Sildenafil Decreases BACE1 and Cathepsin B Levels and Reduces APP Amyloidogenic Processing in the SAMP8 Mouse.

The senescence-accelerated mouse-prone 8 (SAMP8), used as a model of aging, displays many established pathological features of Alzheimer's disease. Cognitive impairments and increased levels of hyperphosphorylated tau are found in the hippocampus of SAMP8 mice along with an increased ß-secretase activity and amyloid-ß (Aß) depositions that increase in number and extent with age. Based on a previous study from our laboratory showing an amelioration of cognitive impairments and tau pathology by sildenafil, in this study we tested whether this drug could also modulate the amyloid precursor protein amyloidogenic processing in this mouse model. Our results show that the protein levels of the ß-secretases ß-site amyloid precursor protein cleaving enzyme 1 and cathepsin B are higher in the hippocampus of 9-month-old SAMP8 mice than those of age-matched senescence-resistant-1. Sildenafil (7.5mg/kg for 4 weeks) attenuated learning and memory impairments shown by SAMP8 mice in the passive avoidance test. The increased expression of ß-site amyloid precursor protein cleaving enzyme 1 was also reduced by sildenafil, an effect paralleled to decreases in the activities of two ß-site amyloid precursor protein cleaving enzyme 1 modulators, calpain and cyclin-dependent kinase 5 protein. Interestingly, sildenafil enhanced both Akt and glycogen synthase kinase-3ß (ser9) phosphorylation, which could be mediating the reduction in cathepsin B levels found in the hippocampus of sildenafil-treated SAMP8 mice. Sildenafil-induced reduction in ß-site amyloid precursor protein cleaving enzyme 1 and cathepsin B expression in SAMP8 mice was associated with a decrease in hippocampal Aß42 levels which, in turn, could mediate the parallel decline in glial fibrillary acidic protein expression observed in these animals. These findings highlight the therapeutic potential of sildenafil in Alzheimer's disease pathogenesis.

Implication of JNK pathway on tau pathology and cognitive decline in a senescence-accelerated mouse model.

The senescence accelerated mouse-prone 8 (SAMP8) strain of mice is an experimental model of accelerated senescence that also shares several pathological features with Alzheimer's disease. Among them, cognitive impairments and abnormal hyperphosphorylation of tau are ameliorated by the phosphodiesterase 5 inhibitor sildenafil, possibly through the modulation of Cdk5/p25 and Akt/GSK-3ß pathways. Here we studied the implication of protein phosphatase 2A (PP2A) and c-Jun N-terminal kinase (JNK) in the therapeutic effects of sildenafil. Results demonstrated that there were no differences in hippocampal PP2A protein levels or activity (measured by its inactive isoform phopho-PP2A Y307) when we compared 6-month old SAMP8 mice and age-matched control, SAMR1 mice, treated with saline or sildenafil (7.5mg/kg i.p. for 4 weeks). However, this same treatment of sildenafil, that had been shown to reverse the cognitive impairment and tau hyperphosphorylation in this animal model, also reversed the increased levels of activated JNK (p-JNK) found in the hippocampus of SAMP8 mice. Moreover, the administration of the JNK inhibitor, D-JNKI-1 (0.2mg/kg i.p. for 3 weeks) also ameliorated the cognitive deficits shown by SAMP8 mice in the Morris water maze and decreased hippocampal levels of phospho-c-Jun(Ser73). When phosphorylated tau (AT8 epitope) was analyzed a significant reduction was observed in the hippocampus of D-JNKI-1 treated SAMP8 mice, providing a plausible explanation for the attenuation of cognitive decline shown by these animals. These findings suggest the involvement of the JNK pathway on tau pathology and cognitive deficits shown by 6-month old SAMP8 mice. They also point to the modulation of this kinase to be among the mechanisms responsible for the beneficial effects shown by sildenafil.

Inhibition of calpain-regulated p35/cdk5 plays a central role in sildenafil-induced protection against chemical hypoxia produced by malonate.

Phosphodiesterase 5 (PDE5) inhibitors have recently been reported to exert beneficial effects against ischemia-reperfusion injury in several organs but their neuroprotective effects in brain stroke models are scarce. The present study was undertaken to assess the effects of sildenafil against cell death caused by intrastriatal injection of malonate, an inhibitor of succinate dehydrogenase; which produces both energy depletion and lesions similar to those seen in cerebral ischemia. Our data demonstrate that sildenafil (1.5mg/kg by mouth (p.o.)), given 30min before malonate (1.5µmol/2µL), significantly decreased the lesion volume caused by this toxin. This protective effect can be probably related to the inhibition of excitotoxic pathways. Thus, malonate induced the activation of the calcium-dependent protease, calpain and the cyclin-dependent kinase 5, cdk5; which resulted in the hyperphosphorylation of tau and the cleavage of the protective transcription factor, myocyte enhancer factor 2, MEF2. All these effects were also significantly reduced by sildenafil pre-treatment, suggesting that sildenafil protects against malonate-induced cell death through the regulation of the calpain/p25/cdk5 signaling pathway. Similar findings were obtained using inhibitors of calpain or cdk5, further supporting our contention. Sildenafil also increased MEF2 phosphorylation and Bcl-2/Bax and Bcl-xL/Bax ratios, effects that might as well contribute to prevent cell death. Finally, sildenafil neuroprotection was extended not only to rat hippocampal slices subjected to oxygen and glucose deprivation when added at the time of reoxygenation, but also, in vivo when administered after malonate injection. Thus, the therapeutic window for sildenafil against malonate-induced hypoxia was set at 3h.

