Are There Common Mechanisms in Neurodegeneration? Yun Joong Kim, MD Lab of Neurodegenerative Diseases Research ILSONG Institute of Life Science The Hallym Academy of Sciences Department of Neurology Hallym University

Common clinical features in neurodegenerative disorders Onset in mid-life ; sporadic cases Progressive over decades Selective regions of involvement Overlapped symptoms and signs

Some neuropathological features are shared Lewy body pathology AD < PD < DLBD -synuclein Lewy body in PD, AD and DLBD Glial and neuronal cytoplasmic inclusion (GCI and NCI) in MSA LB-like inclusions, neuraxonal spheroid and LNs in NBIA 1 NACP in AD Neurofibrillary tangle (tau) AD, FTDP-17, CBGD, PSP and etc Neuronal intranuclear inclusion in polyglutamine disorders

Ub-immunoreactive inclusions in neurodegenerative disorders Disease Gene Mutations Pathology AD APP Missense Amyloid plaques, PS1 neurofibrillary tangle PS2 FTDP Tau Tau inclusions Pick’s Pick bodies ALS SOD Lewy-body-like inclusions PD -synuclein Lewy bodies UCHL1 Parkin DLBD MSA Glial cytoplasmic inclusions (GCI) Prion Prion protein (PrP) plaque DRPLA Atrophin1 Polyglutamine Neuronal nuclear inclusions HD Huntingtin SCA1 Ataxin1 SCA3/MJD Ataxin3 SCA7 Ataxin7

Facts against common mechanism Inclusions do not correlate with neuronal cell death. Inherited cases are caused by mutations in different gene. Specific sites of neurodegeneration within the brain Ubiquitous expression of proteins implicated in inherited neurodegenerative disorders

Genetics of neurodegenerative diseases What we want to know from rare mutation? Familial PD A30P -Syn A53T -Syn WT -Syn Sporadic PD ? Neurodegeneration

Genetics of neurodegenerative diseases What we want to know from rare mutation? Familial PD A30P -Syn A53T -Syn Increased ROS Altered -Syn conformation Accumulation of -Syn Ubiquitin proteasome dysfunction Microglial activation Increased extravesicular dopamine Mitochondrial dysfunction WT -Syn Sporadic PD ? ↓Complex 1 ? ↑ROS ? Neurodegeneration

Cause or effect? Innocent bystander? Familial PD A30P -Syn A53T -Syn WT -Syn Sporadic PD Inclusions? ? Neurodegeneration ?

Alzheimer’s disease Amyloid plagues Neurofibrillary tangle Accumulation of extracellular aggregates of the 42-residue amyloid -protein (A42) Neurofibrillary tangle Intraneuronal aggregates of microtubule-associated phosphoprotein tau

ISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA T V I V ITLVMLKK 1 17 NH2 289 671 770 COOH TM A KPI 670/671 692 693 694 714 715 716 717 723 ISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA T V I V ITLVMLKK N L G O K I M A V P    I F G L -secretase -secretase 18 687 711 or 713 770 C83 APPs -  p3 AICD -secretase -secretase 18 671 711 or 713 770 C99 APPs -  A AICD

A peptides A1-40 A1-42 Less hydrophobic Highly self-aggregating Normally 5-10% of total secreted A peptide

Regulation of A42 production & clearance Either APP or PS mutant increases A42 fraction about 15-40%. Clearance ApoE4 Neprilysin ; a member of metalloproteases Insulin degrading enzyme (IDE) microglia

A Hypothesis A cascade → Accumulation of A42 / A42 oligomerization → ↑production or ↓clearance of A42 → Accumulation of A42 / A42 oligomerization → Synaptic dysfunction Microglial activation / inflammation Oxidative injury Altered kinase/phosphatase / PHF formation (tau) → Wide spread neuronal / synaptic dysfunction, neuronal loss with attendant neurotransmitter deficits

Etiology of Parkinson’s disease Oxidative stress Mitochondrial complex I defect Mutations in -synuclein Parkin UCH-L1 DJ-1

