Analysis of the role of PRK1 in neuroprotection

The main research focus of my lab, over the last decade, has been the analysis of adenosine receptor/purine nucleoside-mediated cellular signaling pathways in hypoxic neurons. The research objective of this proposal is the in depth analysis of the candidate function of pre-selected PRK1 as a direct signaling intermediate in purine nucleoside-mediated rescue mechanisms of hypoxic neuronal cells. Rational basis: Our group has recently identified a candidate role of protein kinase C-related kinase (PRK1; also known as PKN1 amongst other synonyms) in the regulation of neuroprotective processes. Our key findings are: 1) Neurotrophic factors and hypoxia effectively upregulate PRK1 activity. PRK1-deficient neuronal cells show 2) loss of viability and neurite formation and 3) cannot be rescued from hypoxia by neurotrophic factors. Nevertheless, the role of PRK1 and the precise PRK1-associated molecular mechanisms in the control of neuronal protection remain unknown. Major aims and objects: Neuroprotection and neuroregeneration represent the two possible therapeutic approaches to ameliorate neuronal damage in stroke. Growing evidence suggests that purine nucleosides might act as trophic factors in hypoxic brain. Based on our recent experimental work, we have discovered a PRK1 function in the regulation of purine nucleoside-mediated rescue responses of neuronal cells. Nevertheless, the mode of action (MoA) of PRK1 as intrinsic intermediate in this pathway remains unresolved. In order to further validate our findings as well as dissect the signaling mechanisms of this PRK1mediated candidate pathway in neuronal cell biology during hypoxic stress, I propose to address following key questions: As Aim 1 and based on our preliminary results, we shall focus on the study of the `upstream` regulation of PRK1 by adenosine receptor and small G-proteins (RhoA and Rac1) in neuronal cells. As Aim 2 we shall elucidate the downstream effector molecules of PRK1 with specific emphasis on the PRK1 transactivation pathway of the plasticity protein GAP-43. As Aim 3 the necessity of PRK1 and, potentially PRK2, function(s) for neuroprotection of hypoxic neurons will be addressed using cultures from PRK1 and PRK2 single- and double knockout mice. As Aim 4 an in vivo stroke model, a pioneer work for our scientific location, will be established employing PRK1 and optionally also PRK2 single- and double knockout mice. The use of genetic mouse models in combination with tools in biochemistry, siRNA-mediated knockdown and cell biology, including the unique tool list and `know how`, from our collaborators will open new research avenues that represent an innovative paradigm for exploring the role and molecular mechanisms of PRK1 in purine nucleoside- mediated protection of hypoxic neurons.

Our studies on the role of ADORA/purine-nucleoside signaling cascade in hypoxia/ischemia (1-3) suggested a potential link to the serinehreonine protein kinase N (PRK/PKN) (4, 5). PKN1 is highly expressed in the brain, however, little was known about its function in neurons. As part of the FWF project we studied the role and regulation of PKN1. We identified PKN1 as a gatekeeper of neuronal AKT activity and demonstrated the in vivo significance of this interaction during neurodevelopment. We showed that PKN1 expression and AKT phosphorylation are inversely correlated during critical developmental stages of axo-dendritic maturation in the cerebellum. Deletion of PKN1 results in hyperactivated AKT, causing thicker Purkinje cell dendrites and a shifted climbing/parallel fiber homeostasis. This leads to cerebellar shrinkage and an ataxia-like motor-phenotype in adult animals. Our work thus established PKN1 as a compelling new therapeutic candidate target for studies of human brain disorders (6). _______________________ 1.Thauerer B, zur Nedden S, and Baier-Bitterlich G. Vital role of protein kinase C-related kinase in the formation and stability of neurites during hypoxia. J Neurochem. 2010;113(2):432-46. 2.zur Nedden S, Tomaselli B, and Baier-Bitterlich G. HIF-1 alpha is an essential effector for purine nucleoside-mediated neuroprotection against hypoxia in PC12 cells and primary cerebellar granule neurons. Journal of neurochemistry. 2008;105(5):1901-14. 3.Tomaselli B, Nedden SZ, Podhraski V, and Baier-Bitterlich G. p42/44 MAPK is an essential effector for purine nucleoside-mediated neuroprotection of hypoxic PC12 cells and primary cerebellar granule neurons. Mol Cell Neurosci. 2008;38(4):559-68. 4.Mukai H. The structure and function of PKN, a protein kinase having a catalytic domain homologous to that of PKC. J Biochem (Tokyo). 2003;133(1):17-27. 5.Quetier I, Marshall JJ, Spencer-Dene B, Lachmann S, Casamassima A, Franco C, et al. Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion. Cell Rep. 2016. 6.zur Nedden S, Eith R, Schwarzer C, Zanetti L, Seitter H, Fresser F, et al. Protein kinase N1 critically regulates cerebellar development and long-term function. J Clin Invest. 2018;128(5):2076-88.