Sildenafil ameliorates cognitive deficits and tau pathology in a senescence-accelerated mouse model.

Aging is associated with a deterioration of cognitive performance and with increased risk of neurodegenerative disorders. In the present study we tested whether the specific phosphodiesterase 5 inhibitor sildenafil could ameliorate the age-dependent cognitive impairments shown by the senescence-accelerated mouse prone-8 (SAMP8). Sildenafil administration (7.5 mg/kg for 4 weeks) to 5-month-old SAMP8 mice attenuated spatial learning and memory impairments shown by these mice in the Morris Water Maze. Tau hyperphosphorylation (AT8 but not PHF-1 epitope) shown by SAMP8 mice at this age was also decreased in the hippocampus of sildenafil-treated mice, an effect probably related to a decrease in cyclin-dependent kinase 5 protein expression and activity (p25/p35 ratio). Interestingly, sildenafil also phosphorylated Akt, which was associated with an increase of glycogen synthase kinase-3ß phosphorylation, providing a plausible explanation for the reductions in tau hyperphosphorylation (AT8 and PHF-1 epitopes) and attenuation of cognitive deficits shown by 9-month-old SAMP8 mice. Overall, sildenafil might be beneficial in age-related brain dysfunction and could be an emerging candidate for the treatment of other neurodegenerative diseases.

Long-lasting neuroprotective effect of sildenafil against 3,4-methylenedioxymethamphetamine- induced 5-hydroxytryptamine deficits in the rat brain.

Sildenafil, given shortly before 3,4-methylenedioxymethamphetamine (MDMA), affords protection against 5-hydroxytryptamine (5-HT) depletions caused by this amphetamine derivative by an acute preconditioning-like mechanism. Because acute and delayed preconditionings do not share the same mechanisms, we investigated whether sildenafil would also protect the 5-HT system of the rat if given 24 hr before MDMA. For this, MDMA (3 × 5 mg/kg i.p., every 2 hr) was administered to rats previously treated with sildenafil (8 mg/kg p.o.). One week later, 5-HT content and 5-HT transporter density were measured in the striatum, frontal cortex, and hippocampus of the rats. Our findings indicate that sildenafil afforded significant protection against MDMA-induced 5-HT deficits without altering the acute hyperthermic response to MDMA or its metabolic disposition. Sildenafil promoted ERK1/2 activation an effect that was paralleled by an increase in MnSOD expression that persisted 24 hr later. In addition, superoxide and superoxide-derived oxidants, shown by ethidium fluorescence, increased after the last MDMA injection, an effect that was prevented by sildenafil pretreatment. Similarly, MDMA increased nitrotyrosine concentration in the hippocampus, an effect not shown by sildenafil-pretreated rats. In conclusion, our data demonstrate that sildenafil produces a significant, long-lasting neuroprotective effect against MDMA-induced 5-HT deficits. This effect is apparently mediated by an increased expression of MnSOD and a subsequent reduced susceptibility to the oxidative stress caused by MDMA.

Sildenafil protects against 3-nitropropionic acid neurotoxicity through the modulation of calpain, CREB, and BDNF.

In this study we tested whether phosphodiesterase 5 (PDE5) inhibitors, sildenafil and vardenafil, would afford protection against 3-nitropropionic acid (3NP), which produces striatal lesions that closely mimic some of the neuropathological features of Huntington's Disease (HD). The neurotoxin was given over 5 days by constant systemic infusion using osmotic minipumps. Animals treated with PDE5 inhibitors (sildenafil or vardenafil) showed improved neurologic scores, reduced the loss of striatal DARPP-32 protein levels and lesion volumes, and decreased calpain activation produced by 3NP. This protective effect was independent of changes in 3NP-induced succinate dehydrogenase inhibition. Furthermore, striatal p-CREB levels along with the expression of BDNF were significantly increased in sildenafil-treated rats. In summary, PDE5 inhibitors protected against 3NP-induced striatal degeneration by reducing calpain activation and by promoting survival pathways. These data encourage further evaluation of PDE5 inhibitors in transgenic mouse models of HD.