The concomitant inhibition of HDAC and PDE5 has a synergistic effect on histone acetylation
Using primary neuronal cultures, we tested whether the combination of vorinostat (a class I and class IIb HDAC inhibitor) and tadalafil (a PDE5 inhibitor) had a synergistic effect on the induction of histone 3 acetylation at lys9 (AcH3K9), an epigenetic mark implicated in memory enhancement in mice [13, 14]. When we exposed the cultured neurons to different concentrations of vorinostat and tadalafil for 2 h (10, 100 and 500 nM), we detected a strong induction of AcH3K9 by vorinostat at concentrations of 100 and 500 nM, whereas no effect was found with tadalafil (Fig. 1a). Interestingly, we observed a synergistic effect on this epigenetic mark when vorinostat (50 nM) was administered in combination with tadalafil (50 and 200 nM: Fig. 1b). This synergistic effect was confirmed in the SH-SY5Y neuroblastoma cell line and using alpha technology, where AcH3K9 was induced significantly by vorinostat (50 nM) combined with tadalafil (from 160 nM: Fig. 1e; compared with each compound alone in Fig. 1c, d). These data suggest that PDE5 inhibition may provoke CREB phosphorylation and, in turn, the recruitment of CBP, thereby enhancing histone acetylation and memory-related gene transcription (Fig. 1f).
Concomitant inhibition of HDAC and PDE5 had a synergistic effect on long-term potentiation in APP/PS1 transgenic mice
To evaluate the physiological consequences of vorinostat and tadalafil treatment, we first investigated their effects by monitoring long-term synaptic plasticity (LTP), a well-known cellular mechanism that underlies memory processes [15]. Extracellular synaptic activity was recorded in hippocampal slices from APP/PS1 adult mice (7–9 months), showing that the LTP induction protocol evoked similar synaptic potentiation after a brief application of vorinostat (2 μM) to that of control slices treated with the vehicle alone. Likewise, tadalafil (50 nM) preincubation did not significantly affect the degree of LTP in APP/PS1 slices (Fig. 2a, b), yet incubation with a combination of vorinostat and tadalafil did produce robust potentiation of synaptic transmission (Fig. 2a, b), significantly beyond that obtained by application of each inhibitor alone. By contrast, the combination of both inhibitors did not affect LTP in APP/PS1-negative slices compared with control conditions (Fig. 2c, d). Therefore, HDAC and PDE5 inhibitors would appear to have a synergistic effect on LTP in AD mice.
The effects of vorinostat and tadalafil combination therapy on memory function in aged Tg2576 mice
A 2–3-week treatment with vorinostat (50 mg/kg) restored contextual memory deficits in the APPswe/PS1dE9 AD mouse model [16], and likewise, tadalafil (15 mg/kg) improves memory deficits in an aged AD mouse model [17]. Thus, we tested whether the combined administration of lower doses of both of these compounds could restore cognitive deficits in the Tg2576 AD mouse model. To test this hypothesis, we treated 14–16-month-old Tg2576 mice for 4 weeks with tadalafil (1 mg/kg, p.o.) and vorinostat (12.5 mg/kg, i.p.), with either inhibitor alone (tadalafil [1 mg/kg, p.o.] or vorinostat [12.5 mg/kg, i.p.]), or with the vehicle alone (10 % DMSO) daily. We performed an initial pharmacokinetic study of the administration of vorinostat, tadalafil or combined vorinostat and tadalafil at these doses in plasma and brain samples. The brain/plasma ratios 20 min after combined vorinostat and tadalafil administration were 11 % for tadalafil and 5.5 % for vorinostat, corresponding to brain concentrations of 30 and 345.5 nmol/kg (Additional file 1: Figure S1 and Table S1).
Memory function was assessed during the second and third week of treatment using two hippocampal-dependent tasks, contextual fear conditioning (FC) and Morris water maze (MWM) tests, respectively (see scheme in Fig. 3a). Compared to wild-type (WT) mice, Tg2576 mice exhibited severe disruption in freezing behaviour in the FC test 24 h after training (t(19,2) = 5.3, p ≤ 0.001) (Additional file 1: Figure S2a). Memory impairment was rescued to a significant extent by a combined 2-week treatment with vorinostat and tadalafil (F(3,44) = 2.7, p ≤ 0.05), whereas neither of these drugs alone restored fear memory (Fig. 3b). In the MWM, no significant differences were observed among the experimental groups during the visible-platform training phase, indicating that the animals had a comparable ability to perform the task (Additional file 1: Figure S2b). However, Tg2576 animals (14–16 months old) showed severe cognitive impairment in both the acquisition phase (significant main effect of genotype F(1,29) = 13, p ≤ 0.001) and during the probe test on days 7 (t(29) = 2, p = 0.05) and 9 (t(29) = 2.2, p ≤ 0.05: Additional file 1: Figure S2c and 2d). Notably, the combination of vorinostat and tadalafil rescued these memory deficits, evident through the lower escape latencies in the acquisition phase (F(3,316) = 21.3, p ≤ 0.001: Fig. 3c). Interestingly, the group receiving only vorinostat also had lower escape latencies compared to the mice that received the vehicle alone (F(3,316) = 21.3, p ≤ 0.001: Fig. 3c). Nevertheless, during the retention phase on day 9, only the animals that received the combination of vorinostat and tadalafil spent significantly more time than vehicle-treated animals in the correct quadrant (F(3,36) = 2.9, p ≤ 0.05), demonstrating a synergistic effect of the two drugs on spatial memory retention (Fig. 3d).
