Abstract
A novel, potent, and highly specific inhibitor of calcium-calmodulin-dependent phosphodiesterases (PDE) of the PDE1 family, ITI-214, was used to investigate the role of PDE1 in inflammatory responses. ITI-214 dose-dependently suppressed lipopolysaccharide (LPS)-induced gene expression of pro-inflammatory cytokines in an immortalized murine microglial cell line, BV2 cells. RNA profiling (RNA-Seq) was used to analyze the impact of ITI-214 on the BV2 cell transcriptome in the absence and the presence of LPS. ITI-214 was found to regulate classes of genes that are involved in inflammation and cell migration responses to LPS exposure. The gene expression changes seen with ITI-214 treatment were distinct from those elicited by inhibitors of other PDEs with anti-inflammatory activity (e.g., a PDE4 inhibitor), indicating a distinct mechanism of action for PDE1. Functionally, ITI-214 inhibited ADP-induced migration of BV2 cells through a P2Y12-receptor-dependent pathway, possibly due to increases in the extent of cAMP and VASP phosphorylation downstream of receptor activation. Importantly, this effect was recapitulated in P2 rat microglial cells in vitro, indicating that these pathways are active in native microglial cells. These studies are the first to demonstrate that inhibition of PDE1 exerts anti-inflammatory effects through effects on microglia signaling pathways. The ability of PDE1 inhibitors to prevent or dampen excessive inflammatory responses of BV2 cells and microglia provides a basis for exploring their therapeutic utility in the treatment of neurodegenerative diseases associated with increased inflammation and microglia proliferation such as Parkinson's disease and Alzheimer's disease.
Highlights
A novel, potent, and highly specific inhibitor of calcium-calmodulin-dependent phosphodiesterases (PDE) of the PDE1 family, ITI-214, was used to investigate the role of PDE1 in inflammatory responses
The PDE1 phosphodiesterases (i.e., PDE1A, 1B, and 1C isoforms) are dual cAMP/cGMP phosphodiesterase activated by calcium/calmodulin, and function to dampen cyclic nucleotide signaling in excitatory cells such as neurons, cardiac myocytes, and possibly microglia
As human and mouse microglial cells express PDE1B, one of the 3 main isoforms of this enzyme, at significant levels (Zhang et al, 2014), we reasoned that inhibition of PDE1 in inflammatory cells, microglia, might alter cyclic nucleotide signaling and be therapeutic in diseases associated with excessive inflammatory signaling
Summary
A novel, potent, and highly specific inhibitor of calcium-calmodulin-dependent phosphodiesterases (PDE) of the PDE1 family, ITI-214, was used to investigate the role of PDE1 in inflammatory responses. ITI-214 inhibited ADP-induced migration of BV2 cells through a P2Y12-receptor-dependent pathway, possibly due to increases in the extent of cAMP and VASP phosphorylation downstream of receptor activation. This effect was recapitulated in P2 rat microglial cells in vitro, indicating that these pathways are active in native microglial cells. As human and mouse microglial cells express PDE1B, one of the 3 main isoforms of this enzyme, at significant levels (Zhang et al, 2014), we reasoned that inhibition of PDE1 in inflammatory cells, microglia, might alter cyclic nucleotide signaling and be therapeutic in diseases associated with excessive inflammatory signaling. We propose that PDE1 inhibition may reduce inflammatory responses through reductions in cellular motility
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