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More than two decades ago, amyloid-beta (Aβ) was first identified as a potential marker of Alzheimer's disease (AD) and, since then, this small self-aggregating peptide has been only considered responsible for neurotoxicity in this pathology (1). Aβ derives from the sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases (2–4) and takes two prevalent forms in humans, Aβ40 and Aβ42. Conversely, alternative processing of APP by α-secretase precludes Aβ formation because this enzyme cleaves the precursor protein within the Aβ sequence. Since α- and β-secretase compete for the same substrate, APP, the imbalance between these two proteolytic pathways is believed to play a crucial role in the pathogenesis of AD.
However, the presence of Aβ in the cerebrospinal fluid of nondemented individuals (5), and in media from neuronal cell cultures (6), has led to the idea that this peptide may have a function in the normal physiology of the central nervous system (CNS) (7). As a matter of fact, recent data have demonstrated that hippocampal long term potentiation (LTP), the neurochemical substrate of learning and memory processes, can be enhanced by low amounts of exogenous Aβ and that the endogenous peptide is necessary for hippocampal LTP induction. Furthermore, the Aβ-mediated modulation of LTP is paralleled by the evident effects on memory functions, as assessed in specific cognitive tasks (8, 9).
At a molecular level, the second messenger cyclic adenosine monophosphate (cAMP) seems to be essentially involved in the complex mechanisms regulating memory (10, 11) since LTP has been found to be required for the activation of the cAMP/cAMP-dependent protein kinase A (PKA)/cAMP response element-binding (CREB) pathway, and its genetic or pharmacological manipulation can affect cognitive functions (12–14).
In addition, impairment of PKA/CREB activity has been observed in the brain of AD patients and AD mouse models (15–17), thus leading to the belief that cAMP-enhancing strategies may be beneficial for the treatment of memory loss in AD and other neurodegenerative diseases.
cAMP is generated from ATP by adenylyl cyclase and is rapidly degraded by phosphodiesterases (PDEs), a superfamily of enzymes classified into 11 families, namely PDE1 to PDE11. Cerebral levels of cAMP critically depend on the activity of PDE4 and it has been extensively demonstrated that PDE4 inhibitors, such as rolipram, can facilitate LTP and improve memory in a variety of behavioral tests, both under physiological and pathological conditions, including AD (18).
Interestingly, scattered evidence indicates that cAMP pathway effectors can stimulate either APP synthesis (19) or its amyloidogenic and nonamyloidogenic processing (20, 21). In this context, to gain further insights into the role of cAMP on the biology of APP, we investigated whether the blockade of endogenous cAMP degradation by the selective PDE4 inhibitor rolipram was able to affect the metabolism of APP in neuronal cultured cells. Our results show, for the first time, that rolipram stimulates APP expression and the secretion of sAPPα, Aβ40, and Aβ42 through the elevation of endogenous cAMP given that its effects are mimicked by the membrane-permeant analog 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt (8Br-cAMP) and by the adenylyl cyclase activator forskolin. Moreover, the rolipram-induced effects were not affected by H-89, suggesting that endogenous cAMP may act through a PKA-independent pathway.
Cell Culture and Treatments
The cells used in this study (mouse Neuro-2a [N2a], stably expressing wild-type human AβPP695) were obtained from Peter Davies (Albert Einstein College of Medicine, Bronx, NY) and grown in 50% Dulbecco modified Eagle's medium, 50% OptiMEM with 0.1 mM nonessential amino acids, 200 μg/ml geneticin, and 5% fetal bovine serum.
8Br-cAMP (prepared as a 187 mM stock solution in sterile water), forskolin (100 mM in dimethyl sulfoxide, DMSO), rolipram (50 mM in DMSO), and H-89 dihydrochloride hydrate (2 mM in sterile water) were obtained from Sigma-Aldrich (Italy) and diluted to the indicated final concentrations immediately before use.
