Do pathological changes in tau protein isoforms manifest in cerebrospinal fluid of tauopathy patients?

Transcription

1 Do pathological changes in tau protein isoforms manifest in cerebrospinal fluid of tauopathy patients? Development and validation of sensitive immuno-pcr assays Barcelona, November 2012 Rohan de Silva, DPhil Reta Lila Weston Institute UCL Institute of Neurology London

2 UCL Institute of Neurology/ National Hospital of Neurology and Neurosurgery Queen Square 1 Wakefield Street 2

3 The need for diagnostic biomarkers in neurodegenerative disorders Definitive diagnosis is done post mortem Accurate diagnosis important for treatment options Identification of prodromal cases crucial for preventive intervention 3

4 The tau protein and neurodegenerative disorders 4

5 The tau protein and neurodegenerative disorders Current tau-based CSF assays 5

6 The tau protein and neurodegenerative disorders Current tau-based CSF assays Sensitive immuno-pcr assays for CSF tau isoforms 6

7 Plaques β-amyloid (βa) peptides derived from β-amyloid precursor protein (APP) Neurofibrillary tangles (NFT) Hyperphosphorylated tau protein. Dr Simon Lovestone, IoP, London From: 7

8 The tauopathies Alzheimer s disease ALS/parkinsonism-dementia complex Argyrophilic grain disease Corticobasal degeneration Creutzfeld-Jakob disease Dementia pugilistica Diffuse neurofibrillary tangles with calcification Down s syndrome FTDP-17T Gerstmann-Sträussler-Scheinker disease Hallervorden-Spatz disease Myotonic dystrophy Niemann-Pick disease Non-Guamanian motor neuron disease with neurofibrillary tangles Pick s disease Postencephalitic parkinsonism Prion protein cerebral amyloid angiopathy Progressive subcortical gliosis Progressive supranuclear palsy Supacute sclerosing panencephalitis Tangle only dementia Dr Simon Lovestone, IoP, London From: /on-line/brain/28.asp 8

9 Tau protein: Multi-domain, multi-function 1 N1 N2 R1 R2 R3 R4 441 Acidic domain Projection Domain Interaction with: Neuronal plasma membrane Cytoskeleton Signal Transduction (PLC-γ/Src-kinases) Proline-rich domain Repeat domain Microtubule-binding Domain Microtubule polymerisation and stabilisation Binding with other proteins (PS1, PP2A) Axonal transport Regulation of motor-protein mediated vesicle transport along the microtubules modified from Buée et al. Brain Res Rev 2000 From Amos (2004) Org Biomol Chem 9

10 Tau protein: Multi-domain, multi-function 1 N1 N2 R1 R2 R3 R4 441 Acidic domain Projection Domain Interaction with: Neuronal plasma membrane Cytoskeleton Signal Transduction (PLC-γ/Src-kinases) Proline-rich domain Repeat domain Microtubule-binding Domain Microtubule polymerisation and stabilisation Binding with other proteins (PS1, PP2A) Axonal transport Regulation of motor-protein mediated vesicle transport along the microtubules modified from Buée et al. Brain Res Rev 2000 From Amos (2004) Org Biomol Chem 10

11 Tau protein: Multi-domain, multi-function 1 N1 N2 R1 R2 R3 R4 441 Acidic domain Projection Domain Interaction with: Neuronal plasma membrane Cytoskeleton Signal Transduction (PLC-γ/Src-kinases) Proline-rich domain Repeat domain Microtubule-binding Domain Microtubule polymerisation and stabilisation Binding with other proteins (PS1, PP2A) Axonal transport Regulation of motor-protein mediated vesicle transport along the microtubules modified from Buée et al. Brain Res Rev 2000 From Amos (2004) Org Biomol Chem Public domain/adear 11

12 Exon A MAPT: Alternative splicing of exons 2, 3 and N,4R 1N,4R Tau protein: Six isoforms in CNS N,4R 2N,3R = N,3R N,3R Disturbances in tau isoform homeostasis, specially the 3R-tau/4R-tau ratio form an important mechanistic basis in the tauopathies.

13 Exon A MAPT: Alternative splicing of exons 2, 3 and FTDP-17T mutations: Exon 10 splicing mutations pre-mrna 2N,4R 1N,4R 0N,4R 2N,3R 1N,3R 0N,3R = Δ280K N279K Tau protein: Six isoforms in CNS Ex10+3 Ex10+12 Ex10+13 Ex10+14 Ex10+16 S305N Ex10-10 L284L P301L 5 3 Intron 9 Exon 10 Intron Exon 1 R5H/L Missense mutations Exon 9 K257T I260V L266V G272V Exon 10 N279K/Δ280K L284L N296N/H/Δ296N P301L/S S305N/S/I Exon 11 L315R S320F Exon 12 Q336R V337M E342V S352L/V K369I Exon 13 G389R R406W 13

14 from Buée et al. Brain Res Rev 2000 Different tau isoform composition in neurofibrillar pathology in different tauopathies.

15 from Buée et al. Brain Res Rev 2000 RD3 and RD4 monoclonal antibodies recognise 3R- and 4R-tau, respectively.

