(Europe Section Alessandro Volta Medal) Alzheimer Disease and Oxidative Stress: Thou Shalt not Breathe nor Think!
Authors: Amatore Christian Andre 
Published: February 2020

Oxidative stress is an essential metabolic outcome in aerobic organisms due to the activity of mitochondria in providing the basic energy of cells or during the operation of several enzymatic pools (NADH-oxidases, NADPH-oxidases, NO-synthases, etc.) in recycling or producing important molecules. It also serves to regulate the size and shape of organs or restructure them during foetal development by apoptosis. The same occurs in organs (e.g., liver, bones) at the adult stage to control cell populations. Oxidative stress is also indispensable to the immune system by allowing macrophages to eliminate virus, bacteria and impaired or dead cells through phagocytosis. In fact, no aerobic organism could live without oxidative stress.

Albeit these numerous benefits of oxidative stress explain why evolution maintain these essential mechanisms in aerobic organisms, they are associated to highly negative issues. Indeed, oxidative stress mechanisms provide a variety of life-harmful radicals and species called generically Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) whose fluxes need to be finely controlled to avoid the destruction of most organic molecules (e.g., lipids in cell membranes, enzymes, etc.) and biological ones (DNA, proteins, etc.) in cells. Thus, under normal conditions, a panoply of dedicated antioxidants and specific enzymatic systems (e.g., superoxide dismutase, catalase, etc.) is present in cells and ensures a fine homeostatic balance. However, rupture of this delicate balance is frequent and may provoke severe damages in cells and tissues that are important causative factors in many human pathologies (aging, cancers, AIDS, hearth and brain strokes, Parkinson and Alzheimer’ diseases, etc.) when their rates of occurrence exceed the capabilities of repairing systems.

All these important issues are well documented in medicine and biology but only through their long and middle terms outcomes in cells, tissues and organisms. However, investigations of the primary nature and effects of ROS and RNS remained impossible for long time because of the lack of analytical methods with adequate time and space resolutions. Using platinized carbon fiber ultramicroelectrodes positioned in an “artificial synapse” configuration we could establish that the so-called “oxidative stress cellular bursts” consist into the release of a reproducible cocktail involving a few femtomoles of hydrogen peroxide, nitric oxide, peroxynitrite and nitrite ions per cell. Our results have quantitatively confirmed that these species stem from the simultaneous and coupled initial production of superoxide and NO° by two separated pre-existing enzymatic pools: NADPH-oxidases (O2°-) and NO-synthases (NO°).

The same methodology also allows a precise kinetic and analytical characterization of the nature of functional hyperemia, viz., of the fine-tuning mechanism coupling neuronal activity and local blood flux supply in the brain. Besides its functions in supplying oxygen, glucose, etc., to the active brain areas, this mechanism underlies the present imaging methods (f-IRM, PET scans) used in investigating the working brain or for medical purposes. We investigated this process on single stellate neurons from perfused rat cerebellum slices and could establish for the first time that active neurons signal their need for increased blood supplies by emitting pulses of locally important concentrations of NO° (up to 500 nM). These fluxes were shown to develop over ca 50 µm around the active neuron to induce the opening of vascular constrictions in nearby capillaries allowing them to deliver oxygen and nutrients to the active brain cells. This process is extremely intense but specifically tailored to only activate blood capillaries in the near vicinity of active neurons. Since neurons act cooperatively along chains, it is inferred that under normal physiological conditions, a whole brain area may be irrigated by larger blood flows while being active, which is precisely what is reported by f-IRM or PET-scans.

Therefore, two fluxes (O2 and NO°) coexist in active parts of the brain. As such, this is not a problem. However, when free copper(II)-containing Amyloid-beta and ascorbate molecules are also present this ought to initiate a dangerous radical-chain process leading to the fast Cu(I)/Cu(II)-catalyzed production of protonated peroxynitrite (HO-ONO). This lipophilic species has enough stability to diffuse over several tens of a micrometer. When penetrating into neuron membranes, it spontaneously decomposes into OH° and NO2°. OH° leads to the fast creation of nanopores in the cell membrane, disrupting it and provoking apoptosis. We could fully establish the validity of this mechanism based on our analytical measurements and DFT investigations. Though overseen by the medical community, this mechanism may thus contribute to installing Alzheimer disease.