Research Scholarships 2019: Project 1

Targeting prolyl hydroxylase domain (PHD) therapy in the central nervous systems

PI: Professor John O'Connor

Introduction

Recent promising new research has demonstrated that inhibition of the prolyl hydroxylase enzyme (PHD) and activation of hypoxia inducible factors (HIF) protects brain neurons in ischemia and hypoxia. Recently we have produced data to indicate that this enzyme also has significant acute effects on synaptic transmission and plasticity in the hippocampus (Lanigan & O’Connor, 2018; Corcoran et al., 2013; Wall et al., 2014). It has been shown recently in C. elegans that under hypoxic conditions and reduced prolyl hydroxylase activity there is reduced AMPA receptor subunit trafficking and changes in behavior (Park et al., 2012), also implicating important effects of these agents on synaptic plasticity. Recently we have shown that application of the non-specific PHD inhibitor DMOG has inhibitory effects on synaptic transmission (Lanigan & O’Connor, 2018; Wall et al., 2014) and that DMOG, DFO and JNJ42041935 (a more specific PHD inhibitor) have inhibitory effects on long-term potentiation (LTP) in rat and mouse hippocampus (Batti et al., 2010; Corcoran & O’Connor, 2013). Furthermore we have also demonstrated that these inhibitory effects can be ablated in PHD2 KO mice (Corcoran et al., 2013). We have also previously demonstrated a role for p38MAP kinase in synaptic plasticity modulation by immune cytokines (Wall et al, 2015; Mukandala et al., 2016). Park & Rongo, (2016) have now shown a role for p38 MAP kinase in hypoxia in C. elegans.  These data provide strong evidence for a role for PHD2 in modulating synaptic plasticity.  In this project we will investigate the actions of PHD2 inhibitors on long-term potentiation (LTP) in vitro in control and PHD2 KO mice, investigate, the mechanisms by which PHD inhibition affects AMPA and NMDA currents in isolated neurons and also investigate the role of the p38MAPK in hypoxia in hippocampal neurons. Therefore our overall aim is to decipher the molecular mechanisms of PHD2 function in hypoxia and synaptic transmission and plasticity.

Why is this question important?

PHDs are O2 dependent enzymes, which hydroxylase target molecules. Hypoxia inducible factors (HIF) are regulated by four hydroxylases: PHD-1, -2, -3 and factor inhibiting HIF (FIH). To date there has been little research into the role and function of the specific enzyme isoforms in hypoxia in neurons. In fact different PHD isoforms differentially contribute to specific pathophysiological processes, including angiogenesis, erythropoiesis, tumorigenesis and cell growth (e.g. Appelhoff et al., 2004; Takeda et al., 2011). Our understanding of O2 sensing has been revolutionized over the past 10 years. The possibility of simulating the body’s coordinated response to hypoxia with small molecule prolyl hydroxylase (PHD) inhibitors offers enormous potential in the treatment of a wide range of oxygen-deprivation-related disorders such as stroke. PHD inhibiting drugs have recently been shown to have applications in ischemic diseases and proof of principle demonstrated by major pharmaceutical companies in some treatments. However the role for the different isoforms of PHD in the CNS is still unclear. Some of the first evidence to appear showing a neuronal phenotype for PHDs came from KO PHD3-/- mice where PHD3 was shown to be essential for proper sympathoadrenal development (Bishop et al., 2008). Siddiq et al., (2009) has shown that RNA interference to PHD1 but not PHD2 and 3 isoforms, prevents oxidative death independent of HIF and CREB activation but with a role for HIF-2 and not HIF-1. Recently there have been a number of papers showing that inhibition of PHDs after a stroke model reduces ischemic brain injury (Nagel et al., 2011; Walmsley et al., 2011; Ogle et al., 2012; Shang et al., 2012; Kunze et al., 2012). Cortical neurons cultured in 1% O2 or which have PHD inhibited are highly resistant to glutamate-induced NMDA receptor-dependent excitotoxic injury (Li et al., 2011).  It is now clear that besides their effects on HIFs, PHDs also regulate other downstream targets. There is now clear evidence for HIF-independent pathways during hypoxia (Wong et al., 2012). Park et al., (2012) have demonstrated a novel role for PHD sensing in AMPA receptor trafficking in C elegans. Here the PDZ and PTB domain-containing protein LIN-10 is required both for the synaptic localization of the AMPAR subunit GLR-1. Also of interest, Huo et al., (2013) have shown that PHD2 can regulate intracellular cyclic AMP levels and phosphodiesterase 4D in cardiomyocytes. Shao et al., (2009) showed also in C Elegans that EGL-9 regulates HIF-1 via two distinct pathways: oxygen-dependent degradation of HIF-1 and an uncharacterized vhl-1-independent pathway in which EGL-9 represses HIF-1 transcriptional activity. Since these inhibitors are now in clinical trials for the prevention of stroke induced neuronal damage it is therefore of utmost importance to decipher the mechanisms of action of PHD2 in synaptic plasticity in the CNS and open new horizons for research in this field.

Methods

In this proposal we will use three complementary research models using state of the art technologies,  in vitro extracellular recording of LTP from wild type and PHD2 KO mice, in vitro whole cell recordings from hippocampal neurons and protein analysis in isolated brain slices using ELISA and western blot techniques.

Enquiries and applications (to include cover letter and CV) to Professor John O'Connor. Email: john.oconnor@ucd.ie

Closing date: Friday 31st May, 2019.