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Inhibition of phosphodiesterase 4D (PDE4D) enzymes has been investigated as therapeutic strategy to treat memory problems in Alzheimer's disease (AD). Although PDE4D inhibitors are effective in enhancing memory processes in rodents and humans, severe side effects may hamper their clinical use. PDE4D enzymes comprise different isoforms, which, when targeted specifically, can increase treatment efficacy and safety. The function of PDE4D isoforms in AD and in molecular memory processes per se has remained unresolved. Here, we report the upregulation of specific PDE4D isoforms in transgenic AD mice and hippocampal neurons exposed to amyloid-beta. Furthermore, by means of pharmacological inhibition and CRISPR-Cas9 knockdown, we show that the long-form PDE4D3, -D5, -D7, and -D9 isoforms regulate neuronal plasticity and convey resilience against amyloid-beta in vitro. These results indicate that isoform-specific, next to non-selective, PDE4D inhibition is efficient in promoting neuroplasticity in an AD context. Therapeutic effects of non-selective PDE4D inhibitors are likely achieved through actions on long isoforms. Future research should identify which long PDE4D isoforms should be specifically targeted in vivo to both improve treatment efficacy and reduce side effects. This work was supported by Alzheimer Nederland [Grant WE.03–2016-07]. TV and JP 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 rofumilast for the treatment of cognitive impairment as well as PDE4D inhibitors for the treatment of Alzheimer’s disease. OB, CB, EF, RR, and JP have a proprietary interest in GEBR32a compound.

O1 Applying translational approaches for the nonclinical and clinical evaluation of the sGC stimulator CY6463 in CNS diseases Christopher J. Winrow Cyclerion Therapeutics, Cambridge Massachusetts, USA Correspondence: Christopher J. Winrow (cwinrow@cyclerion.com) J Transl Med 2022, 21(1):O1 Introduction: The NO-sGC-cGMP pathway plays a critical role in central nervous system (CNS) function and is impacted across a range of neurological and psychiatric diseases. NO is recognized as a key neu-rotransmitter that is produced on-demand within the CNS and can act through sGC and cGMP to govern a range of downstream effects. We have identified CY6463, a CNS-penetrant sGC stimulator, with demonstrated pharmacological effects in nonclinical and clinical studies. By acting as a selective positive allosteric modulator of sGC, CY6463 can amplify endogenous NO signaling while maintaining upstream spatial and temporal regulation. This enables the on-demand production of cGMP and propagation of downstream signals within the CNS. Methods: A range of nonclinical studies were conducted to understand the in vitro and in vivo properties of CY6463 and supported advancement into clinical development. Phase 1 clinical studies included single-ascending dose, multiple-ascending dose and food interaction studies along with a translational pharmacology study in healthy elderly participants. Results: This presentation will describe the nonclinical pharmacology of CY6463, along with clinical data from Phase 1 studies including the pharmacokinetic, safety, and pharmacodynamic results of our clinical translational pharmacology study in elderly participants. Furthermore, we will discuss our translational biomarker strategy that has been carried through into clinical studies in three separate patient populations and provide outlines of these clinical studies and updates on progress to date. Conclusions: Applying a translational biomarker based approach to the development of CY6463 has enabled advancement of clinical studies in well-defined patient populations to help understand the potential opportunity for modulating sGC function in neuropsychiatric and neurodegenerative diseases. Acknowledgements: CJW is an employee of Cyclerion Therapeutics and gratefully acknowledges the contributions of the Cyclerion team members and collaborators to this project. O2 sGC modulators as cognitive enhancers: neuronal and/ or vascular? Correspondence: Jos Prickaerts (jos.prickaerts@maastrichtuniversity.nl) J Transl Med 2022, 21(1):O2 Introduction: Cognitive impairment is one of the main symptoms of Alzheimer's disease or Vascular dementia, which negatively impacts the quality of life of patients. Therefore, a pharmacological intervention that has memory enhancing effects would be beneficial to patients. Vascular dementia is characterized by impairments in cer-ebral blood flow, endothelial function and blood-brain barrier integrity. These processes are all physiologically regulated by the soluble guanylate cyclase (sGC)-cGMP signaling pathway in blood vessel cells. Additionally, neuronal cGMP signaling plays an important role in long-term potentiation underlying memory formation. Therefore, targeting the NO-sGC-cGMP pathway may be a therapeutic strategy for treating neuronal-and/or vascular-based dementias. Methods: sGC stimulators acting on heme-bound sGC and one sGC activator acting on heme-free sGC were tested in the object location task (OLT) on acquisition memory processes, in healthy rodents and in deficit models. Vascular function and neuroplasticity were assessed. Funding was provided by a restricted research grant from Bayer AG, Pharmaceuticals.