Oxidative modifications of -synuclein promote accumulation & aggregation : sporadic PD 8M Urea 6M guanidinium chloride No treatment Peroxinitrite/CO2 treatment Anti-nitrated--synuclein-specific Ab

Accumulation of -synuclein in PD brain A53T -synuclein transgenic mouse Anti -synuclein Ab

Rotenone causes -synuclein accumulation in rontenone-induced murine PD model Anti--synuclein Ab Midbrain

Ubiquitin proteasome pathway for protein degradation ATP ADP E1; Ub activating enzyme E1 E1 + Ub Substrate; misfolded or unfolded protein Ub E2 Ub Ub E1; Ub conjugating enzyme Ub E2 Ub + + Ub E3 E3; Ub ligating enzyme “substrate specific” ADP Ub ATP Unfolding, degradation & translocation Proteasome

Decreased proteasome activity after overexpression of A30P -synuclein Decreased proteasome activity after overexpression of A30P -synuclein Tanaka et al., 2001

Role of Parkin mutation in PD UbcH7/6, UBC6/7 Ub Ub-like RING1 IBR RING2 1 Sp22 Ub 76 238 293 Ub Synphilin CDCrel-1 Pael-R 314 377 418 449 455 Parkin Autosomal recessive juvenile parkinsonism (AR-JP) Serves as E3 ligase of O-glycosylated isoform of -synuclein (Sp22), CDCrel-1, synphilin & Pael-R Parkin mutation → impairment of Ub-proteasome pathway → Accumulation of substrate proteins

Altered proteasomal function in sporadic PD

Genes/loci associated with familial ALS Gene/Loci Comments References Adult onset SOD1 Located on Ch 21; Mostly missense mutations responsible for ~20% familial ALS Rosen et al., 1993 NF-H Located on Ch 22; codon deletions in KSP repeats found in ~1% cases of sporadic ALS Figlewicz et al., 1994 Al-Chalabi et al., 1999 EAAT2 RNA processing errors Mutations in one ALS case Lin et al., 1998 Aoki et al., 1998 Cytochrome c oxidase Mitochondrial DNA microdeletion in one ALS case Borthwick et al., 1999 Ch 9q21-q22 Hosler et al., 2000 Juvenile onset Ch 2q33-q35 Autosomal recessive juvenile ALS Hentati et al., 1998 Ch15q15-q24 Ch 9q34 Autosomal dominant juvenile ALS Chance et al., 1998

Proposed mechanisms of pathogenesis in ALS Julien JP, Cell 2001 Autoimmunity Astrocyte damage SOD1 mutation Oxidative damage Anti-Ca2+ channel Ab Anti-Fas Ab Aberrant catalysis? ↓EAAT2 Excitotoxicity Misfolding Damage to mitochondria ↑Ca2+ Aggregates Organelle trapping Chaperone dysfunction Ubiquitin proteasome dysfunction Axonal transport defects Mitochondrial vacuolization Neuron Death ↑Bax, Bad IF inclusions ↓Bcl-2, Bcl-xl ? Caspase-1 Caspase-3 ↑ Peripherin Inflammation Neurofilament mutation Gliosis Asymptomatic stage Disease onset Disease spreading

Polyglutamine diseases: (CAG)n Repeat size Repeat Location Disease Gene (protein) Normal SBMA Androgen Receptor (AR) 9-36 38-62 Coding/N–ter HD Huntingtin (3144 aa) 6-37 38-121 DRPLA Atrophin-1 6-35 49-88 SCA1 Ataxin-1 6-34 39-82 SCA2 Ataxin-2 15-31 36-63 SCA3 Ataxin-3 12-40 55-84 Coding/C–ter SCA6 1A-volt-dep calcium channel 4-18 21-33 SCA7 Ataxin-7 4-35 37-306 SCA17 TATA binding protein 25-42 47-63 Coding/N-ter

Inclusions in HD brain striatum cb ctx striatum cb ctx G pallidus Cbll Ab 1 striatum cb ctx striatum cb ctx internal capsule G pallidus Cbll

