Preconditioning / Hormesis

• Concept of invariable ‘Milieu Intérieur’ (internal medium) as definedby Claude Bernard (1865)

• Homeostasis as defined by Walter Bradford Cannon (1926)designates all the processes involved in ensuring the stability ofphysiological parameters (body temperature, pH, osmolarity, etc…)of the internal medium

• ‘Allostasis’ and allostatic load’ were later defined by Bruce McEwen(1998). It applies to the response to stress by the HPA axis

The HPA (hypothalamus-pituitary-adrenocorticalgland) axis allows to cope with stress thanks tothe release of different hormones in cascade.However, chronic stress may become harmful tothe body because of an overactivation of thisaxis. Indeed, chronic exposure to the stresshormones can endanger cellular, especiallyneuronal viability (that‘s what is called the‘allostatic load’). The allostatic load couldcontribute to the progression ofneurodegenerative diseases.

• ‘Hormesis’ as defined by Southam and Ehrlich (1943) applies to short- or long-lasting beneficial effects activated in cells after a ‘moderate’ stress (or harmful stimulus). ‘What does not kill you will make you stronger!’.

• Hormesis was first defined by toxicologists who observed beneficial effects of very low doses of poisonous substances (‘poisons’). As you will see in the next slides, this applies to a lot of substances and hormesis has been evidenced in a lot of living beings, including plants.

• ‘Only the dose makes the poison’ (Paracelsus)

• Hormetic mechanisms are currently observed in aerobic organisms in order to counteract oxidative stress. In this line, physical exercise, moderate hypoxia, and naturally-occurring antioxidants are triggers of hormetic mechanism enabling cells to resist harmful oxidative stress.

• ‘Cardiac preconditionning’ designates a medical procedure leading to a light hypoxia of the heart during recovery after heart infarct.

• Hormesis is also observed in the brain (Mattson et Calabrese 2003).

Hormesis = Adaptative homeostasis

Homeostasis

No effect

Beneficial effect

Toxic effect

concentration

‘Bell-shaped’ curve which is a hallmark of hormetic mechanisms

Schematic ‘Time course’ of biological mechanisms involved in hormetic events.(resistance to oxidative stress)

Examples of hormetic adaptations

• Hormesis is also observed in the plant kingdom, especially with environmentally spread molecules:

– Herbicides, including glyphosate, display positive effects on plant growth at very low doses…

– Pharmaceutical agents (medicines discarded in the soil or water (see next slide)

Triticum aestivum:wheat

Zea mays:corn

Hormetic adaptations of the redox status

• Oxygen consumption by the mitochondria leads to the production of oxygenated radical species (so-called ROS).

• The accumulation of ROS leads to ‘oxidative stress’ as ROS may directly bind to cell biomolecules (proteins, lipids, nucleic acids) and impair their activity .

• Cells have different pathways to regulate ROS concentration: non-enzymatic antioxidants (GSH, Vitamine E) and enzymatic antioxidants (SOD-1, Glutathione peroxydase, …)

• There are many intracellular pathways which will activate enzymatic defenses against ROS: this is the case of the Nrf2 pathway which is activated by many ways, including ROS.

• Other pathways (activated by natural substances, exercise, caloric restriction) linked to the metabolic status of the cell many also impact on the redox status.

Just a reminder…

Oxygen radicals production and regulation

Complex III

Complex III

O2.- H2O2

O2.-

O2.-

HO2H2O2

H2O2

LOOH

H2O

LOH

O2.- H2O2 H2O

Peroxiredoxines (Prx)

Glutathione peroxidase (Gpx1)Catalase

Prx3

Glutathione peroxidase 3 (Gpx3)

EcSOD(Sod3)

Glutathione peroxidase 4 (Gpx4)

Lipidic peroxydation

CuZnSOD (Sod1)

O2.- H2O2

CuZnSOD (Sod1)

Gpx4

Thioredoxine 1(réduite)

Thioredoxine 1(oxydée)

Méthionine oxydée

Met=OMéthionine

Met

Methionine Sulfoxide Reductase A (MsrA)

NADP+ NADPH

Thioredoxine 1 reductase

Complex I

MnSOD (Sod2)

O2.-

8-oxodG

8-oxoguanine DNAGlycosylase (Ogg1)

Production and regulation of ROS in cells(O2

.- : NADPH oxydase; NO: NOsynthase)

H2O2

2H2O

2GSH

GS-SG NADPH + H+

NADP+ ROOH

2ROH

2GSH

GS-SG NADPH + H+

NADP+

Glutathione cycle with associated enzymes

Glutathione utilization to remove peroxydes from cells

rapport GSH/GSSG

Under resting conditions, Nrf2 is associated with Keap1. Keap1 can recruit a proteic complexCul3/Rbx1/E2.The ubiquitin ligase E2 can ubiquitinylate Nrf2 for proteolytic degradation. In the presence of (ROS) or antioxidants or after phosphorylation, Keap1 releases Nrf2 which then translocates to the nucleus and bind Antioxidant Responsive Element . This triggers the transcription of antioxydants enzymes: Hemeoxygenase (HO1), NADPH Quinone oxydoreductase (NOQ1), Glutathion-S-transferase (GST), Glutathion peroxydase, Glutathion reductase,g-glutamate-cysteine synthetase (g-GCS)…It is noticeable that the expression of enzymes involved in GSH synthesis is prominently stimulated after Nrf2 activation. This ensures a quick response to regulate oxidative stress.

