Translational and mechanistic studies on carbon monoxide as a modulator of neuroinflammation and neuroprotection – MITO-CO

We are utilizing a chemical approach to design and synthesize a novel class of hybrid compounds with dual bioactivity in order to maximize the delivery of precise amounts of CO to cells and tissues. We have also developed a device for measuring in real time the effects of CO on mitochondrial function under physiological conditions of oxygen. These two technologies will enable us to better define the mechanism(s) of action of CO in the context of neuroinflammation and neuroprotection.

We have designed and synthesized the first example of novel “hybrid CO-RMs” that possess a dual activity as they are capable of simultaneously release small quantities of CO and stimulate the production of CO within the cells. In collaboration with a scientific group in Portugal, we will have the opportunity to test the potential therapeutic effects of these compounds in models of cerebral ischemia and inflammation. Preliminary data have confirmed that our approach is feasible. Using a fluorescent probe highly sensitive to CO, we have demonstrated that indeed these hybrid molecules gradually liberate CO in a controlled fashion. When tested in microglia, the inflammatory cells of the brain, the hybrid CO-RMs showed an improved ability to reduce inflammation and activate intracellular cytoprotective pathways. We have also conducted experiments showing that CO-RMs have the ability at low concentrations to increase oxygen consumption by mitochondria. The French and Portuguese partners have the opportunity to foster their research findings as they are both management committee members of a COST Action on gasotransmitters.

Our final goal is to optimize the pharmacological properties of hybrid CO-RMs and maximize their therapeutic potential for the treatment of neuroinflammatory and neurodegenerative disorders. Using novel methodological approaches we are searching to identify the mechanism of action by which CO exerts anti-inflammatory and cytoprotective effects in mammals. The implications of this study are important as they may lead to the discovery of a class of drugs that divert from classical pharmaceuticals.

Preliminary data on the effectiveness of new CO-RMs as anti-inflammatory agents in microglia cells have been already presented as a poster at the 5th International Symposium on Nutrition, Oxygen Biology & Medicine held in Paris in June 2013. We are currently negotiating with SATT Ile-de-France Innovation for the submission of a patent on the use of our new hybrid molecules as therapeutic agents against inflammation.

Exacerbated neuroinflammation and neural cell death are complex processes leading to the progressive decline of neuronal activities associated with the development of neurodegenerative diseases and the pathogenesis of ischemic stroke. There is an urgent need to investigate the cross-talk between inflammation and neuronal cell injury with innovative conceptual and methodological approaches that may lead to novel therapies. Emerging and perhaps counterintuitive new findings point to carbon monoxide (CO) as an effective anti-inflammatory and cytoprotective agent. This gas produced endogenously during the degradation of heme by heme oxygenase-1 (HO-1) is a ubiquitous signalling molecule necessary for cells to counteract oxidative stress and injury. The advent of CO-releasing molecules (CO-RMs), a class of compounds that deliver precise amounts of CO to tissues and exert beneficial effects, have reinforced the concept that CO can be exploited therapeutically. Although the mode of action of CO and its intracellular targets are unknown, recent work revealed that CO can increase mitochondrial respiration and bioenergetic thereby directly controlling the production of oxidant species (ROS) by these organelles. Whether these properties of CO could be exploited for targeting mitochondria in cells with consequent modulation of neuroinflammation and neuroprotection is a timely topic that will be investigated in this proposal.

Here we propose a comprehensive research approach that develops around two major areas, namely the use of novel chemical entities targeting the heme oxygenase-1/carbon monoxide (CO) pathway and the study of these substances in protection against neuroinflammation and neuronal damage. The following objectives will be pursued. In Objective 1 we will design and test new molecules targeting the HO-1/CO pathway as possible anti-inflammatory and neuroprotective agents. These molecules, synthesized in collaboration with chemists, will be examined in astrocytes for metabolic changes and microglia cells for their anti-inflammatory action. Co-culture systems of astrocytes/neurons and microglia/neurons will establish whether modulation of glial function by CO affects neuronal protection. In Objective 2 we will study the effect of CO and the HO-1 pathway on mitochondria as possible targets in the modulation of neuroinflammation and neuroprotection. Mitochondrial function, biogenesis, metabolism and bioenergetic will be investigated in living cells and isolated mitochondria after treatment with CO-RMs and HO-1 inducers. A dedicated hypoxic chamber for the culture of cells at controlled oxygen tension and an XF Analyzer that enables real time assessment of glycolysis and oxidative phosphorylation in living cells will be used. In Objective 3 validation of CO and HO-1 inducers as novel therapeutic agents in neuroprotection will be performed using an in vivo model of cerebral ischemia.

The multidisciplinary nature and the knowledge of the partners in disciplines such as chemistry, cell biology and pharmacology clearly represent the strength and added value of this proposal. Dr. Motterlini and Dr. Vieira complement each other because of their mutual interest in CO biology and their aim to understand more deeply how CO and mitochondria interact to mediate the positive effects of this gas. This is certainly a novel concept to tackle and one of the major tasks of the proposal. Furthermore, this joint proposal is well balanced since it combines the complementary expertise of Dr. Motterlini on the anti-inflammatory properties of CO and that of Dr Vieira on cell death and metabolism. Together they possess the know-how necessary for the success of this research proposal and represent the only two group leaders in Europe who demonstrated independently a clear role of CO on mitochondria bioenergetics and ROS signalling. Thus, their international collaboration not only represents an added value but also a true competitive advantage in the field.