16 from Buée et al. Brain Res Rev 2000 AT8 (ptau S 199 /S 202 or T 205 /S 208 ) RD3 (3R-tau) RD4 (4R-tau) PSP: tufted astrocyte AD Temporal neocortex (x20) PSP Griseum pontis (x10) Pick s Disease Granule cell layer (x20) PSP: oligodendroglial coiled body

17 The question: Are changes in brain 3R-tau and 4R-tau isoforms reflected in CSF? The need: CSF assay(s) for the differential diagnosis of tauopathies and parkinsonian disorders.

18 CSF biomarkers in AD Decreased Aβ 42 due to: Increased sequestration Aβoligomerisation Unknown matrix effects (Slemmon et al 2012 J Neurochem) Increased tau and phospho-tau (p-thr 181 ) due to: Axonal degeneration Active release 18

19 CSF biomarkers in AD Decreased Aβ 42 due to: Increased sequestration Aβoligomerisation Unknown matrix effects (Slemmon et al 2012 J Neurochem) Increased tau and phospho-tau (p-thr 181 ) due to: Axonal degeneration Active release 19

20 20

21 CSF α-synuclein is reduced selectively in synuclienopathies Training cohort Validation cohort CSF α-synuclein (pg/ml) CSF total-tau (pg/ml) 21

22 CSF biomarkers in AD Decreased Aβ 42 due to: Increased sequestration Aβ oligomerisation Unknown matrix effects (Slemmon et al 2012 J Neurochem) Increased tau and phospho-tau (p-thr 181 ) due to: Axonal degeneration Active release 22

23 CSF biomarkers in AD Decreased Aβ 42 due to: Increased sequestration Aβoligomerisation Unknown matrix effects (Slemmon et al 2012 J Neurochem) Increased tau and phospho-tau (p-thr 181 ) due to: Axonal degeneration Active release 23

24 24 Sunderland et al 2003

25 Scatter plot of AD v Controls Aβ42 (pg/ml) A Beta 1-42Ta Tau (pg/ml) Control AD courtesy of Nick Fox & Ross Paterson 25

26 AD markers and cognitive impairment (MCI) conversion to AD Cut-off values for pathological CSF : Total-tau: >350 pg/ml Aβ 42 : <530 pg/ml Concentrations of total-tau, p-tau 181, and Aβ 42 in CSF are strongly associated with future development of Alzheimer s disease in patients with MCI. 26

27 27 Yoshiyama, Lee & Trojanowski, JNEN, 2012

28 CSF-tau in other parkinsonian disorders and tauopathies 28

29 Shoji et al

30 Total CSF-tau levels in tauopathies (Arai et al, 1997, Biochem Biophys Res Comm.) n=6 Total CSF-tau increased in AD, DLB and FTD. n=8 n=6 n=3 n=? 30

31 Aerts et al

32 32 Süssmuth et al 2010

33 RS and PSP-P Sruljies et al J Neur Transm

34 Would 3R-tau and 4R-tau levels in CSF discriminate between tauopathies? Are changes in tau isoform homeostasis (pathology or gene mutations) reflected in CSF? Would PSP and CBD (and FTDP17 with exon 10 splicing mutations) have elevated 4R-tau in CSF? Does PiD have elevated 3R-tau? 34

35 Tau isoform sandwich ELISA Standard curves Assay ranges: 3R-tau: pg/ml 4R-tau: pg/ml Parallelism 35

36 36

37 HRP DNA Quantitative real-time PCR (Imperacer ) Detection antibody (pan-tau) Antigen (tau isoforms) Capture antibody (RD3/RD4) 37

38 Assay performance 38

39 Parallelism 39

40 Sensitivity Sensitivity: 10pg/ml Dynamic range: pg/ml 40

41 Samples Cohort A Cohort B Cohort C Cohort D 41

42 3R-tau in CSF No change compared to controls 42

43 4R-tau in CSF Significant reductions of 4R-tau in PSP, PDD and AD 43

44 Combined cohorts 3R-tau 4R-tau 44

45 Combined cohorts 45

46 Conclusions 1 No reliable biomarker for differential diagnosis of parkinsonian disorders with tau pathology No consistent changes in total- and phospho-tau measures. Disturbed 3R-/4R-tau isoform homeostasis in many tauopathies (FTLD-tau mutations and tau pathology) Immuno-PCR assays allow measurement of 3R- and 4R-tau in CSF showing selective decrease of 4R-tau in tauopathies. Reduction of 4R-tau not restricted to 4R-tauopathies (PSP and CBS). 46

47 Interpretations? True reduction of 4R-tau e.g. due to increased retention in brain in tau inclusions (à la Aβ 42 and α synuclein!) Disease associated conformational masking. from Jeganathan et al (2008) J Biol Chem

48 Acknowledgements Reta Lila Weston Institute/UCL Institute of Neurology Dr Connie Luk Mark Spengler Sven Schulz Ron Wacker Beena Punnamoottil Andrew Lees John Hardy Tamas Revesz Nick Fox Roberto Simone Victoria Kay Fidel Anaya Nadia Magdalinou Geshanthi Hondhamuni Sylvia Agathou Khawar Hussain Alan Pittman Amanda Myers Sample contributions Barcelona Yaroslau Compta Maria José Martí Ana Cámara Gothenburg Henrik Zetterberg Kaj Blennow Radu Constantinescu Amsterdam Yolande Pijnenburg Rotterdam John Van Swieten Wan Zheng Chiu Brescia Barbara Borroni Göttingen Brit Mollenhauer Claudia Trenkwalder Tübingen Walter Mätzler Melbourne David Williams Perdita Cheshire 48