Clash Of The Titans! Convergence Of Tau And Alpha-Synuclein Amyloid In Neurodegenerative Diseases John Q. Trojanowski, M.D., Ph.D. Institute on Aging Center for Neurodegenerative Disease Research Department of Pathology and Laboratory Medicine University of Pennsylvania Philadelphia, PA

Neurodegenerative Diseases Characterized by Brain Amyloidosis Disease Lesions Components Parkinson’s Disease LBs  -Synuclein Dementia with Lewy Bodies LBs  -Synuclein Multiple System Atrophy GCIs  -Synuclein Alzheimer’s Disease SPs Aβ (Most common synucleinopathy!) NFTs Tau LBs α-Synuclein Prion diseases SPs Prions Tauopathies NFTs Tau Trinucleotide Inclusions Expanded Repeat Expansion PolyQ tracts

High Median Low Millions Year US Population Demographics 85 Years of Age or Older:

Synucleinopathies Parkinson’s disease - familial and sporadic* Dementia with Lewy bodies Multiple system atrophy Neurodegeneration with brain iron accumulation-1 (formerly H-SD) Pure autonomic failure REM sleep behavior disorder Down syndrome* Alzheimer’s disease* (The most common α- synuclein brain amyloidosis!) * These disorders are “triple” brain amyloidoses with tau, Aβ and α-synuclein amyloid deposits

Normal Alpha Synuclein An abundant low Mr synaptic protein, present to a lesser extent in perikarya and axons, but also in oligodendroglia Other members of the synuclein family of synaptic proteins include beta- and gamma-synuclein Function is unknown but may play roles in synaptic transmission Is a phosphoprotein, but role of alpha- synuclein phosphorylation in its normal function is unknown

Alpha-Synuclein Mutations Cause Familial Parkinson’s Disease H2NH2N COOH Hydrophobic Middle Negatively Charged NAC peptide (aa 61-95) A30P mutation A53T mutation = 6 imperfect repeats of 11 amino acids with the conserved core KTKEGV Carboxy-terminus Section Mutations promote protein aggregation and filament formation

Pathological Alpha-Synuclein Forms insoluble filamentous aggregates with the properties of amyloid Amino acids in the NAC domain are the minimal, essential sequences required for fibrilization Filamentous alpha-synuclein inclusions form in neuronal perikarya, processes and in gliaI cells Is abnormally phosphorylated, nitrated and ubiquitinated

Alpha-Synuclein Pathological Inclusions Lewy bodies Electon microscopy Lewy neurites Lewy bodies Glial cytoplasmic inclusions Glial cytoplasmic inclusions Electron microscopy

Environmental and/or Genetic Risk Factors Synuclein Dysfunction and/or Aggregation Genetic Factors  -Synuclein Mutations or Duplications APP, PS1, PS2 Mutations, DS Neurodegeneration ? Alpha-synuclein Dysfunction And Aggregation Play Central Roles in Mechanisms of Neurodegenerative Disease

But hold on! Tau and Alpha-Synuclein Pathology Commonly Co-occurr in Neurodegenerative Diseases – So what does this mean? Diseases with tau, α-synuclein and abundant A  deposits –Lewy body variant of Alzheimer’s disease –Alzheimer’s disease –Familial Alzheimer’s disease –Down’s syndrome Diseases with tau, α-synuclein and scant/no A  deposits –Parkinson’s disease/Dementia with Lewy bodies –Multiple system atrophy –Guam ALS/PDC –Neurodegeneration with brain iron accumulation type 1 –Some cases of PSP, CBD, and Pick’s disease

Normal Tau Proteins Abundant low molecular weight microtubule (MT) associated proteins localized mainly in axons Promote MT polymerization, bind to MTs and stabilize MTs in the polymerized state Normally is phosphorylated at a range of Ser and Thr residues Phosphorylation negatively regulates binding of tau to MTs

4R2N 4R1N 4R0N 3R2N 3R1N 3R0N E2 R1 R2R3 R4 R1R2R3R R2 R1 R2 R3R E3 R3 R R1 R3R R1R3R R1 E10 SDS-PAGE 67kDa 62kDa 59kDa 54kDa 52kDa 48kDa Six Human Brain Tau Isoforms Are Generated By Alternative Splicing

PHF-Tau Proteins Insoluble Form filamentous amyloid deposits in neuronal cell bodies/processes and glia Aberrantly hyperphosphorylated at Ser/Thr; ubiquitinated Unable to bind to MTs unless dephosphorylated in vitro

AAUAAGAAGCUGGAUCUU CCGGGAGGCGGCAG-U 4411 Exon 10 N279K  280K Intron 9 gu u ga g a c c u L284L P301L/S S305N/S V337M R406W G C U A   AAG cca u N279K  280K P301LS305N S305SL284L a u ac g u Intron u/t +16 g +13 C G272V K257T I260V G389R t +12 T P301S Mutations Impairing Tau Protein Function -G272V -  280K-P301L -P301S-V337M -G389R-R406W Mutations Altering Exon 10 Splicing -N279K -  280K-L284L -S305N-S305S -Intron 10 mutations E342V Tau Mutations Cause FTDP-17 By Different Mechanisms And Aggregation Of Tau Disrupts Axonal Transport Leading to Neurodegeneration Mutations Promote Tau Aggregation -G272V -P301L –P301S -V337M