Finally, the drug treatments were followed by a washout period of 4 weeks, after which mice were re-trained in a reversal phase of the MWM test, placing the platform in the opposite quadrant. The hidden platform training was carried out over 5 days (four trials per day) and followed by a memory retention probe test on day 6. Tg2576 mice treated with the vehicle performed worse than WT mice in both the acquisition (F(1, 28) = 7.0, p ≤ 0.01: Additional file 1: Figure S2e) and the retention phase on day 6 (t(29) = 2.8, p ≤ 0.01: Additional file 1: Figure S2f). By contrast, these transgenic mice treated with the combination therapy displayed a significantly shorter escape latency than the control Tg2576 mice that received the vehicle alone (F(3,196) = 10.5, p ≤ 0.001: Fig. 3e). Once again, the animals that received vorinostat alone also showed lower escape latencies than the Tg2576 mice that receive the vehicle alone (F(3,196) = 10.5, p ≤ 0.05: Fig. 3e). During the probe test, only the group of animals receiving the combination of vorinostat and tadalafil spent significantly more time in the target quadrant compared with the vehicle-treated mice (F(3,36) = 4.0, p ≤ 0.05: Fig. 3f). Together, these results indicate that combination therapy of vorinostat and tadalafil during 4 weeks restored memory impairment in aged Tg2576 mice whose cognition was severely affected. Furthermore, this effect was maintained after a 4-week washout period. Interestingly, the treatment with vorinostat enhances the learning capacity of transgenic mice, yet memory was not consolidated by this drug alone since no differences were detected between Tg2676 mice receiving the vehicle alone or vorinostat during the probe tests.
Effects of vorinostat and tadalafil combination therapy on pathological AD markers in aged Tg2576 mice
Aβ42 was assessed in protein extracts from the parieto-temporal cortex of Tg2576 treated mice by ELISA, and a marked decrease in Aβ42 was observed in the mice that received the combination therapy compared to those that received the vehicle alone (F(3,36) = 3.3, p ≤ 0.05: Fig. 4a). By contrast, no significant differences were found between the mice that received vorinostat or tadalafil and those treated with the vehicle alone (Fig. 4a). Based on the significant decrease in Aβ42 in the animals that received the combination therapy, we explored APP processing in Western blots of the same extracts. No significant differences were found in APP (Fig. 4b) or in the APP C-terminal C99 fragment (CTF: Fig. 4c) between the Tg2576 mice that received the combination therapy or the vehicle alone. We also analyzed the tau phosphorylation in the same extracts using a pTau-specific (Ser-202/Thr-205) antibody normalized to total tau (T46). Compared to WT mice, there was a significant increase in pTau levels (t(7,1) = −3.3, p ≤ 0.01: Additional file 1: Figure S3a) in Tg2576 mice, which decreased following vorinostat treatment (F(3,11) = 8.7, p ≤ 0.05: Fig. 4d). This effect was even more pronounced in the mice that received tadalafil or the combination therapy (F(3,11) = 8.7, p ≤ 0.01). No significant changes were observed in total tau relative to actin among the experimental groups.
We also tested whether the behavioural recovery induced by the combination therapy reflected structural changes, such as the density of dendritic spines. The density of spines on hippocampal CA1 pyramidal neurons was analyzed in Tg2576 mice and their WT littermates using an optimized Golgi impregnation method. In line with previous findings [18], a significantly lower density of apical dendrites was found on CA1 pyramidal neurons in Tg2576 mice than in WT mice (t(70) = 3.9, p ≤ 0.001: Additional file 1: Figure S3b), while treatment with tadalafil, vorinostat or the combination of these drugs reversed the deficit in spine density on apical CA1 dendrites, which returned to control values (F(3,141) = 12.6, p ≤ 0.001: Fig. 4e). These results suggest that the reduction in spine density is reversible even long after disease onset, which might account for the memory improvement observed and maintained for 4 weeks after washout. However, other mechanisms may influence memory recovery since the effect on dendritic spine density in the mice that receive tadalafil alone was not correlated with the behavioural data.
Gene expression induced by vorinostat, tadalafil and the combination of the two in the hippocampus of Tg2576 mice
To better define the pathways and networks enhanced by the different treatments, we adopted a non-parametric approach implemented in the Gene Set Enrichment Analysis (GSEA) software to determine the genes upregulated by these drugs [19]. GSEA allowed us to determine the pathways and gene signatures enriched among 95 gene sets selected on the basis of memory function and related to AD. GSEA was carried out on the ranked lists reflecting the changes in the microarray studies performed, and the enriched gene sets included those participating in aging and AD-related cellular processes and pathways (reactome amyloids, aging brain, highly calcium permeable postsynaptic nicotinic acetylcholine receptors, among others), providing evidence for an AD-related phenotype. Except for the mice that received vorinostat, several of the gene sets enriched by the other treatments were connected directly with CREB-dependent genes (M15359, combination versus saline, p = 0.008 and tadalafil versus saline, p = 0.002; M11370, combination versus saline, p = 0.010 and tadalafil versus saline, p = 0.045; M19129, combination versus saline, p = 0.023). Interestingly, the analysis identified enriched pathways and gene signatures associated with synaptic transmission (M2923) through the genes upregulated in the hippocampus of mice administered the combined therapy (vorinostat and tadalafil, p = 0.042), but not the individual treatments alone (vorinostat or tadalafil). This suggests that the transcriptional consequences of targeting HDAC and PDE5 together may underlie the recovery of memory function observed in the treated animals.