Preclinical evaluation of PKN1 as stroke-target

Stroke is a major global cause of death and disability. The only treatment approved by the FDA is tissue- plasminogen-activator; its time window, however, restricts its benefit to a minority of patients. Targeting molecular events during the sub-acute phase of stroke may provide a longer therapeutic window and greater clinical success. Despite promising preclinical data on therapeutics promoting endogenous neurorepair, numerous clinical trials have failed to show the efficacy of drugs in patients. Hopeful new targets are phosphoinositide-3- kinase/AKT and adenosine receptor (ADORA-mediated neuroprotective signaling-pathways). Elevated levels of purine nucleosides including adenosine produced in response to hypoxia/ischemia act as powerful neuroprotectants and intra-/intercellular messengers during ischemia and help prevent additional injury by inhibiting activated immune cells. During our studies on the role of ADORA/purine-nucleoside signaling cascade in hypoxia/ischemia, we discovered a link to protein kinase N (PKN), a family mapped at the heart of signaling networks governing differentiation and cell survival. Since then, we have been studying (FWF- grant-P26002) the function and regulation of PKN1, the predominant isoform in brain. Results revealed PKN1 as a critical gatekeeper of the AKT-survival-signaling pathway, exerting physiological and non-redundant functions in murine brain development (zur Nedden et al., Manuscript in preparation). Additionally, we provide strong experimental evidence that PKN1 has a critical function in the regulation of protective mechanisms during ischemia, as shown in the in vivo mouse model of MCAo-filament stroke (zur Nedden et al., Manuscript submitted). However, there are considerable gaps in our understanding of the molecular processes of neuron-intrinsic PKN1 regulation and function, for instance, in our knowledge of (i) autophosphorylation sites on PKN1 of fundamental importance for the recruitment and activation of PKN1 and (ii) mechanistic insights into how PKN1 physically and functionally interacts with neuronal AKT and how the PKN1/AKT-complex formation selectively fine tunes neuron functionality. Central hypothesis: In the current proposal, we want (1) to validate our hypothesis of human PKN1/AKT cross- talk as candidate pathway critical for neuroprotection/neuroregeneration in acute stroke and (2) address its human relevance by analysis of expression and activation regulation of PKN1 and AKT in a human neuronal cell line and in clinical biopsies of stroke patients.

Stroke, a global cause of death and neurological disability, remains one of the least treatable diseases, thus necessitating the urgent search for new treatments. We have identified Protein kinase N1 (PKN1) as an important regulator of brain development 1-4. To validate PKN1 as a potential stroke target, the project focused on the physiological and pathophysiological role of PKN1 in young and adult brain. I) PKN1 in early brain development The developing cerebellum is specifically vulnerable to hypoxia-ischemia, which occurs during hypoxic-ischemic encephalopathy (HI), a condition typically caused by oxygen deprivation during or shortly after birth. In this context, activation of the pro-survival protein kinase AKT has emerged as a promising new target for neuroprotective interventions. The project investigated the role of PKN1 in an in vitro model of HI, utilising postnatal cerebellar granule neurons derived from Pkn1 wildtype and mice with a Pkn1 deletion (Pkn1-/- knockout). The results obtained demonstrated that Pkn1-/- cells exhibited significantly higher AKT phosphorylation (pAKT), resulting in improved survival after HI, suggesting a protective phenotype of Pkn1 knockout after HI 4 . II) Physiological and pathophysiological role of PKN1 in the adult brain To this end, an in vivo stroke model was established using Pkn1 wild-type and knockout mice. The data showed reduced brain lesion volume and improved functional recovery in adult Pkn1-/- mice after stroke. The outcome of this study is therefore consistent in showing that the absence of PKN1 results in a protective phenotype 5. However, PKN1-mediated inhibitory regulation of pAKT is reduced in the adult brain 2,3, prompting investigations into additional physiological and pathophysiological functions of PKN1 in adult neurons. Our research has shown that PKN1 acts as a central regulator of cerebral energy metabolism, as Pkn1-/- brain tissue had higher glycolytic flux, pyruvate-induced mitochondrial oxygen consumption rates and adenine nucleotide levels 5,6. The significant metabolic changes in the Pkn1-/- brain resulted in a highly protective phenotype in in vitro and in vivo stroke models 5. Thus, our groundbreaking findings identify PKN1 as a novel key regulator of cerebral energy metabolism under both physiological and pathophysiological conditions and establish post-ischemic PKN1 inhibition as a promising new focus for future stroke therapies. References: 1.Brunner M, et al. Int J Mol Sci. 2024;25. doi: 10.3390/ijms25052848 2.Safari MS, et al. Front Synaptic Neurosci. 2021;13:640495. doi: 10.3389/fnsyn.2021.640495 3.zur Nedden S, et al. J Clin Invest. 2018;128:2076-2088. doi: 10.1172/JCI96165 4.Zur Nedden S, et al. Biomolecules. 2023;13. doi: 10.3390/biom13111599 5.Zur Nedden S, et al. Metabolism. 2024;161:156039. doi: 10.1016/j.metabol.2024.156039 6.Safari MS, et al. Mol Metab. 2024;88:102018. doi: 10.1016/j.molmet.2024.102018