Tau Positive Inclusions in Neurons DEM PEG CBD GUAM AD PSP FTDP17 Pick’s disease

Paired Helical Filaments (PHFs) Formed By Tau Are Building Blocks Of NFTs In AD Brain Two twisting strands with an apparent periodicity of 80nm and an alternating width between 8 and 20nm Molecular composition tau From Lee et al. Science (1991) 251, 675-8

2N4R 2N3R 1N4R 1N3R 0N4R 0N3R 72kDa 68kDa 64kDa 60kDa Dephos kDa 62kDa 59kDa 54kDa 52kDa 48kDa AD ALS/PDC Down’s syndrome FTDP-17 (G272V, V337M,etc.) GSS Nieman-Pick disease type C FTDP-17 (  K280) Pick’s disease CBD FTDP-17 (mutations in I10, L284L, etc.) PSP Sarkosyl-insoluble Tau Bands Before and After Dephosphorylation

Tau Dysfunction And The Pathogenesis Of AD And Related Neurodegenerative Tauopathies Genetic Factors Tau Mutations APP, PS1, PS2 Mutations Environmental Factors Perturbation of 4R/3R Ratio Loss of Tau Function Gain of Toxic Function Hyperphosphorylation DeP-Tau Kinases Phophotases P-Tau Tau Dysfunction Tau Aggregation/ MT Loss Impaired Transport & Neurodegeneration ? ?

Tau Pathology in Patients with the A53T Alpha-Synuclein Mutation  -syn Tau

Summary of  -Synuclein Assembly Studies  -Synuclein readily assembles into 10 nm diameter filaments A53T mutation facilitates  -synuclein assembly  -Synuclein is incapable of fibrillogenesis Residue is required for  - synuclein filament assembly

HSP70 PROTECTS AGAINST ALPHA- SYNUCLEIN INDUCED DOPAMINERGIC NEURON DEGENERTATION (Auluck et al., Science 2002)

Characterization of  -Synuclein Transgenic Mice (Giasson et al., Neuron, 2002)

Abundant  -synuclein Inclusions in A53T  -synuclein Transgenic Mice SNL-4 Syn 303 Syn 505 Syn 506 Syn 303 Syn 505 Spinal cord Raphe pons midbrain pons locus ceruleus cerebellum

Accumulation of Detergent Insoluble  -synuclein in the Spinal Cord of A53T Tg Mice

α-Synuclein Filaments can be Isolated from A53T Transgenic Mice

a-syn tau overlay Tau Pathology in the Spinal Cord and Midbrain of A53T a-Syn Tg Mice

Lessons From Nicoll, et al. (Nat. Med., 2003) For Therapy Of Brain Amyloidoses Subtraction of Aβ from the brains of patients with diseases having abundant tau, alpha-synuclein & A  pathologies –Lewy body variant of Alzheimer’s disease –Sporadic/Familial AD –Down’s syndrome Will convert them to phenocopies of diseases with abundant tau and/or alpha-synuclein, but scant/no A  pathologies –Parkinson’s disease/Dementia with Lewy bodies –Guam Marianna dementia/PDC

Model of Fibrilization in Neurodegenerative Disease =  -helix = random coil =  -pleated sheet ^^ ^

mRNA Ribo- somes N N chaperone C NN C Misfolded Small oligomers Inclusions Cell Death N C Cell Death Proteasome The Deleterious Consequences Of Protein Misfolding

 -synuclein tau vesicles Improper trafficking Increased abundance Tau hyperphosphorylation Microtubules Pathological aggregates More Deleterious Consequences Of Protein Misfolding

Five Year Delay in AD Onset Halves Prevalence & Incidence

It Takes Great A Team! Mark Forman Benoit Giasson Makoto Higuchi Hiro Uryu Bin Zhang CNDR Members Virginia Lee N. Bonini, J. Duda J. Galvin, D. Galasko L. Golbe, M. Grossman H. Hurtig, T. Iwatsubo C. Lippa, B. Miller D. Murphy, M. Stern Nat’l AD Coordinating Center NIA Alzheimer Disease Centers Penn Head Injury Center Supported by Grants from the NIA, the Alzheimer’s Association, Michael J. Fox Foundation and the Families of our Patients

DISCLAIMER If you attended to the content of this lecture, you may have participated in effortful mental activity, and this may reduce your risk for dementia. * However, this lecture is not intended as a therapeutic intervention and there is no guarantee that this lecture has therapeutic benefit. * Wilson et al. JAMA 287: , 2003; Verghese et al. NEJM, 34: , 2003