Nrf2

P

Keap1-SH

-SHKeap1

SH-

SH-

Nrf2

P

MAF

AREARE

Transcription

ROSPollutantsAntioxydantsphosphorylation

protéasome

Activation of the Nrf2 pathway

Antioxydant effect of (HO-1)

In the CNS, the Nrf2 pathway is mainly found in astrocytes: they can protect neurons by fuelling them with GSH.This pathway is now widely studied in order to find new therapeutic approaches to treat neurodegenerative diseases which progression is associated with oxidative stress.

NrF2 is activated by numerous naturally-occurring antioxydants found in food (sulforaphane, curcumin, EGCG, …). It is believed that it is the pathway associated to the beneficial effects of a ‘healthy lifestyle’ !These antioxydants may also regulate intracellualr pathways involved in energy production (see next slides).

GSH level in neurons is crucial for their survival. All brain diseases including neurodegerative diseases and neurdevelopmental diseases (forms of schizophrenia, autism) are linked to reduced GSH concentration in the brain. Therefore, pathways leading to GSH synthesis may have protective effects against these diseases.

resvératrol

FOXO pathway activation increases lifespan (C. Elegans) by stimulating the expression of antioxydant enzymes. This pathway is activated JNK and AMPK.

Hypoxia-induced hormesis

• Moderate hypoxia leads to the expression of factors which will contribute to oxygen supply (i.e. vascularization) and will protect cells against a more severe hypoxia/ischémia.

• Cardiac preconditionning (1986). This is observed in many organs including the brain.

• Preconditionning involves a lot of factors including HIF leading to genomic effects.

POS: another example of hormesis ?

• Concept of ‘POS’: preparation for oxidative stress.

• In the environment, living organisms experience many stresses (oxygen deprivation, hypothermia, dehydration,…) which promote adaptations in these organisms.

• Oxygen deprivation leads to decreased transcription/traduction, reduced of metabolic rate, increased antioxydant defenses and metabolic rewiring to fermentation.

• These phenomena correlate with the activation of intracellular pathways NrF2, HIF,…

• POS: preparation to oxidative stress occurring during reperfusion?

Mechanisms of POS

Examples of POS

• Anoxia and hypothermia stimulate the expression of antioxydant enzymes in muscle cells of different species of snakes.

• Dehydration leads to the activation of NrF2 and FOXO1 in frogs.

• Dehydration (8h) leads to reversible expression of SOD, catalase and GST in molluscs.

Activation of HIF1a

Activation of HIF1a : under normoxic condition, HIF1a is engaged in a proteiccomplex leading to its ubiquitinylation and degradation. Under hypoxia, HIF1a is released and translocates to the nucleus.

Gene expression stimulated by HIF (in purple those activated by HIF1) leading to increased vascularization

HIF and cancer?

• HIFs play a major role in tumor progression…

Relationship between HIF and Alzheimer’s disease?

• HIF have a very controversal role in neurodegenerative diseases: they can activate both pro- and anti-Alzheimer factors…

Effect of physical exercise

Physical exercise increases cellular ROS content. This leads to genomic actions resulting in improved mitochondrial activity, in muscle cells first.

‘Mitohormesis’ concept : adaptative mechanisms of mitochondria to stress

Exercise, mitochondrial health and cycle

Cellular responses to mitochondrial stress MTS=mitochondrial targeting sequence

ATF5 (mammals)

ATF5

The mitochondrial Unfolded Protein Response (UPRmt) pathway (1).

The mitochondrial Unfolded Protein Response (UPRmt) pathway (2).

‘Dark side’ of mitohormesis: UPRmt activation in cancer cells

• Recent work evidences that endogenous mitohormesis is found in cancer cells (breast cancer).

• Mitohormesis leads to increased basal UPRmt which render cancer cells more invasive and metastatic.

• Breast cancer patients with increased UPRmt levels have worse clinical outcomes.

Mitochondria-to-nucleus retrograde signalling pathways in mammals

Article

Measurement of cell viability PC-12 (pheochromocytoma): model of catecholamine releasing cells. 6-OHDA: toxic to cells producing and releasing catecholamines.

Measurement of apoptosis: anti-apoptotic effect of berberine

Activation of PI3K, AKT and Nrf2

Effect of a PI3K blocker of the effect of berberine (1)

Effect of a PI3K and a HO-1 blocker of the effect of berberine (2)

Effect of berberine on dopaminergic neurons of zebra fish. Dopaminergic neurons are positive for tyrosine hydroxylase (TH); the limiting enzyme for dopamine synthesis.

Effect of berberine on locomotor activity of zebra fish.

Summarizing scheme