Aβ Enzyme-Linked Immunosorbent Assay (ELISA)
AβX-40 and AβX-42 ELISA kits (Wako Chemicals GmbH, Germany) were used to determine the levels of Aβ peptides released into supernatant media from cultured cells, as described previously (22). Briefly, at the end of treatments, the conditioned media were collected, spun at 1,000g for 10 min at 4 °C to remove cell debris, and stored at −80 °C until use. Immediately after thawing, conditioned media were diluted 30 times with 1× dilution buffer and 100 μl from each sample was used. ELISA tests were carried out following the manufacturer protocols, and the levels of Aβ peptides were calculated according to the standard curves prepared on the same ELISA plates.
Preparation of Protein Extracts and Immunoblot Analysis
Total proteins from cell cultures were extracted as described previously (23). Immunoblots were done according to standard methods, using the following antibodies: monoclonal mouse anti-human APP (22C11 and 6E10, Millipore, Italy), monoclonal mouse anti-β-actin (Sigma-Aldrich), and anti-mouse secondary antibody coupled to horseradish peroxidase (GE Healthcare, UK). Proteins were visualized with an enzyme-linked chemiluminescence detection kit according to the manufacturer's instructions (GE Healthcare). Chemiluminescence was monitored by exposure to films, and signals were analyzed under nonsaturating condition with an image densitometer (Bio-Rad, CA).
N2a cells were grown overnight on culture slides and then treated as indicated. After fixing and permeabilization with ice-cold methanol, nonspecific binding sites were blocked with 3% BSA. Cells were subsequently incubated with the anti-cAMP monoclonal antibody (Abcam, UK, diluted 1:200 in PBS) and subsequently labeled with the secondary antibody Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, Italy) at a 1:500 dilution. Immunostained cells were observed with the appropriate filter on a Bio-Rad 1024 confocal microscope (planapochromat ×60 oil-immersion objective, numerical aperture 1.4).
cAMP Enzymatic Immunoassay
Quantification of intracellular cAMP was performed with DetectX Direct Cyclic AMP Enzyme Immunoassay Kit (Arbor Assay, MI), following the manufacturer's protocol. cAMP levels were calculated according to the standard curves prepared on the same enzymatic immunoassay (EIA) plates.
Results are expressed as means ± S.D. Data were analyzed by two-tailed Student's t test.
In N2a Cells, Rolipram Increases Both Protein Expression and Processing of APP
In the amyloidogenic pathway, processing of APP by β- and γ-secretases leads to the production of Aβ40 and Aβ42, whereas in the alternative nonamyloidogenic pathway, cleavage of APP by α-secretase results in the shedding of nearly the entire ectodomain, yielding a large soluble AβPP derivative called sAPPα Therefore, changes in Aβ or sAPPα levels closely reflect variations of β/γ- or α-mediated cleavage, respectively.
In this context, we analyzed the effects of rolipram on these different aspects of APP metabolism in a mouse neuronal cell line (N2a) stably transfected to express human APP. Conditioned media from N2a exposed for 24 h to increasing rolipram concentrations (0.1–10 μM) were subjected to Aβ-specific sandwich ELISA. The results shown in Figure 1A indicate that the PDE4 selective inhibitor significantly stimulated the release of both Aβ40 and Aβ42 in a concentration-dependent manner.
As shown in Figure 1B, 24 h treatment with rolipram also induced a robust concentration-dependent increase in sAPPα, measured in the conditioned media by immunoblot. Moreover, the enhancement of secreted Aβ and sAPPα was accompanied by a consistent elevation in the content of intracellular APP holoprotein (Fig. 1C).
To verify the possible involvement of PKA in the stimulation of APP metabolism induced by rolipram, we pretreated the cells with H-89, an isoquinolinesulfonamide that acts as a competitive antagonist of ATP at its binding site on the PKA catalytic subunit (24). As shown in Figure 2, 1 μM H-89 was unable to influence the rolipram-mediated effects on either APP expression level or its cleavage products. Higher concentrations of H-89 were not applied to avoid nonselective effects of this compound (25, 26).
