Brain Research Center Medical University of Vienna

Oxidative Injury to Glial Cells and Neurons as a Basis for Progressive Disease in MS

Hans Lassmann (MD) Disclosures

• HL received honoraria for lectures from Novartis, Biogen, Sanofi Aventis, TEVA, Roche, The Role of Oxidative Injury in Multiple Sclerosis Lesions

• MS lesions specific changes in expression indicate a major role of oxidative injury in demyelination and neurodegeneration • Neurons and different glia cells react differently to • Oxidative stress is initiated through activated microglia and macrophages and amplified by mitochondrial injury and iron liberation Mechanism of Tissue Injury in MS

White Matter Lesions Subpial Cortical Lesions

Demyelinated (EA) lesions MS TB AD Initial lesions Normal appearing white matter

RNA isolation from Pathway analysis micro-dissected Lesion Immunohistochemical Areas Validation in a large Genome wide Sample of Different MS microarrays Lesions MS Specific in Progressive MS

Gene expression changes in active white and grey matter lesions identify production of reactive oxygen species and oxidative Up-regulated Down-regulated tissue injury as ROS Production CYBA, CYBB, NCF1 NOS1, NOS2A, NOS3, dominant pathways MPO, EPX, PTGS1, PXDN NOX5, , MIOX, RAC1, RAC3, of tissue injury in ROS detoxification GPX4, PRDX1, 2, 4 GPX3, GPX5, PRDX3 active MS Induced by ROS ALOX12, ATOX1, EPHX2, APOE; CYGB, PNKP, GPR156, MSRA, STK25, SCARA3, SFTPD, SIRT2, OSGIN, GLRX2, PRG3, SRXN1 Fischer et al 2012, 2013 SEPP1, SGK2, TXNRD2 Oxidative Injury in Active White Matter Lesions (Haider et al 2011)

Cells with oxidized DNA (8- OHdG) are mainly present in the zone of ongoing activity 8-OHdG is found in apototic nuclei of olgodendrocytes, in some astrocytes and in phagocytosed nuclei in macrophages Oxidative Injury in Active White Matter Lesions (Haider et al 2011)

Astrocytes 8-OHdG E06

Oligodendrocytes Severe oxidative Damage in Oligodendrocytes associated with cell death and demyelination, Emperipolesis of oligodendrocytes with oxidized DNA in astrocytes, Oxidative injury in astrocytes sequestered in autophagic vacuoles Oxidative Injury in Active Cortical Lesions (Fischer et al 2013)

Oxidized Phospholipids (Oxidative Injury)

Dendrite Fragments Apoptotic Neurons Central Chromtolysis Dystrophic Axons

DNA Damage AIF Liberation

DNA Injury AIF normal AIF Apoptosis Neurons with Oxidative Injury in the MS Cortex

Neurons with oxidative injury are present in lesions and NAGM

Correlate with meningeal inflammation in MS

(Haider et al 2016) Differential Reaction of Brain Cells to Oxidative Stress • Basic Pattern – Distal (dying back) cytopathy; cell processes more affected than cell bodies • Neuron (Fischer et al 2013, Wegner et al 2006, Jürgens et al 2016) – Dendritic degeneration, axonal degeneration, loss of dendritic spines, synapse loss – neuronal apoptosis • Oligodendrocytes (Aboul Enein et al 2003) – Distal oligodendrogliopathy; Loss of myelin associated glycoprotein; – OG apoptosis, demyelination • Astrocytes (Sharma et al 2010) – Loss of processes, loss of glia limitans and aquaporins, a-dystroglycan – autophagic granules • Microglia / Macrophages (Lopes e al 2008, Hametner et al 2013) – Radical production, phagocytosis of damaged cells – Microglia dystrophy and senescence after iron loading Consequences of Oxidative Injury in MS Lesions • Mitochondrial Injury (Mahad et al 2008, Campbell et al 2010) • Functional disturbance of respiratory chain • Damage and degradation of respiratory chain proteins • Mitochondrial DNA mutations / deletions • Amplification of oxidative damage • Histotoxic „virtual“ hypoxia / Energy Deficiency (Trapp & Stys 2009) • Neurodegeneration • Chronic cell stress (ER stress / apoptosis) • Ionic imbalance Mitochondrial Injury in MS