Traumatic spinal cord injury (SCI) is characterized by severe neuroinflammation and hampered neuroregeneration, which often leads to permanent neurological deficits. Current therapies include decompression surgery, rehabilitation, and in some instances, the use of corticosteroids. However, the golden standard of corticosteroids still achieves minimal improvements in functional outcomes. Therefore, new strategies tackling the initial inflammatory reactions and stimulating endogenous repair in later stages are crucial to achieving functional repair in SCI patients. Cyclic adenosine monophosphate (cAMP) is an important second messenger in the central nervous system (CNS) that modulates these processes. A sustained drop in cAMP levels is observed during SCI, and elevating cAMP is associated with improved functional outcomes in experimental models. cAMP is regulated in a spatiotemporal manner by its hydrolyzing enzyme phosphodiesterase (PDE). Growing evidence suggests that inhibition of cAMP-specific PDEs (PDE4, PDE7, and PDE8) is an important strategy to orchestrate neuroinflammation and regeneration in the CNS. Therefore, this review focuses on the current evidence related to the immunomodulatory and neuroregenerative role of cAMP-specific PDE inhibition in the SCI pathophysiology. The APC was funded by FWO (G042121N and 1209123N)

O1 Applying translational approaches for the nonclinical and clinical evaluation of the sGC stimulator CY6463 in CNS diseases Christopher J. Winrow Cyclerion Therapeutics, Cambridge Massachusetts, USA Correspondence: Christopher J. Winrow (cwinrow@cyclerion.com) J Transl Med 2022, 21(1):O1 Introduction: The NO-sGC-cGMP pathway plays a critical role in central nervous system (CNS) function and is impacted across a range of neurological and psychiatric diseases. NO is recognized as a key neu-rotransmitter that is produced on-demand within the CNS and can act through sGC and cGMP to govern a range of downstream effects. We have identified CY6463, a CNS-penetrant sGC stimulator, with demonstrated pharmacological effects in nonclinical and clinical studies. By acting as a selective positive allosteric modulator of sGC, CY6463 can amplify endogenous NO signaling while maintaining upstream spatial and temporal regulation. This enables the on-demand production of cGMP and propagation of downstream signals within the CNS. Methods: A range of nonclinical studies were conducted to understand the in vitro and in vivo properties of CY6463 and supported advancement into clinical development. Phase 1 clinical studies included single-ascending dose, multiple-ascending dose and food interaction studies along with a translational pharmacology study in healthy elderly participants. Results: This presentation will describe the nonclinical pharmacology of CY6463, along with clinical data from Phase 1 studies including the pharmacokinetic, safety, and pharmacodynamic results of our clinical translational pharmacology study in elderly participants. Furthermore, we will discuss our translational biomarker strategy that has been carried through into clinical studies in three separate patient populations and provide outlines of these clinical studies and updates on progress to date. Conclusions: Applying a translational biomarker based approach to the development of CY6463 has enabled advancement of clinical studies in well-defined patient populations to help understand the potential opportunity for modulating sGC function in neuropsychiatric and neurodegenerative diseases. Acknowledgements: CJW is an employee of Cyclerion Therapeutics and gratefully acknowledges the contributions of the Cyclerion team members and collaborators to this project. O2 sGC modulators as cognitive enhancers: neuronal and/ or vascular? Correspondence: Jos Prickaerts (jos.prickaerts@maastrichtuniversity.nl) J Transl Med 2022, 21(1):O2 Introduction: Cognitive impairment is one of the main symptoms of Alzheimer's disease or Vascular dementia, which negatively impacts the quality of life of patients. Therefore, a pharmacological intervention that has memory enhancing effects would be beneficial to patients. Vascular dementia is characterized by impairments in cer-ebral blood flow, endothelial function and blood-brain barrier integrity. These processes are all physiologically regulated by the soluble guanylate cyclase (sGC)-cGMP signaling pathway in blood vessel cells. Additionally, neuronal cGMP signaling plays an important role in long-term potentiation underlying memory formation. Therefore, targeting the NO-sGC-cGMP pathway may be a therapeutic strategy for treating neuronal-and/or vascular-based dementias. Methods: sGC stimulators acting on heme-bound sGC and one sGC activator acting on heme-free sGC were tested in the object location task (OLT) on acquisition memory processes, in healthy rodents and in deficit models. Vascular function and neuroplasticity were assessed. Funding was in part supported by a restricted grant from Bayer AG and Merck Sharp & Dohme Corp.