Proteolysis of huntingtin is important in the pathogenesis of HD Wt huntingtin (htt) – 3144 aa N-terminal htt fragments Nuclear and cytoplasmic inclusions contain mutant N-terminal huntingtin fragment. Readily form inclusions Harmful when expressed in vitro and in vivo ; The smaller fragments are more toxic. Most interactions

Altered of conformation Unfolded Monomer -sheet transition (genetic, environment) -structured monomer oligomerization Oligomeric intermediate linear addition oligomer association Protofibril Fibril Fibers Inclusions Altered of conformation Accumulation of “potentially toxic misfolded protein, oligomeric intermediate or protofibril” due to Impaired clearance mechanism Resistance to degradation

Inclusion or aggregation ≠ accumulation or oligomerization SCA1 154Q/2Q KI SCA1 TG E6-AP KO NII Cb cortical neurons CA1 hippocampal neurons early Purkinje cells Ant horn neurons later Neurodegeneration intensified ↓ Neuronal intranuclear inclusion (NII) Aggregation (inclusion) formation may be cell’s way to sequester mutant protein, thereby to curtail its toxic effect.

Genetic ? ? ? Neuron Death Accumulation of oligomeric peptides / protofibril ? ? ? ? ? ? Environments What are the deleteriou effects of the accumulation of oligomeric peptides/protofibril?

SDS-stable oligomers of human A block hippocampal LTP in vivo Intracerebroventricular microinjection of conditioned media containing A monomer, dimers, oligomers (7PA2) in rat

-secretase inhibitors block oligomer formation Pretreatment of cells with -secretase inhibitors does not block LTP.

Interaction between -synuclein protofibrils & DA Leakage of dopamine from vesicle Vesicle permeabilization by protofibrillar -synuclein Vesicle permeabilization by protofibrillar -synuclein is sensitive to Parkinson's disease-linked mutations and occurs by a pore-like mechanism Annular alpha-synuclein protofibrils are produced when spherical protofibrils are incubated in solution or bound to brain-derived membranes Dopamine dependent neurotoxicity of -synuclein Accumulation of -synuclein in dopaminergic neurons results in apoptosis that requires endogenous production and is mediated ROS. -synuclein is not toxic in non-dopaminergic neurons. “-synuclein protofibril → DA leakage → ↑ ROS”

Impairment of ubiquitin proteasome system by expanded polyglutamine Ub-proteasome system reporter – GFP-degron/CL1 (GFPU) HEK293 cells stably expressing GFPU a b c d e

Altered transcription in polyQ disorders An abnormal conformation of mutant polyQ associate with short polyQ stretches in other critical cellular elements. Depletion of CREB-binding protein (CBP), a transcriptional coactivator  Interference with CBP-activated gene transcription

Anti-amyloid compound, Congo red prevent polyQ induced cell death

Mutant full-length polyQ-containing protein proteolysis polyQ containing fragments polyQ oligomers Degradation by UPP or lysosome protein recruitment accumulation trascriptional dysregulation proteasome inhibition ? caspase activation Neuronal death

Reversal of neuropathology and motor dysfunction in a conditional model of HD Tet-OFF transgenic HD mouse P0 18 wks 2 mg/ml Doxycycline 34 wks Caudate Putamen GFAP counts D1 receptor * ** * *** Control HD94 (18 wks) Gene ON (34 wks) Gene OFF (34 wks) *, p<0.05; **, p<0.001; ***, p<0.005

Summary & Conclusion Multiple mechanisms may be involved in the pathogenesis of neurodegeneration. Proteasomal dysfunction accumulates misfolded proteins linked with neurodegenerative diseases. Accumulation of misfolded protein leading to oligomerization and protofibril formation appears linked to neuronal death in some neurodegenerative diseases. Turning off transgene (mutant huntingtin) expression reverses neurodegeneration in HD transgenic mouse. Understanding of pathogenic mechanisms may be useful in developing therapy in the future.

Acknowledgement Marian DiFiglia (Harvard Univ) Ellen Sapp (Harvard Univ) Chulhee Choi (Ewha Univ) Jinho Kim (Chosun Univ) and Authors in many excellent papers