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Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4Dand PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS. This work has been supported by FWO (12G0817N, 1S57521N, G041421N, and 12G0817N), Fondation Charctot Stichting (ID2020- 0019), Nationale Belgische Multiple Sclerose Liga (Charco18VT), MS Liga Vlaanderen and Stichting MS Research (18-1016 MS). MS, EP, JP and TV have a proprietary interest in selective PDE4D inhibitors for the treatment of demyelinating disorders and neurodegenerative disorders. JP has a proprietary interest in the PDE4 inhibitor roflumilast for the treatment of cognitive impairment as well as PDE4D inhibitors for the treatment of Alzheimer’s disease. We thank Prof. Dr. O.N. Viacheslav (University Medical Center Hamburg-Eppendorf, German Center for Cardiovascular Research) and Prof. Dr. M. Conti (University of California), for providing the PDE4B KO animals. Furthermore, we thank Rewind Therapeutics for providing the visual evoked potential equipment.

The phosphodiesterase 4 (PDE4) enzyme family plays a pivotal role in regulating levels of the second messenger cAMP. Consequently, PDE4 inhibitors have been investigated as a therapeutic strategy to enhance cAMP signaling in a broad range of diseases, including several types of cancers, as well as in various neurologic, dermatological, and inflammatory diseases. Despite their widespread therapeutic potential, the progression of PDE4 inhibitors into the clinic has been hampered because of their related relatively small therapeutic window, which increases the chance of producing adverse side effects. Interestingly, the PDE4 enzyme family consists of several subtypes and isoforms that can be modified post-translationally or can engage in specific protein-protein interactions to yield a variety of conformational states. Inhibition of specific PDE4 subtypes, isoforms, or conformational states may lead to more precise effects and hence improve the safety profile of PDE4 inhibition. In this review, we provide an overview of the variety of PDE4 isoforms and how their activity and inhibition is influenced by post-translational modifications and interactions with partner proteins. Furthermore, we describe the importance of screening potential PDE4 inhibitors in view of different PDE4 subtypes, isoforms, and conformational states rather than testing compounds directed toward a specific PDE4 catalytic domain. Lastly, potential mechanisms underlying PDE4-mediated adverse effects are outlined. In this review, we illustrate that PDE4 inhibitors retain their therapeutic potential in myriad diseases, but target identification should be more precise to establish selective inhibition of disease-affected PDE4 isoforms while avoiding isoforms involved in adverse effects. SIGNIFICANCE STATEMENT: Although the PDE4 enzyme family is a therapeutic target in an extensive range of disorders, clinical use of PDE4 inhibitors has been hindered because of the adverse side effects. This review elaborately shows that safer and more effective PDE4 targeting is possible by characterizing 1) which PDE4 subtypes and isoforms exist, 2) how PDE4 isoforms can adopt specific conformations upon post-translational modifications and protein-protein interactions, and 3) which PDE4 inhibitors can selectively bind specific PDE4 subtypes, isoforms, and/or conformations.

Pattern separation is a hippocampal process in which highly similar stimuli are recognized as separate representations, and deficits could lead to memory impairments in neuropsychiatric disorders such as schizophrenia. The 5-HT1A receptor (5-HT1AR) is believed to be involved in these hippocampal pattern separation processes. However, in the dorsal raphe nucleus (DRN), the 5-HT1AR is expressed as a somatodendritic autoreceptor, negatively regulates serotonergic signaling, and could thereby counteract the effects of hippocampal postsynaptic 5-HT1A receptors. Therefore, this study aims to identify how pre- and post-synaptic 5-HT1AR activity affects pattern separation. Object pattern separation (OPS) performance was measured in male Wistar rats after both acute and chronic treatment (i.p.) with 5-HT1AR biased agonists F13714 (0.0025 mg/kg acutely, 0.02 mg/kg/day chronically) or NLX-101 (0.08 mg/kg acutely, 0.32 mg/kg/day chronically), which preferentially activate autoreceptors or postsynaptic receptors respectively, for 14 days. Body temperature - a functional correlate of hypothalamic 5-HT1AR stimulation - was measured daily. Additionally, 5-HT1AR density (DRN) and plasticity markers (hippocampus) were assessed. Acute treatment with F13714 impaired OPS performance, whereas chronic treatment normalized this, and a drop in body temperature was found from day 4 onwards. NLX-101 enhanced OPS performance acutely and chronically, and caused an acute drop in body temperature. Chronic NLX-101 treatment increased doublecortin positive neurons in the dorsal hippocampus, while chronic treatment with F13714 resulted in a downregulation of 5-HT1A autoreceptors, which likely reversed the acute impairment in OPS performance. Chronic treatment with NLX-101 appears to have therapeutic potential to improve brain plasticity and OPS performance.

