Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer’s disease prevention

Recently, we have found that various intracellular cyclic adenosine monophosphate (cAMP)-elevating agents, both pharmacological (dibutyryl-cAMP, forskolin, and rolipram) and physiological (pituitary adenylate cyclase-activating polypeptide), decrease cell-surface levels of 67-kDa laminin receptor (67LR) and cellular prion protein (PrP^C). Thereby, they inhibit the internalization of amyloid-β oligomer (AβO) and attenuate AβO-induced neuronal death (Figure 1; Gopalakrishna et al., 2022). We postulate that the 67LR-PrP^C-mediated AβO mechanism may be important in understanding the Alzheimer’s disease (AD)-preventive actions of green tea polyphenol, epigallocatechin-3-gallate (EGCG), which is known to bind 67LR at a site within the PrP^C-binding site and induce 67LR internalization. This mechanism may also be relevant in understanding the anti-AD actions of dietary agents such as resveratrol and quercetin as well as synthetic drugs (including the ones in clinical trials) that elevate intracellular cAMP by inhibiting cyclic nucleotide phosphodiesterases (PDEs).

Figure 1.

Schematic presentation of a possible mechanism by which cAMP decreases PrP^C-67LR-mediated uptake of AβO and neuronal death and confers protection against AD.

AβO binds to cell-surface associated PrP^C and the resultant AβO-PrP^C complex binds 67LR and internalizes without the need for elevated cAMP. This action leads to the hyperphosphorylation of tau and elevations of both intracellular Ca²⁺ and ROS, which induce neuronal cell death. AβO inhibits PKA and CREB activation. Green tea polyphenol (EGCG), by binding to 67LR at a site within the PrP^C-binding site, antagonizes the AβO-PrP^C binding to 67LR and thus inhibits neuronal uptake of AβO. In addition, EGCG induces endocytosis of 67LR and an elevation of intracellular cAMP. Neuropeptide PACAP and diterpenoid forskolin by activating AC, dietary agents such as resveratrol and quercetin, and synthetic agents such as rolipram by inhibiting PDE elevate intracellular cAMP. In turn, cAMP induces endocytosis of 67LR/PrP^C and depletes these receptors from the cell surface, thus suppressing the internalization of AβO. Inhibition of AβO uptake also facilitates cAMP-dependent activation of PKA/CREB signaling, leading to synaptic plasticity and long-term memory. This proposed anti-AβO mechanism may be relevant in the anti-AD actions of some hormonal, dietary, and synthetic agents which elevate intracellular cAMP. AC: Adenylyl cyclase; AD: Alzheimer’s disease; AβO: Amyloid-β oligomers; cAMP: cyclic adenosine monophosphate; CREB: cyclic adenosine monophosphate-response element-binding protein; EGCG: epigallocatechin-3-gallate; LR: 67-kDa laminin receptor; PACAP: pituitary adenylate cyclase-activating polypeptide; PDE: cyclic nucleotide phosphodiesterase; PKA: protein kinase A; PrP^C: cellular prion protein; ROS: reactive oxygen species. Created with ChemDraw.

Soluble AβO plays an important role in the pathogenesis of AD (Hardy and Selkoe, 2002). AβO are internalized into neurons, causing tau hyperphosphorylation, dysregulation of intracellular Ca²⁺ homeostasis, and increased production of reactive oxygen species. All these detrimental actions lead to synaptic loss and neuronal death. Therefore, the mechanisms by which AβO enters neurons and intracellular signaling that influences neuronal vulnerability versus resistance to AβO-induced cell death are of utmost importance to developing therapeutic agents for the prevention and treatment of AD.

Lipid raft-associated PrP^C is a major high-affinity receptor for AβO (Smith et al., 2019). The resulting AβO-PrP^C complex then binds to various cell-surface co-receptors to initiate signaling for its internalization into neurons to induce synaptic toxicity and neuronal death (Smith et al., 2019). Another lipid raft-associated protein, 67LR is one of the co-receptors for the AβO-PrP^C complex. This AβO-PrP^C complex mediates the internalization of AβO and subsequent neuronal death (Pinnock et al., 2016). Additional receptors for AβO also play important roles in the pathogenesis of AD. AβO binding to the N-methyl-D-aspartate receptor NR2B subunit induces an influx of Ca²⁺ (Liu et al., 2019). AβO also binds with high affinity to microglial TREM2 and induces DAP12-mediated activation of protein tyrosine kinase SYK (Wang et al., 2022). Intracellular signals influencing the cell-surface expression of PrP^C, 67LR, and other AβO receptors are largely unknown.

67LR as a co-receptor for AβO-PrP^C mediating neuronal cell death: Our study using neuroscreen-1 cells, alongside other reported studies done on Neuro 2a cells, show that 67LR is a major co-receptor for AβO-PrP^C complex and is involved in the internalization of AβO into neuronal cells (Pinnock et al., 2016; Gopalakrishna et al., 2022). The blocking of this receptor with antibodies results in a substantial decrease in AβO uptake and AβO-induced neuronal cell death. Since AβO and AβO-PrP^C complex bind to additional receptors, one could expect that 67LR-blocking antibody only moderately inhibits AβO uptake and AβO-induced cell death. We do not know whether 67LR is the most crucial co-receptor or it influences other AβO co-receptors clustered in the lipid rafts.

