An experimental medicine study of the phosphodiesterase-4 inhibitor, roflumilast, on working memory-related brain activity and episodic memory in schizophrenia patients.

RATIONALE: Schizophrenia is associated with impairments in cognitive functioning yet there are no approved drugs to treat these deficits. OBJECTIVES: Based on animal models, we investigated the potential for roflumilast, a selective inhibitor of phosphodiesterase type 4 (PDE4), to improve cognition, which may act by increasing intracellular cyclic adenosine monophosphate in brain regions underlying cognitive deficits in schizophrenia. METHODS: This study consisted of a randomised, double-blind, placebo-controlled, crossover design involving 15 schizophrenia patients. In 3 treatment periods, patients were given 8 days of placebo or one of the two doses of roflumilast (100 and 250 µg daily) with 14 days of washout between treatments. The primary endpoints were dorsolateral prefrontal cortex (DLPFC) activation during a visuospatial working memory task measured with fMRI on dosing day 8 and verbal memory and working memory performance change from baseline to day 8. Least square mean change scores were calculated for behavioural outcomes; fMRI data were analysed in SPM12 with bilateral DLPFC as regions of interest. RESULTS: Verbal memory was significantly improved under 250 µg roflumilast (effect size (ES) = 0.77) compared to placebo. fMRI analyses revealed that increasing dose of roflumilast was associated with reduction of bilateral DLPFC activation during working memory compared to placebo, although this was not statistically significant (ES = 0.31 for the higher dose). Working memory was not improved (ES = 0.03). CONCLUSIONS: Results support the mechanistic validation of potential novel strategies for improving cognitive dysfunction in schizophrenia and suggest that PDE4 inhibition may be beneficial for cognitive dysfunction in schizophrenia. TRIAL REGISTRATION: NCT02079844 .

Acute treatment with the PDE4 inhibitor roflumilast improves verbal word memory in healthy old individuals: a double-blind placebo-controlled study.

There is ample evidence that phosphodiesterase 4 (PDE4) inhibition can improve memory performance in animal studies. In the present study, we examined the acute effects of the PDE4 inhibitor roflumilast on memory performance in healthy individuals (60-80 years of age). We tested the effects of acute roflumilast administration (100, 250, 1000 µg) in a double-blind, placebo-controlled, 4-way crossover design. Participants were first screened for their verbal word memory performance to ensure normal memory performance (within 0.5 standard deviation from norm score; n = 20) Drug effects on memory performance were tested in a verbal memory test and a spatial memory test. Reported side effects of drug treatment were registered. Roflumilast (100 µg) improved the delayed recall performance of the participants (Cohen's d, 0.69). No effects were observed in the spatial memory task. Roflumilast was well tolerated at this low dose. Although no clear adverse side effects were reported at the low dose, mild adverse events (including headache, dizziness, insomnia, and diarrhea) were reported after the 1000 µg dose. The present study provides first evidence that the PDE4 inhibitor roflumilast improves verbal memory performance in old participants. The current data encourage further development of PDE4 inhibitors for improving memory.

The melatonin receptor MT1 is required for the differential regulatory actions of melatonin on neuronal 'clock' gene expression in striatal neurons in vitro.

Through inhibitory G protein-coupled melatonin receptors, melatonin regulates intracellular signaling systems and also the transcriptional activity of certain genes. Clock genes are proposed as regulatory factors in forming dopamine-related behaviors and mood and melatonin has the ability to regulate these processes. Melatonin-mediated changes in clock gene expression have been reported in brain regions, including the striatum, that are crucial for the development of dopaminergic behaviors and mood. However, it is not known whether melatonin receptors present in striatum mediate these effects. Therefore, we investigated the role of the melatonin/melatonin receptor system on clock gene expression using a model of primary neuronal cultures prepared from striatum. We found that melatonin at the receptor affinity range (i.e., nm) affects the expression of the clock genes mPer1, mClock, mBmal1 and mNPAS2 (neuronal PAS domain protein 2) differentially in a pertussis toxin-sensitive manner: a decrease in Per1 and Clock, an increase in NPAS2 and no change in Bmal1 expression. Furthermore, mutating MT1 melatonin receptor (i.e., MT1 knockouts, MT1(-/-)) reversed melatonin-induced changes, indicating the involvement of MT1 receptor in the regulatory action of melatonin on neuronal clock gene expression. Therefore, by controlling clock gene expression we propose melatonin receptors (i.e., MT1) as novel therapeutic targets for the pathobiologies of dopamine-related behaviors and mood.

Melatonin signaling in mouse cerebellar granule cells with variable native MT1 and MT2 melatonin receptors.

Although G protein-coupled MT1 and MT2 melatonin receptors are expressed in neurons of the mammalian brain including in humans, relatively little is known about the influence of native MT1 and MT2 melatonin receptors on neuronal melatonin signaling. Whereas human cerebellar granule cells (CGC) express only MT1 receptors, mouse CGC express both MT1 and MT2. To study the effects of altered neuronal MT1/MT2 receptors, we used CGC cultures prepared from immature cerebella of wild-type mice (MT1/MT2 CGC) and MT1- and MT2-knockout mice (MT2 and MT1 CGC, respectively). Here we report that in MT1/MT2 cultures, physiological (low nanomolar) concentrations of melatonin decrease the activity (phosphorylation) of extracellular-signal-regulated kinase (ERK) whereas a micromolar concentration was ineffective. Both MT1 and MT2 deficiencies transformed the melatonin inhibition of ERK into melatonin-induced ERK activation. In MT1/MT2 CGC, 1 nM melatonin inhibited serine/threonine kinase Akt, whereas in MT1 and MT2 CGC, this concentration was ineffective. Under these conditions, both MT1 and MT2 deficiencies prevented melatonin from inhibiting forskolin-stimulated cAMP levels and cFos immunoreactivity. We demonstrated that selective removal of native neuronal MT1 and MT2 receptors has a profound effect on the intracellular actions of low/physiological concentrations of melatonin. Since the expression of MT1 and MT2 receptors is cell-type-specific and species-dependent, we postulate that the pattern of expression of neuronal melatonin receptor types in different brain areas and cells could determine the capabilities of endogenous melatonin in regulating neuronal functioning.

Valproate administration to mice increases histone acetylation and 5-lipoxygenase content in the hippocampus.

Gene expression can be regulated by chromatin remodeling induced by the opposing actions of histone acetyltransferases and histone deacetylases (HDAC). HDAC inhibitors are considered putative anti-cancer drugs, but may also alter gene expression in the brain. Valproic acid (valproate; VPA), a drug used for treatment of bipolar disorder, has been characterized as a HDAC inhibitor. In neuronal cultures, VPA increases the expression of 5-lipoxygenase (5-LOX). Here we show that in vivo treatment of mice with intraperitoneal VPA injections increases the acetylation of histone H3 and the content of 5-LOX immunoreactive protein in the hippocampus. Since the extent of 5-LOX expression may alter mouse behavior, we suggest that VPA-altered chromatin remodeling and 5-LOX expression in the brain may be functionally important.