Processing of APP in 8Br-cAMP-Treated N2a Cells
To evaluate whether the effects of rolipram were due to cAMP, we analyzed the ability of 8Br-cAMP, a well-known membrane-permeant analog of the second messenger, to influence APP processing.
Figure 1 shows that, when N2a cells were exposed to 8Br-cAMP (1–1,000 μM) for 24 h, a significant, concentration-dependent increase of Aβ40, Aβ42 (Fig. 1A), and sAPPα Fig. 1B) was observed in the culture media. Similar to rolipram, 8Br-cAMP was able to also enhance the protein expression of APP, as measured by immunoblotting of the cell extracts (Fig. 1C).
In line with previous studies performed on non-neuronal cells (19, 20), these data indicate that neuronal cultures increase the expression of APP and both its amyloidogenic and nonamyloidogenic processing in response to 8Br-cAMP treatment.
APP Expression and Processing in Forskolin-Stimulated N2a Cells
We then examined the effect of the adenylyl cyclase activator forskolin to further support the involvement of endogenous cAMP in the modulation of APP metabolism. The results shown in Figure 1 indicate that Aβ40, Aβ42 (Fig. 1A), and sAPPα levels (Fig. 1B), as well as the protein expression of APP (Fig. 1C), were stimulated by forskolin (1 and 10 μM).
Rolipram Increases the Intracellular Levels of cAMP
Finally, to verify the capability of rolipram enhancing the accumulation of cAMP in our cell culture model, we used confocal microscopy analysis. As indicated in Figure 3A, N2a cells exposed for 30 min to 10 μM rolipram showed a substantial increase in cAMP immunofluorescence over the controls (DMSO-treated cells). The efficacy of rolipram on cAMP elevation was further confirmed and quantified by cAMP-specific EIA, which revealed that intracellular cAMP levels had increased by 50% and 100% after a 30-min treatment with 10 and 100 μM rolipram, respectively (Fig. 3B).
This study demonstrates that, in neuronal cultures, the selective PDE4 inhibitor, rolipram, stimulates APP expression and both its amyloidogenic and nonamyloidogenic processing, resulting in the increase of Aβ40, Aβ42, and sAPPα. The effect exerted by rolipram on APP metabolism is likely to be mediated by cAMP, a conclusion supported by these three lines of evidence: 1) the effects of rolipram are mimicked by the cAMP analog 8Br-cAMP; 2) the effects of rolipram are reproduced in increasing endogenous cAMP by way of forskolin-induced activation of its synthesizing enzyme adenylyl cyclase; and 3) intracellular levels of endogenous cAMP are increased in our cell model after rolipram treatment.
Consistent with our findings, previous studies reported that the activation of adrenergic receptors coupled to increased cAMP formation enhances both APP mRNA and holoprotein in cortical astrocytes (19), and that forskolin stimulates the secretion of sAPPα in a pheochromocytoma cell line (27). In addition, cAMP elevation triggered by forskolin was found to activate the α- and β/γ-secretase pathways in HEK293 cells that overexpressed either wild-type or Swedish mutated APP (20).
Our preliminary results with H-89 seem to suggest that PKA is not involved in the rolipram-induced effects on APP and its metabolism. Actually, H-89 was devoid of any effect on basal production of sAPPα in HEK293 cells expressing wild type or mutated forms of APP and presenilin 1, although it decreased Aβ peptide levels (28).
In our experiments, we have used H-89 at 1 μM to maintain its selectivity toward PKA, as higher concentrations of this compound have been reported to inhibit a variety of protein kinases (26). However, this concentration is 20 times higher than the Ki of H-89 reported to inhibit PKA (24) and is similar to those (1–3 μM) found effective in many recent studies investigating the PKA-mediated effects in intact cell systems (29–31). Beside PKA, intracellular cAMP signaling can also be mediated by other effectors, such as the exchange protein activated by cAMP (Epac). Interestingly, a PKA-independent increase of sAPPα has been reported to occur in neuronal cells following stimulation of serotonin 5-HT4 receptors and activation of the cAMP-dependent Epac/Rap1/Rac secretory pathway (32). Experiments are currently underway to find out which molecular mechanisms act downstream of the rolipram-induced accumulation of cAMP and are involved in the stimulation of APP expression and processing.