cAMP-elevating agents decrease cell-surface expression of 67LR and PrP^C, uptake of AβO, and neuronal death: Others have shown that cAMP can protect cortical neurons from death induced by Aβ(25–35) by mechanisms that are unknown (Parvathenani et al., 2000). Previously we had shown that various synthetic cAMP-elevating agents induce endocytosis of 67LR in a protein kinase A (PKA)-dependent manner. Our recent study shows that these agents also decrease cell-surface expression of PrP^C in a PKA-dependent manner (Gopalakrishna et al., 2022). 67LR has a PrP^C-binding site at its “peptide G” region (amino acid residues 161–179). Thus, PrP^C may pre-associate with 67LR in the lipid rafts, enabling cAMP to induce co-internalization of 67LR and PrP^C in a PKA-dependent manner. This action would result in the depletion of the cell surface of PrP^C available for binding with AβO. Consistent with these observations, we found that various synthetic cAMP-elevating agents such as cell-permeable dibutyryl-cAMP, adenylyl cyclase activator forskolin, and PDE inhibitor rolipram all substantially decreased AβO uptake and AβO-induced neuronal cell death. In addition, the physiological neuropeptide, pituitary adenylate cyclase-activating polypeptide also decreased cell-surface levels of both PrP^C and 67LR and attenuated neuronal uptake of AβO and AβO-induced neuronal death in a cAMP/PKA-dependent manner. At this juncture, we do not know whether cAMP also internalizes other co-receptors that bind the AβO-PrP^C complex. Previously, we found that cAMP also induces the internalization of the cell-surface Nogo-A receptor, NgR1, which directly binds AβO (Gopalakrishna et al., 2020). Therefore, cAMP may have a profound suppressive effect on neuronal AβO uptake and subsequent AβO-induced neuronal death.

A decrease in internalization of AβO may enhance cAMP-induced activation of cAMP response element-binding protein (CREB): cAMP/PKA signaling induces phosphorylation and activation of CREB, a transcription factor regulating the expression of genes involved in synaptic plasticity and long-term memory. A decrease in the neuronal uptake of AβO by cAMP and an increase in CREB phosphorylation by cAMP may cooperate to promote neuro- and synaptoprotection against AβO toxicity effectively. Previous studies have shown that AβO decreases PKA activity and CREB activation in neurons (Vitolo et al., 2002). Therefore, initial cAMP signaling leading to a decrease in neuronal uptake of AβO will limit AβO-induced inhibition of PKA and CREB, thus facilitating cAMP-mediated activation of CREB and subsequent transcriptional activation of cognitive and neuroprotective genes. It is likely that cAMP-induced activation of CREB may also protect neurons from the toxic effects of internalized AβO by 67LR-independent pathways.

Dysregulation of cAMP signaling in Alzheimer’s disease: The cAMP-induced AβO-counteractive mechanism may be particularly relevant in preventing the pathogenesis of AD as cAMP-related signaling is dysregulated in AD. A previous study has found lower cAMP, adenylyl cyclase, and PKA levels in various brain regions, including the hippocampus in AD patients and mouse models compared to healthy age-matched controls (Kelly, 2018). Pituitary adenylate cyclase-activating polypeptide, which elevates intracellular cAMP, is lower in AD patients relative to age-matched healthy people.

The implication of cAMP-mediated anti-AβO mechanism in the therapeutic actions of some dietary and synthetic agents targeting AD: In AD animal models, green tea polyphenol, EGCG, was shown to improve cognition (Youn et al., 2022). EGCG binds with high affinity to 67LR at its peptide G region (amino acid residues 161–170) (Fujimura et al., 2022). This EGCG-binding site is located within the PrP^C-binding site on 67LR (amino acid residues 161–179). Therefore, EGCG may effectively antagonize the binding of the AβO-PrP^C complex with 67LR. In addition, EGCG induces the endocytosis of 67LR, elevates intracellular cAMP, and triggers the activation of CREB. It is intriguing that the same 67LR, which serves as a co-receptor for AβO-PrP^C to induce neurotoxicity, also serves as a receptor for EGCG to elicit neuroprotection. Thus, the proposed anti-AβO mechanism may be very relevant for understanding the protective role of EGCG in AD.

Other dietary agents, such as resveratrol and quercetin, are protective against AD in various transgenic mouse models. These agents also induce an elevation of cAMP by inhibiting PDEs. In preclinical models of AD, numerous synthetic PDE inhibitors have been shown to cause remarkable improvement in cognition. Currently, various PDE inhibitors are under clinical trials for treating AD and mild cognitive impairment.

Excess pharmacological global activation of cAMP pathways can cause cognitive decline, hyperexcitability, and hyperalgesia (Gopalakrishna et al., 2022). However, by selecting the appropriate isoenzyme-specific PDE inhibitor, it is possible to enhance cAMP levels in specific cellular compartments to induce internalization of 67LR and PrP^C and thus decrease AβO uptake. Alternatively, dietary agents which moderately elevate cAMP may have desired therapeutic benefits in AD.

In summation, besides the rate of production and clearance of Aβ in the brain, the intrinsic neuronal susceptibility/resistance to AβO may influence the onset and progression of AD. Our studies show that by elevating intracellular cAMP, it is possible to decrease the cell-surface expression of AβO receptors and co-receptors (PrP^C and 67LR) to suppress neuronal uptake of AβO and AβO-induced neuronal death while enhancing cAMP signaling for CREB activation to protect from synaptotoxicity induced by AβO. Therefore, the above cAMP-dependent dual mechanism may underlie the AD-targeted therapeutic potential of several hormonal, dietary, and synthetic agents that elevate intracellular cAMP.

This work was supported by NIH grants, NINDS R21 NS116720 and NINDS/NIA RF1 NS130681 (to RG and WJM) and NIA R21 AG059422 (to NRB).