Neuroprotective signaling mechanisms in the mammalian brain

Summary Alzheimer’s, Parkinson’s disease and other degenerative disorders are characterized by increased neuronal death. Deregulation of several physiological processes, as a consequence of the neuronal death, occurs especially in the regions of the central nervous system involved in learning and memory. Therefore, preserving normal neuronal functions is of great importance. Design and implementation of new therapeutics in clinical trials in order to prevent and stop neuronal death is quite challenging since neurodegenerative processes underlie complex signaling. Therefore, studying and revealing these neuronal processes would lead to more effective therapeutics. One model to study in vitro the molecular mechanisms underlying neurodegeneration is the glutamate-induced excitotoxicity paradigm, where neurons are treated with glutamate to induce neuronal death and several substances are tested for their potency to prevent this neuronal death. Hitherto, a number of studies showed that the cytokine TNF-a is able to promote neuroprotection against glutamate-induced excitotoxicity in cortical neurons. TNF-a is a known cytokine with increased expression in the brain and in the cerebrospinal serum of human patients with neurodegenerative disorders. The main focus of this thesis was to investigate TNF-a-mediated neuroprotective molecular mechanisms in the mammalian brain. Chapters 2 and 4 of this thesis investigate the neuroprotective mechanisms by which TNF-a enhances neuronal resistance against glutamate-induced excitotoxicity. First, the kinetics of the neuroprotective effect of TNF-a in primary cortical neurons was studied. Short-term exposure of neurons for 2-4 h to TNF-a did not rescue neurons from the glutamate-induced cellular death. However, after 6-8 h or longer of TNF-a treatment neuronal survival was significantly increased. It was previously shown that long term (24 h) TNF-a treatment causes an increase of PKB/Akt phosphorylation. PKB/Akt phosphorylation in cortical neurons is associated with increased cellular survival. Chapter 2 describes the PKB/Akt kinetics upon TNF-a treatment. Since PTEN is the major negative regulator of PKB/Akt activation the PTEN expression upon TNF-a treatment was investigated. In previous studies, an increase of PTEN expression in cortical neurons was shown to induce neuronal death. Short term TNF-a treatment (2-4 h) induced an increase of PTEN expression and a decrease of PKB/Akt phosphorylation. However, long term TNF-a treatment (6-8 to 24 h) resulted in a decrease of PTEN expression and augmented PKB/Akt phosphorylation in a time dependent manner. The fact that high levels of PTEN coincided with low levels of PKB/Akt phosphorylation and vice versa is in accordance with the inverse relationship between PTEN and PKB/Akt activation. To assess the contribution of both TNF-receptors, TNF-R1 and/or TNF-R2 to the neuroprotective TNF-a mediated pathway, PKB/Akt and PTEN expression in TNF-R1-/- and TNF-R2-/- neurons was investigated. It appeared that in TNF-R1-/- neurons, PKB/Akt was phosphorylated after long term TNF-a treatment with visible effects after 6-8 h, while in TNF-R2-/- PKB/Akt phosphorylation was down regulated in a TNF-a treatment dependent manner (Chapter 2). Taken together, these results suggested that TNF-a minimizes the consequences of excitotoxic events through PKB/Akt signaling. Since PKB/Akt pathway could be regulated by cyclic adenosine monophosphate (cAMP)-dependent signaling as well, chapter 3 investigated the effect of two mainstream pathways initiated by cAMP, cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) on PKB/Akt phosphorylation in primary cortical neurons. PKA activation led to a decrease of PKB/Akt phosphorylation, whereas activation of Epac increased PKB/Akt phosphorylation. PKA, PKB/Akt and Epacs were all shown to be complexed with the neuronal A-kinase anchoring protein 150 (AKAP150). Particularly, activation of Epac2 increased phosphorylation of PKB/Akt complexed to AKAP150, whereas silencing of cellular Epac2 diminished PKB/Akt phosphorylation. From experiments using PKA binding deficient AKAP150 and PKA disrupting anchoring to AKAP150 peptides, AKAP150 was found to act as a key regulator in the two cAMP pathways that control PKB/Akt phosphorylation. Although the involvement of the TNF-R2, PKB/Akt and NF-kB were suggested to be important in TNF-a induced neuroprotective mechanisms, data on potential downstream targets of these signalling pathways were not available. Therefore, in Chapter 4 we investigated SK channels as possible downstream molecules of TNF-a signaling, since NF-kB regulates the expression of SK2 channels. SK channels modulate synaptic excitability and neuronal activity by contributing to afterhyperpolarization processes. In our studies, we investigated the action of highly specific agonists (NS309, CyPPA) and antagonists (apamin) of SK channels on cortical neurons. The finding that NS309 and CyPPA mediated increased cellular survival against glutamate toxicity, while apamin partially reverted the neuroprotective effect elicited by TNF-a treatment demonstrated the crucial role of SK channel activity in the TNF-a induced neuroprotective mechanisms. Interestingly, a commercially available drug, lovastatin commonly used for coronary artery disease specifically enhanced TNF-R2 expression in HUVEC cells. Lovastatin was proposed as neuroprotective agent for several neurodegenerative disorders. Although both lovastatin and TNF-a were shown to be neuroprotective, so far their signaling pathways had been studied independently. The study described in chapter 5 shows that lovastatin can induce TNF-R2 expression in primary cortical neurons. It was demonstrated that NF-kB is responsible for the lovastatin-dependent increase of TNF-R2 expression (Chapter 5). Since in TNF-R1-/- neurons inhibition of PKB/Akt activity reverted the protective effect induced by lovastatin it was concluded that lovastatin-induced TNF-R2 expression contributes to neuronal survival through PKB/Akt activation. Chapter 6 consolidates the role of PKB/Akt as a downstream molecule in lovastatin-mediated neuroprotective signaling. Using an in vivo model, in which NMDA was infused in the nucleus basalis of Meynert (MNB), located in the basal forebrain cholinergic complex, the effect of lovastatin on cortical cholinergic fibers was investigated. Since the basal forebrain provides the major input in cholinergic projections to the cerebral cortex and hippocampus the cortical cholinergic target area was quantified to determine the magnitude of cholinergic fiber loss produced by NMDA lesions in MNB. Lovastatin treatment significantly protected cholinergic neurons and their cortical projections against NMDA induced cell death in MNB. Lovastatin-mediated neuroprotection was shown to be dependent on protein kinase B (PKB)/Akt signaling since inhibition of PKB/Akt with LY294002 blocked the lovastatin-induced neuroprotective effect (Chapter 6). In addition, lovastatin treatment improved memory deficits in NMDA-lesioned mice. Taken together, our studies suggest potential for therapeutic manipulation of TNF-a signaling in the treatment of neurodegenerative disorders.