Synapses are the functional units of the brain. They form specific contact points that drive neuronal communication and are highly plastic in their strength, density, and shape. A carefully orchestrated balance between synaptogenesis and synaptic pruning, i.e., the elimination of weak or redundant synapses, ensures adequate synaptic density. An imbalance between these two processes lies at the basis of multiple neuropathologies. Recent evidence has highlighted the importance of glia-neuron interactions in the synaptic unit, emphasized by glial phagocytosis of synapses and local excretion of inflammatory mediators. These findings warrant a closer look into the molecular basis of cell-signaling pathways in the different brain cells that are related to synaptic plasticity. In neurons, intracellular second messengers, such as cyclic guanosine or adenosine monophosphate (cGMP and cAMP, respectively), are known mediators of synaptic homeostasis and plasticity. Increased levels of these second messengers in glial cells slow down inflammation and neurodegenerative processes. These multi-faceted effects provide the opportunity to counteract excessive synapse loss by targeting cGMP and cAMP pathways in multiple cell types. Phosphodiesterases (PDEs) are specialized degraders of these second messengers, rendering them attractive targets to combat the detrimental effects of neurological disorders. Cellular and subcellular compartmentalization of the specific isoforms of PDEs leads to divergent downstream effects for these enzymes in the various central nervous system resident cell types. This review provides a detailed overview on the role of PDEs and their inhibition in the context of glia-neuron interactions in different neuropathologies characterized by synapse loss. In doing so, it provides a framework to support future research towards finding combinational therapy for specific neuropathologies.

Soluble guanylate cyclase (sGC) - cyclic guanosine monophosphate (cGMP) signalling is important for healthy memory function and a healthy vascular system. Targeting sGC-cGMP signalling can therefore be a potential strategy to enhance memory processes. sGC can be targeted by using agonists, such as sGC stimulator riociguat. Therefore, this study aimed to target sGC using riociguat to investigate its acute effects on memory function and neuronal plasticity in mice. The effects of riociguat on long-term memory and a biperiden-induced memory deficit model for assessing short-term memory were tested in the object location task, and working memory was tested in the Y-maze continuous alternation task. Pharmacokinetic measurements were performed within brain tissue of mice, and hippocampal plasticity measures were assessed using western blotting. Acute oral administration with a low dose of 0.03 mg/kg riociguat was able to enhance working-, short-, and long-term spatial memory. Under cerebral vasoconstriction higher doses of riociguat were still effective on memory. Pharmacokinetic measurements revealed poor brain penetration of riociguat and its metabolite M-1. Increased activation of VASP was found, while no effects were found on other memory-related hippocampal plasticity measures. Memory enhancing effects of riociguat are most likely regulated by vascular peripheral effects on cGMP signalling. Yet, further research is needed to investigate the possible contribution of hemodynamic or metabolic effects of sGC stimulators on memory performance.

Phosphodiesterases (PDEs) have been an interesting drug target for many diseases. Although a vast number of mainly preclinical studies demonstrates beneficial effects of PDE inhibitors for central nervous system (CNS) diseases, no drugs are currently available for CNS indications. In this review, we discuss the rationale of PDE4 inhibitors for different CNS diseases, including memory impairments, striatal disorders, multiple sclerosis (MS), and acquired brain injury (ABU). However, clinical development has been problematic due to mechanism-based adverse effects of these drugs in humans. Our increased understanding of factors influencing the conformational state of the PDE4 enzyme and of how to influence the binding affinity of PDE4 subtype inhibitors, holds promise for the successful development of novel selective PDE4 inhibitors with higher efficacy and fewer adverse effects.

Pattern separation is a hippocampal process in which highly similar stimuli are recognized as separate representations, and deficits could lead to memory impairments in neuropsychiatric disorders such as schizophrenia. The 5-HT

Phosphodiesterases (PDE) are the only enzymes that degrade cAMP and cGMP which are second messengers crucial to memory consolidation. Different PDE inhibitors have been developed and tested for their memory-enhancing potential, but the occurrence of side effects has hampered clinical progression. As separate inhibition of the PDE2 and PDE4 enzyme family has been shown to enhance memory, we investigated whether concurrent treatment with a PDE2 and PDE4 inhibitor can have synergistic effects on memory consolidation processes. We found that combined administration of PF-999 (PDE2 inhibitor) and roflumilast (PDE4 inhibitor) increases the phosphorylation of the AMPA receptor subunit GluR1 and induces CRE-mediated gene expression. Moreover, when combined sub-effective and effective doses of PF-999 and roflumilast were administered after learning, time-dependent forgetting was abolished in an object location memory task. Pharmacokinetic assessment indicated that combined treatment does not alter exposure of the individual compounds. Taken together, these findings suggest that combined PDE2 and PDE4 inhibition has synergistic effects on memory consolidation processes at sub-effective doses, which could therefore provide a therapeutic strategy with an improved safety profile.