Background Cholesterol metabolism contributes as a risk factor for aortic valve stenosis (AS), but pharmacological approaches remained unsatisfying. The liver-X-receptor (LXR) is a key regulator in cholesterol metabolism, though its clinical use is limited due to unwanted side effects. The seaweed-derived oxysterol saringosterol is an agonist of the LXRβ, promising a more favourable tolerability. Purpose This study aimed to better understand the pathophysiology of aortic valve stenosis and to assess the potential of saringosterol as a targeted pharmacotherapy. Methods Tissue samples from aortic valves were collected from patients with AS or aortic valve regurgitation (AR). Transcriptomics were performed and gene ontology (GO) analysis was used to determine pathways and genes that are relevant to AS, and then validated using qPCR. In vivo, mice received a wire-induced aortic valve stenosis and were either fed a diet supplemented with saringosterol or control diet. Haemodynamic characteristics were assessed using echocardiography. Additionally, hepatic concentrations of saringosterol, expression of LXRβ regulated genes as well as aortic valve thickness and composition were assessed. In vitro, human aortic valve interstitial cells (VIC) were cultured in a procalcifing medium and stimulated with saringosterol to investigate the underlying molecular mechanisms. Results Transcriptomic analysis of AS samples revealed the regulation of several GO-terms related to cholesterol- or lipid metabolism. Many of the genes identified were regulated by LXRβ, suggesting its pathophysiological relevance in AS. We validated this assumption by performing qPCR from aortic valves for the most prominent downstream targets of LXRβ, ABCA1 and ABCG1, with both being differentially regulated. In vivo, treatment with saringosterol for six weeks resulted in a significant accumulation of saringosterol in liver tissue as well as induction of LXRβ-regulated genes. Furthermore, treatment with saringosterol strikingly reduced the development of AS after wire injury as assessed by echocardiographic and histological measurements. In vitro, the differentiation of VIC into osteoblastic and myofibroblastic phenotypes was abolished by saringosterol, which reduced the expression of the procalcifying mediators RUNX-2 and ACTA-2 in a dose-dependent manner. Conclusion We identified LXRβ-signalling as a key regulator in the pathophysiology of AS. Transcriptomic analyses revealed that cholesterol metabolism was altered in human AS, and many of the genes involved were linked to the LXRβ. In a murine model, we demonstrated that oral application of saringosterol induced LXRβ-activity and mitigated the development of AS. In vitro experiments demonstrated that saringosterol prevents adverse cell differentiation of VIC, which provides a mechanistic explanation. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): BONFOR Universität Bonn

Introduction In the progressive phase of multiple sclerosis (MS), the hampered differentiation capacity of oligodendrocyte precursor cells (OPCs) eventually results in remyelination failure. We have previously shown that DNA methylation of Id2/Id4 is highly involved in OPC differentiation and remyelination. In this study, we took an unbiased approach by determining genome-wide DNA methylation patterns within chronically demyelinated MS lesions and investigated how certain epigenetic signatures relate to OPC differentiation capacity.Methods We compared genome-wide DNA methylation and transcriptional profiles between chronically demyelinated MS lesions and matched normal-appearing white matter (NAWM), making use of post-mortem brain tissue (n=9/group). DNA methylation differences that inversely correlated with mRNA expression of their corresponding genes were validated for their cell-type specificity in laser-captured OPCs using pyrosequencing. The CRISPR-dCas9-DNMT3a/TET1 system was used to epigenetically edit human-iPSC-derived oligodendrocytes to assess the effect on cellular differentiation.Results Our data show hypermethylation of CpGs within genes that cluster in gene ontologies related to myelination and axon ensheathment. Cell type-specific validation indicates a region-dependent hypermethylation of MBP, encoding for myelin basic protein, in OPCs obtained from white matter lesions compared to NAWM-derived OPCs. By altering the DNA methylation state of specific CpGs within the promotor region of MBP, using epigenetic editing, we show that cellular differentiation can be bidirectionally manipulated using the CRISPR-dCas9-DNMT3a/TET1 system in vitro.Conclusion Our data indicate that OPCs within chronically demyelinated MS lesions acquire an inhibitory phenotype, which translates into hypermethylation of crucial myelination related genes. Altering the epigenetic status of MBP can restore the differentiation capacity of OPCs and possibly boost (re)myelination.Competing Interest StatementThe authors have declared no competing interest.