Amalia M. Dolga

Statins: Mechanisms of neuroprotection

Clinical trials report that the class of drugs known as statins may be neuroprotective in Alzheimers and Parkinsons disease, and further trials are currently underway to test whether these drugs are also beneficial in multiple sclerosis and acute stroke treatment. Since statins are well tolerated and have relatively few side effects, they may be considered as viable drugs to ameliorate neurodegenerative diseases. However, the mechanism of their neuroprotective effects is only partly understood. In this article, we review the current data on the neuroprotective effects of statins and their underlying mechanisms. In the first section, we detail the mechanisms by which statins affect cellular signalling. The primary action of statins is to inhibit cellular cholesterol synthesis. However, the cholesterol synthesis pathway also has several by-products, the non-sterol isoprenoids that are also important in cellular functioning. Furthermore, reduced cholesterol levels may deplete the cholesterol-rich membrane domains known as lipid rafts, which in turn could affect cellular signalling. In the second section, we summarize how the effects on signalling translate into general neuroprotective effects through peripheral systems. Statins improve blood-flow, reduce coagulation, modulate the immune system and reduce oxidative damage. The final section deals with the effects of statins on the central nervous system, particularly during Alzheimers and Parkinsons disease, stroke and multiple sclerosis.

Inflammation and NF-κB in Alzheimer's Disease and Diabetes

Inflammatory processes are a hallmark of many chronic diseases including Alzheimers disease and diabetes mellitus. Fairly recent statistical evidence indicating that type 2 diabetes increases the risk of developing Alzheimers disease has led to investigations of the potential common processes that could explain this relation. Here, we review the literature on how inflammation and the inducible nuclear factor NF-kappaB might be involved in both diabetes mellitus and Alzheimers disease and whether these factors can link both diseases.

Inflammation and NF-kappa B in Alzheimer's Disease and Diabetes

Inflammatory processes are a hallmark of many chronic diseases including Alzheimers disease and diabetes mellitus. Fairly recent statistical evidence indicating that type 2 diabetes increases the risk of developing Alzheimers disease has led to investigations of the potential common processes that could explain this relation. Here, we review the literature on how inflammation and the inducible nuclear factor NF-kappaB might be involved in both diabetes mellitus and Alzheimers disease and whether these factors can link both diseases.

Interleukin-6 upregulates neuronal adenosine A1 receptors: implications for neuromodulation and neuroprotection.

The immunological response in the brain is crucial to overcome neuropathological events. Some inflammatory mediators, such as the immunoregulatory cytokine interleukin-6 (IL-6) affect neuromodulation and may also play protective roles against various noxious conditions. However, the fundamental mechanisms underlying the long-term effects of IL-6 in the brain remain unclear. We now report that IL-6 increases the expression and function of the neuronal adenosine A1 receptor, with relevant consequences to synaptic transmission and neuroprotection. IL-6-induced amplification of A1 receptor function enhances the responses to readily released adenosine during hypoxia, enables neuronal rescue from glutamate-induced death, and protects animals from chemically induced convulsing seizures. Taken together, these results suggest that IL-6 minimizes the consequences of excitotoxic episodes on brain function through the enhancement of endogenous adenosinergic signaling.

TNF-alpha-mediates neuroprotection against glutamate-induced excitotoxicity via NF-kappa B-dependent up-regulation of K(Ca)2.2 channels

Previous studies have shown that tumor necrosis factor‐alpha (TNF‐α) induces neuroprotection against excitotoxic damage in primary cortical neurons via sustained nuclear factor‐kappa B (NF‐κB) activation. The transcription factor NF‐κB can regulate the expression of small conductance calcium‐activated potassium (KCa) channels. These channels reduce neuronal excitability and as such may yield neuroprotection against neuronal overstimulation. In the present study we investigated whether TNF‐α‐mediated neuroprotective signaling is inducing changes in the expression of small conductance KCa channels. Interestingly, the expression of KCa2.2 channel was up‐regulated by TNF‐α treatment in a time‐dependent manner whereas the expression of KCa2.1 and KCa2.3 channels was not altered. The increase in KCa2.2 channel expression after TNF‐α treatment was shown to be dependent on TNF‐R2 and NF‐κB activation. Furthermore, activation of small conductance KCa channels by 6,7‐dichloro‐1H‐indole‐2,3‐dione 3‐oxime or cyclohexyl‐[2‐(3,5‐dimethyl‐pyrazol‐1‐yl)‐6‐methyl‐pyrimidin‐4‐yl]‐amine‐induced neuroprotection against a glutamate challenge. Treatment with the small conductance KCa channel blocker apamin or KCa2.2 channel siRNA reverted the neuroprotective effect elicited by TNF‐α. We conclude that treatment of primary cortical neurons with TNF‐α leads to increased KCa2.2 channel expression which renders neurons more resistant to excitotoxic cell death.

Neuronal AKAP150 coordinates PKA and Epac-mediated PKB/Akt phosphorylation

In diverse neuronal processes ranging from neuronal survival to synaptic plasticity cyclic adenosine monophosphate (cAMP)-dependent signaling is tightly connected with the protein kinase B (PKB)/Akt pathway but the precise nature of this connection remains unknown. In the current study we 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 and HT-4 cells. We demonstrate that PKA activation leads to a reduction of PKB/Akt phosphorylation, whereas activation of Epac has the opposite effect. This effect of Epac on PKB/Akt phosphorylation was mediated by Rap activation. The increase in PKB/Akt phosphorylation after Epac activation could be blocked by pretreatment with Epac2 siRNA and to a somewhat smaller extent by Epac1 siRNA. PKA, PKB/Akt and Epac were all shown to establish complexes with neuronal A-kinase anchoring protein150 (AKAP150). Interestingly, activation of Epac increased phosphorylation of PKB/Akt complexed to AKAP150. From experiments using PKA-binding deficient AKAP150 and peptides disrupting PKA anchoring to AKAPs, we conclude that AKAP150 acts as a key regulator in the two cAMP pathways to control PKB/Akt phosphorylation.