COX1 < COX4 < Complex I or II < Porin

Mitochondria

COX1 Mahad et al, Brain Radical Production 2008 Energy Deficiency Mitochondrial Injury in MS (Campbell et al 2010, 2011)

Clonal expansion of mitochondrial defects

ROS ROS initial ROS injury normal increased vulnerability spontaneous death Mitochondrial Injury and Neurodegeneration and its Prevention

Mitochondrial Injury leads to energy deficiency and subsequent ionic imbalance Neuroprotective effects of ion-channel blockers (Na+ Channel Blockers: Lamotrigine, Phenytoin; Acid sensing Na+ Channel Blockers: Amiloride; Glutamate receptor / Na+ Channel Blockers: Riluzole)

Na+ GluR VGCC

Calpain Dissolution of ++ Na+ Ca Cytoskeleton Ca++ Axonal Destruction Disturbance of Na+ Axonal Transport Oxidative Injury in Multiple Sclerosis Lesions • Oxidative injury is a major pathway of demyelination and neurodegeneration in MS lesions • Potential source of oxygen radical production: – Oxidative Burst (Inflammation, Macrophage / Microglia Activation, Astrocytes) • NOS 1-3, NOX 1-5 Complexes, – Dyscoupling of the Mitochondrial Respiratory Chain • Complex I Defect – Iron Liberation within the Lesions • Fenton Reaction Oxidative Burst in MS Lesions: Driven by Inflammation

Oxidative burst in MS lesions is mainly driven by oxygen radical production through NADPH oxidases (Nox1 and Nox 2)

No up-regulation of NOS molecules in active MS lesions in comparison to controls NAWM EL LL p22phox

LL NOX2 EL NAWM

NCF1 Cellular iron storage in the healthy brain

White matter Cortex

Oligo- Oligo- dendrocytes dendrocytes + + iron iron

Oligo- Neurons dendrocytes + + iron iron

Deep grey matter Deep grey matter

Haider L et al., JNNP 2014, Hametner S et al., Ann Neurol 2013 Iron: promoter of free radical damage

1. Physiologic function of iron: necessary in neurogenesis, myelin synthesis, neurotransmitter production oxygen transport 2. Iron and oxidative stress: Fe2+: low toxicity Oxidative burst H2O2 : low toxicity

2+ 3+ . − Fenton reaction: Fe + H2O2  Fe + OH + OH Iron Related Proteins in Lesions of Progressive MS

• NAWM: NAWM – Ferritin and iron mainly in oligodendrocytes • Active MS Lesion: – Destruction of oligodendrocytes – Uptake of iron in microglia and L macrophages – Ferritin positive macrophages and microglia degenerate – Liberation of iron from degenerating cells may lead to radical production Amplification of Neurodegeneration by Iron in Progressive MS Lesions

Co-localization of iron and MS active Lesion oxidized phospholipids in active MS lesions

Potentiation of neurodegeneration Ox PL Iron (oligodendroglia) by iron loading in vitro

Reduction of neurodegeneration by iron chelators in vivo (EAE, Trauma) Haider et al., Brain 2011 Hametner et al., Ann Neurol 2013 Tissue Injury in Multiple Sclerosis

Current anti- Inflammation inflammatory Treatments Microglia activation due to Microglia / Astroglia Activation Minocyclin pre-existing CNS Oxidative Burst Laquinimod ? damage Fumarates ? ROS / RNI production Liberation of Free Iron from Cellular Stores

Mitochondrial Injury / Energy PARP / AIF DNA Damage Deficiency

Mitochondrial Protection; Tissue Degeneration High dose Biotin Oligodendrocytes > thin axons > neurons > others) Functional Disturbance Na+, Ca++ Channel Blockers Oxidative Injury in MS Lesions

• Oxidative injury and damage is a major driver for demyelination and neurodegeneration in MS lesions • It is induced by a combination of microglia / macrophage activation, mitochondrial injury and iron liberation in the lesions • Different vulnerability of different brain cells to oxidative damage: – Oligodendrocytes > Neurons & Axons >> astrocytes Centre for Brain Research & Clinical Institute of Neurology, MUW Vienna Simon Hametner, Tobias Zrzavy, Lukas Haider, Isabella Wimmer, Marie-Therese Fischer, Jan Bauer, University of Edinbourg, UK: Don Mahad, Douglas Turnbull Vrije University Amsterdam, NL: Jack van Horssen

Funded by the Austrian Science Fund, European Union Projects