PhD Scholarship project 2

SBBS Research Scholarships 2023: Project 2

An electrophysiological, proteomic and immunohistochemical investigation into the actions of prolyl hydroxylase domain (PHD) inhibition in the rat hippocampus

PI: (opens in a new window)Professor John O'Connor

Introduction

Previous 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 (Takeda et al., 2011; Ogle et al., 2012). We have also produced data to indicate that inhibition of this enzyme also has modulatory effects on synaptic transmission and plasticity in the hippocampus (Puzio et al., 2023; Puzio et al., 2022; Lanigan & O’Connor, 2018). It has been shown 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. We have also recently 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). These data provide strong evidence for a role for PHD2 in modulating synaptic signalling and plasticity. Our aims, using state of the art imaging electrophysiology and proteomics are to 1) Investigate the actions of PHD inhibitors on synaptic plasticity using the model - long-term potentiation (LTP). 2) Identify, the mechanisms by which PHD inhibition affects AMPA receptor GluA1 and 2 and a role for NMDA receptors. 3) Create a comprehensive proteomics profile for these effects that can be used to identify potential targets for therapeutic approaches. Therefore, our overall aim is to decipher the molecular mechanisms of PHD2 function in hypoxia and synaptic transmission and plasticity in the brain and thus its future potential as a therapeutic for stroke patients.

Why is this question significant?

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-2a and not HIF-1a. 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., 2013). 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. These effects are likely mediated through AMPA GluR1-4 modulation although other signalling will also be investigated (see contingency below).  Since these inhibitors are now in clinical trials for the prevention of inflammatory bowel disease (IBD, Cummins et al., 2013), it is therefore of utmost importance to decipher the mechanisms of action of PHD2 in synaptic signalling and plasticity in the CNS and open new horizons for research in this field.

How will the question be addressed?

In this proposal we will investigate our objectives utilizing three complementary research models using state of the art technologies, 1) In vitro extracellular recordings from isolated hippocampal slices and whole cell recordings from single hippocampal neurons. 2) Immunohistochemical analysis of the changes in AMPAR receptor GluR1 and 2 in hippocampal slices post hypoxia and OGD 3) Proteomic analysis of key protein changes before and after PHD inhibition in brain slices exposed to hypoxia and OGD. These three methodologies complement each other and will help elucidate the acute role of PHD2 enzymes in synaptic signalling, synaptic plasticity and thus memory.

References

Appelhoff RJ, et al., The Journal of Biological Chemistry; 2004; 279(37), 38458-65. Batti L, et al., Neuroscience. 2010; 167(4):1014-24. Bishop T, et al., Molecular and cellular biology, 2008; 28(10), 3386-400. Corcoran A, et al., Hippocampus. 2013; 23(10):861-72. Corcoran A, O'Connor JJ. Acta Physiol. 2013; 208(4):298-310. Cummins, E., et al., Lab Invest 93, 378–383 (2013). Huo Z, et al., Biochem Biophys Res Commun. 2012; 427(1):73-9. Kunze R, et al. Stroke. 2012; 43(10):2748-56. Lanigan SM, O'Connor JJ. Neuroscience. 2018, 369:168-182. Li, D., et al., Experimental neurology, 2011; 230(2), 302-10. Nagel S, et al., Journal of cerebral blood flow and metabolism: 2011; 31(1), 132-43. Ogle, M. E., et al., Neurobiology of disease, 2012; 45(2), 733-742. Park EC, et al., EMBO J. 2012 Mar 21;31(6):1379-93. Shang, J., et al., Journal of Neuroscience Research, 2012; 90(3), 648-655. Puzio M., et al., Antioxidants, 2023; 12(4):792. Puzio, M., et al. Brain Disorders, 2022; 5, 100030. Shao Z, et al., Genetics. 2009 Nov;183(3):821-9. Siddiq, A., et al., The Journal of Neuroscience: 2009; 29(27), 8828-38. Takeda Y, et al. Nature. 2011; 479(7371):122-6. Walmsley SR, et al. J Clin Invest. 2011; 121(3):1053-63. Wong BW, et al., Trends Biochem Sci. 2013; 38(1):3-11.

Requirements: Applicants should have, or expect to achieve, a minimum of upper 2nd class honours
degree (or equivalent) in Neuroscience, Pharmacology, Physiology or related biomedical science
discipline. 

Conditions:

1. September/October 2023 start date.
2. Full EU fees + €20,000 per annum stipend over four years; EU/UK applicants only
3. The student will be enrolled in a Structured PhD programme, associated with the School.
4. Each student is required to demonstrate in appropriate laboratory practicals as part of their funded scholarship.  Demonstrating hours and lab practicals are detailed and assigned by the SBBS Demonstrating Committee (maximum hours: 288 per annum) .
5. The student and supervisor are required to submit an application for an IRC Postgraduate Scholarship in the first year of their SBBS-funded research scholarship

Apply: Applications should contain a 1-page cover letter outlining your interest and suitability for the
position, a detailed CV and contact information for 2 referees. Applications should be received by
email to john.oconnor@ucd.ie, no later than July 30th 2023. Informal inquiries may be made by
email prior to the application deadline.