Pharmacological phosphodiesterase 4D (PDE4D) inhibition shows therapeutic potential to restore memory function in Alzheimer's disease (AD), but will likely evoke adverse side effects. As PDE4D encodes multiple isoforms, targeting specific isoforms may improve treatment efficacy and safety. Here, we investigated whether PDE4D isoform expression and PDE4D DNA methylation is affected in AD and whether expression changes are associated with severity of pathology and cognitive impairment. In post-mortem temporal lobe brain material from AD patients (n = 42) and age-matched controls (n = 40), we measured PDE4D isoform expression and PDE4D DNA (hydroxy)methylation using quantitative polymerase chain reaction and Illumina 450k Beadarrays, respectively. Linear regression revealed increased PDE4D1, -D3, -D5, and -D8 expression in AD with concurrent (hydroxy)methylation changes in associated promoter regions. Moreover, increased PDE4D1 and-D3 expression was associated with higherplaque and tau pathology levels, higher Braak stages, and progressed cognitive impairment. Future studies should indicate functional roles of specific PDE4D isoforms and the efficacy and safety of their selective inhibition to restore memory function in AD. (C) 2020 The Authors. Published by Elsevier Inc. This work was financially supported by grants from ISAO/Alzheimer Nederland WE.03-2016-07, Young European Research Universities Network (YERUN), and the Baeter Laeve foundation. Additional funds have been provided by the Internationale Stichting Alzheimer Onderzoek (ISAO)/Alzheimer Netherlands (Award #11532; Funded by the Dorpmans-Wigmans Foundation) (DvdH), and by the Joint ProgrammeeNeurodegenerative Disease Research (JPND) for the EPI-AD consortium (http://www.neurodegenerationresearch.eu/wp-content/uploads/2015/10/Factsheet_EPI-AD.pdf).The project is supported through the following funding organizations under the aegis of JPND; The Netherlands, The Netherlands Organisation for Health Research and Development (ZonMw); United Kingdom, Medical Research Council; Germany, German Federal Ministry of Education and Research (BMBF); Luxembourg, National Research Fund (FNR). This project has received funding from the European Union's Horizon 2020 research and innovation program under Grant Agreement No. 643417. Prickaerts, J (corresponding author), Maastricht Univ, Dept Psychiat & Neuropsychol, Sch Mental Hlth & Neurosci, POB 616, NL-6200 MD Maastricht, Netherlands. jos.prickaerts@maastrichtuniversity.nl

Augmentation of cAMP signaling through inhibition of phosphodiesterases (PDE) is known to enhance plasticity and memory. Inhibition of PDE4 enhances consolidation into memory, but less is known about the role of other cAMP specific PDEs. Here, we tested the effects of oral treatment with a selective inhibitor of PDE7 of nanomolar potency on spatial and contextual memory. In an object location task, doses of 0.3–3 mg/kg administered 3 h after training dose-dependently attenuated time-dependent forgetting in rats. Significant enhancement of memory occurred at a dose of 3 mg/kg with corresponding brain levels consistent with PDE7 inhibition. The same dose given prior to training augmented contextual fear conditioning. In mice, daily dosing before training enhanced spatial memory in two different incremental learning paradigms in the Barnes Maze. Drug treated mice made significantly less errors locating the escape in a probe-test 24 h after the end of training, and they exhibited hippocampal-dependent spatial search strategies more frequently than controls, which tended to show serial sampling of escape locations. Acquisition and short-term memory, in contrast, were unaffected. Our data provide evidence for a role of PDE7 in the consolidation of hippocampal-dependent memory. We suggest that targeting PDE7 for memory enhancement may provide an alternative to PDE4 inhibitors, which tend to have undesirable gastrointestinal side-effects.

There is an urgent need for therapies that could reduce the disease burden of preterm hypoxic-ischemic encephalopathy. Here, we evaluate the long-term effects of multipotent adult progenitor cells (MAPC) on long-term behavioral outcomes in a preterm rat model of perinatal asphyxia. Rats of both sexes were treated with two doses of MAPCs within 24 h after the insult. Locomotor, cognitive and psychiatric impairments were evaluated starting at 1.5 (juvenile) and 6 months (adult). Hypoxia-ischemia affected locomotion, cognition, and anxiety in a sex-dependent manner, with higher vulnerability observed in males. The MAPC therapy partially attenuated deficits in object recognition memory in females of all tested ages, and in the adult males. The hypoxic insult caused delayed hyperactivity in adult males, which was corrected by MAPC therapy. These results suggest that MAPCs may have long-term benefits for neurodevelopmental outcome after preterm birth and global hypoxia-ischemia, which warrants further preclinical exploration.

The most commonly used rodent recognition memory tests are based on the novelty-preference paradigm. These tests are derived from the object novelty recognition (ORT) and the object location (OLT) tests. Most of these rodent recognition memory tests have been easily adopted and implemented through manipulations of the test context; number of trials and test phases and the choice of objects, their number and locations. Hence, it has been possible to assess different types of memory (spatial, nonspatial, temporal order and episodic) and processes (short-term, long-term and consolidation) within a single novelty-preference paradigm. The ORT and subsequent variants of this test contributed significantly to our understanding of the neural basis of different forms of memory. In the present chapter, the general principles of ORT and OLT, and the novel variants of these tests are described; they are followed by some considerations and discussions of the test procedures and methodologies.