A large body of evidence demonstrates that cAMP signaling plays a key role in the physiology of memory and suggests that its stimulation can be useful in the treatment of memory loss-related disorders. In particular, elevation of cAMP by rolipram was able to enhance cognitive performances under physiological conditions and has been found effective in ameliorating memory deficits in different experimental models (see ref. 17 for review).
Although it has been suggested that the activation of the PKA/CREB pathway represents the molecular mechanism through which cAMP modulates memory functions, our observation that rolipram and forskolin stimulate the release of sAPPα and Aβ peptides might provide another possible mechanistic explanation. As a matter of fact, sAPPα has been shown to have potent memory-enhancing properties (33–35) and emerging evidence supports the concept that, in the CNS, low (i.e., picomolar) concentrations of Aβ may exert physiological functions on cognitive processes. Indeed, exogenously applied Aβ peptides produce an increase of hippocampal LTP and enhance memory in normal mice (8). Most importantly, endogenously produced Aβ was found to be necessary for the induction of normal LTP and for memory formation in healthy mice (9). Moreover, a neuroprotective role for Aβ peptides has also been reported (36–38).
Therefore, we speculate that, in the normal brain, cAMP favors synaptic plasticity and memory by triggering a physiological production of Aβ and/or sAPPα, a mechanism that may act in concert with, or in addition to, the activation of the PKA/CREB pathway (12).
At first glance, our hypothesis seems difficult to reconcile with the beneficial cognitive effects of rolipram in AD animal models where Aβ is known to impair synaptic plasticity and memory (39–41). However, in AD, Aβ peptides are produced at abnormally elevated concentrations that have been shown to cause the inactivation of PKA, the decrease of CREB phosphorylation in response to glutamate, and the reduction of hippocampal LTP (42). In our opinion, the elevation of cAMP levels by rolipram would normalize the impaired PKA/CREB pathway, thus restoring LTP and improving memory deficits, as shown in a double-transgenic model of AD (17).
Therefore, in AD, the deleterious effects of pathologically high levels of Aβ would prevail over the beneficial ones exerted by the peptides under physiological conditions. In this case, the amelioration of cognitive deficits by low doses of rolipram is likely to be due to the direct rescue of the impaired PKA/CREB pathway.
Memory efficiency, however, seems to require a strictly controlled cAMP production as studies performed on different animal species have shown that dysregulation of adenylyl cyclase or PKA activity can impair memory formation and behavioral performance in cognitive tasks (43). Furthermore, normal rats treated with rolipram at doses of 3 and 30 mg/kg that are much higher than those necessary to improve cognitive performances exhibited memory impairment associated with highly increased cAMP levels (44), an effect that could be due to a cAMP-mediated overstimulation of Aβ production, leading to the well-grounded detrimental effects on LTP and cognitive functions.
In conclusion, we provide further evidence that, in neuronal cells, APP expression and both its amyloidogenic and nonamyloidogenic processing are increased by pharmacological manipulations which elevate the intracellular cAMP levels, including PDE4 inhibition, thus suggesting that this might represent an additional mechanism through which cAMP signaling modulates memory functions. However, excessive elevation of this second messenger could lead to abnormal Aβ production exerting negative effects on cognitive processes. As PDE4 inhibition has been proposed to ameliorate memory decline in AD, our data indicate that detailed dose-escalation studies are necessary to correctly assess the efficacy of this therapeutic strategy in AD patients.
This work was supported by grants from the Alzheimer's Association, Fondazione CARIGE, and PRIN (2009M8FKBB_002 and 2008N9N9KL_002).