Impedance measurement for real time detection of neuronal cell death

Detection of neuronal cell death is a standard requirement in cell culture models of neurodegenerative diseases. Although plenty of viability assays are available for in vitro applications, most of these are endpoint measurements providing only little information on the kinetics of cell death. Here, we validated the xCELLigence system based on impedance measurement for real-time detection of cell death in a neuronal cell line of immortalized hippocampal neurons (HT-22 cells), neuronal progenitor cells (NPC) and differentiated primary cortical neurons. We found a good correlation between impedance measurements and endpoint viability assays in HT-22 cells and NPC, for detecting proliferation, cell death kinetics and also neuroprotective effects of pharmacological inhibitors of apoptosis. In primary neurons we could not detect dendritic outgrowth during differentiation of the cells. Cell death in primary neurons was detectable by the xCELLigence system, however, the changes in the cell index on the basis of impedance measurements depended to a great extent on the severity of the insult. Cell death induced by ionomycin, e.g. shows as a fast paced process involving a strong cellular disintegration, which allows for impedance-based detection. Cell death accompanied by less pronounced morphological changes like glutamate induced cell death, however, is not well accessible by this approach. In conclusion, our data show that impedance measurement is a convenient and reliable method for the detection of proliferation and kinetics of cell death in neuronal cell lines, whereas this method is less suitable for the assessment of neuronal differentiation and viability of primary neurons.

Lovastatin Induces Neuroprotection Through Tumor Necrosis Factor Receptor 2 Signaling Pathways

Statins are widely used as medication to lower cholesterol levels in human patients. In addition, it was recently reported that they also reduce the incidence of stroke and progression of Alzheimers disease when prophylactically administered. To date there is only limited information available on how statins exert this beneficial effect. In this study we investigated the neuroprotective effect of lovastatin in primary cortical neurons. We found that lovastatin protects cortical neurons in a concentration-dependent manner against glutamate-mediated excitotoxicity. Interestingly, lovastatin with or without glutamate and/or tumor necrosis factor-alpha (TNF-alpha) increased TNF receptor 2 (TNF-R2) expression in cortical neurons. It was previously shown that activation of TNF-R2 signaling, which includes phosphorylation of protein kinase B (PKB)/Akt and activation of nuclear factor-kappa B (NF-kappaB), protects neurons against ischemic or excitotoxic insults. To investigate if lovastatin-induced neuroprotection was mediated by TNF-R2 signaling, primary cortical neurons were isolated from TNF-R1(-/-) or TNF-R2(-/-) mice. We could show that lovastatin is neuroprotective in TNF-R1(-/-) neurons, while protection is completely absent in TNF-R2(-/-) neurons. Furthermore, lovastatin-mediated neuroprotection led to an increase in PKB/Akt and NF-kappaB phosphorylation, whereas inhibition of PKB/Akt activation entirely abolished lovastatin-induced neuroprotection. Thus, lovastatin-induced neuroprotection against glutamate-excitotoxicity via activation of TNF-R2-signaling pathways.

A-kinase anchoring protein 150 in the mouse brain is concentrated in areas involved in learning and memory

A-kinase anchoring proteins (AKAPs) form large macromolecular signaling complexes that specifically target cAMP-dependent protein kinase (PKA) to unique subcellular compartments and thus, provide high specificity to PKA signaling. For example, the AKAP79/150 family tethers PKA, PKC and PP2B to neuronal membranes and postsynaptic densities and plays an important role in synaptic function. Several studies suggested that AKAP79/150 anchored PKA contributes to mechanisms associated with synaptic plasticity and memory processes, but the precise role of AKAPs in these processes is still unknown. In this study we established the mouse brain distribution of AKAP150 using two well-characterized AKAP150 antibodies. Using Western blotting and immunohistochemistry we showed that AKAP150 is widely distributed throughout the mouse brain. The highest AKAP150 expression levels were observed in striatum, cerebral cortex and several other forebrain regions (e.g. olfactory tubercle), relatively high expression was found in hippocampus and olfactory bulb and low/no expression in cerebellum, hypothalamus, thalamus and brain stem. Although there were some minor differences in mouse AKAP150 brain distribution compared to the distribution in rat brain, our data suggested that rodents have a characteristic AKAP150 brain distribution pattern. In general we observed that AKAP150 is strongly expressed in mouse brain regions involved in learning and memory. These data support its suggested role in synaptic plasticity and memory processes.

Mitochondrial small conductance SK2 channels prevent glutamate-induced oxytosis and mitochondrial dysfunction

Background: SK2 channels modulate NMDA-dependent neuronal excitability and provide neuroprotection against excitotoxicity. Results: We identify mitoSK2/KCa2.2 channels in neuronal mitochondria and demonstrate their protective function in cells lacking NMDAR. Conclusion: SK2 channels prevent mitochondrial dysfunction and completely restore cell viability independently of NMDAR modulation. Significance: Understanding how mitochondrial SK2 channels operate is crucial to develop novel therapeutic strategies for diseases caused by mitochondrial demise. Small conductance calcium-activated potassium (SK2/KCa2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K+ currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction.