1 inhibits soluble guanylate cyclase activation. Here we examine whether amyloid-β shares this inhibitory activity. Amyloid-β inhibited both drug and nitric oxide-mediated activation of soluble guanylate cyclase in several cell types. Known cGMP-dependent functional responses to nitric oxide in platelets and vascular smooth muscle cells were correspondingly inhibited by amyloid-β. Functional interaction of amyloid-β with the scavenger receptor CD36 was indicated by inhibition of free fatty acid uptake via this receptor. Both soluble oligomer and fibrillar forms of amyloid-β were active. In contrast, amyloid-β did not compete with the known ligand SIRPα for binding to CD47. However, both receptors were necessary for amyloid-β to inhibit cGMP accumulation. These data suggest that amyloid-β interaction with CD36 induces a CD47-dependent signal that inhibits soluble guanylate cyclase activation. Combined with the pleiotropic effects of inhibiting free fatty acid transport via CD36, these data provides a molecular mechanism through which amyloid-β can contribute to the nitric oxide signaling deficiencies associated with Alzheimer's disease.

Figure 1. Fibrillar and soluble Aβ inhibit cellular myristate uptake but not SIRPα binding to CD47.

[³H]-Myristic acid uptake after 5 min into human aortic VSMC (A), HUVEC (B), and microglial cells (C) was determined in the presence of the indicated concentrations of Aβ (soluble or fibrillar) after lysis by liquid scintillation counting. D ¹²⁵I-SIRPα-Fc binding to Jurkat T-cells was measured in the presence of soluble Aβ (0.1-10 µM) or a CD47-specific function-blocking antibody (B6H12) for 1 hour at 25°C.

Figure 2. Aβ inhibits NO-induced cGMP synthesis and function.

A BAEC were pretreated with 10 µM Aβ followed by 10 µM DEA/NO. B HUVEC were pretreated with 10 µM Aβ followed by 90 µM IBMX and 10 µM DEA/NO. C Jurkat cells were pretreated with 10 µM Aβ followed by an NO-independent sGC activator (BAY 41-2272). D Jurkat cells were pretreated with Aβ (0.1–100 µM) followed by 1 µM DEA/NO. E Porcine VSMC were pretreated with Aβ (0.1–10 µM) followed by 10 µM DEA/NO. Following treatment, cells were lysed and assayed for cGMP production. n = 3, * denotes P<0.05. F Porcine VSMC adhesion to collagen coated wells was assessed in the presence of Aβ (0.01–10 µM) for 1 hour. G Thrombin(0.2 U)-induced aggregation of washed human platelets was assessed in the absence (control) or presence of DEA/NO (0.01 µM) and with a 5 min pretreatment of Aβ (10 µM) followed by DEA/NO.

Figure 3. Aβ inhibition of NO signaling is dependent on CD36.

A Jurkat cells or B BAEC were incubated with 10 µM CD36 antisense morpholino or 10 µM 5-mis-splice CD36 control morpholino for 48 hrs. Following CD36 knockdown, cells were pretreated with 10 µM Aβ followed by 10 µM DEA/NO. Following treatment, cell were lysed and assayed for cGMP production. n = 3, * denotes P<0.05.

Figure 4. Aβ inhibition of NO signaling is dependent on CD47.

A Wild type or CD47−/− primary murine lung endothelial cells were pretreated with 10 µM Aβ followed by 10 µM DEA/NO. B Wild type Jurkat cells were incubated with 10 µM CD47 antisense morpholino or 10 µM of a 5 base mismatched CD47 control morpholino for 48 hrs. Following CD47 knockdown, cells were pretreated with 10 µM Aβ followed by 1 µM DEA/NO. Following treatment, cell were lysed and assayed for cGMP production. n = 3, * denotes P<0.05.

Figure 5. Aβ inhibition of NO signaling is not dependent on TSP1.

A Wild type or TSP1−/− primary murine lung endothelial cells were pretreated with 10 µM Aβ followed by 10 µM DEA/NO. Following treatment, cell were lysed and assayed for cGMP production. n = 3, * denotes P<0.05.

Figure 6. Proposed model of Aβ inhibition of cGMP production.

Aβ binds directly to CD36 to inhibit uptake of free fatty acids. In the presence of CD47, CD36 engagement transduces an inhibitory signal to sGC, limiting its activation and production of cGMP.