Likvormarkörer för differentialdiagnostik av demenssjukdomar

Likvormarkörer för differentialdiagnostik av demenssjukdomar Henrik Zetterberg, MD, PhD Docent i neurokemi Avdelningen för psykiatri och neurokemi Göteborgs universitet

Likvoranalyser för diagnostik av Alzheimers sjukdom Study design: Follow-up study on MCI (>4 years) MCI n= 134 57 MCI o AD 56 MCI o MCI 21 MCI o other dementias Cut-off AE42 < 53 pg/ml T-tau > 35 pg/ml Vad har dessa för sjukdomar??? Combination of AE42 and T-tau: Sens. for MCI-AD 95% Spec. non-progr. MCI 83 % +DQVVRQ =HWWHUEHUJ HW DO /DQFHW 1HXURO

Överlapp mot andra demensjukdomar 25 3 2 25 T-tau pg/ml 15 1 P-tau pg/ml 2 15 1 5 5 AD MCI Cont FTD DLB MSA PD PSP AD MCI Cont FTD DLB MSA PD PSP 12 Study design: AD n= 78 MCI-AD n= 15 Cont n= 53 FTD n= 16 DLB n= 15 MSA n= 36 PD n= 46 PSP n= 15 AE42 pg/ml 1 8 6 4 2 AD MCI Cont FTD DLB MSA PD PSP Sensitivity Specificity T-tau 83 % 89 % P-tau 72 % 95 % AE42 91 % 75 % Comb. 87% 92% (2/3 pos.) PPV NPV Comb. 82% 94% 2OVVRQ HW DO &OLQ &KHP

Disease PD versus controls Neurofilament light chain (NFL) Normal NFL in PD (1,11,12,15) Neurofilament heavy chain (NFH) Tau protein (Tau) Beta amyloid 42 (A 42) Glial Fibrillary Acidic Protein (GFAP) Neurofilament Normal NFH in PD (1,15,16) A. Normal Tau in PD (13,58,59,6,61,62). B. Not significantly increased Tau in PD (31) C. Not significantly decreased Tau in PD (1,33) A. Normal A 42 in PD (39,41,42,62). B. Not significantly decreased A 42 in PD (31). Normal GFAP in PD (12,13) MSA, MSA-P, MSA-C versus controls Increased NFL in MSA, MSA-P and in MSA-C (1,12) A. Increased NFH in MSA, MSA-P and in MSA-C (1,15,16); B. Normal NFH in MSA-P and increased NFH in MSA-C but not statistically verified (16). Increased Tau in MSA, MSA-P and MSA-C (1,13,15) A. Decreased A 42 in MSA, Normal GFAP in MSA-P and MSA-C (41); B. MSA, MSA-P and Normal A 42 in MSA, MSA- MSA-C (12,13) P and MSA-C (42). PSP versus controls Increased NFL in PSP (12) Increased NFH in PSP (16) Normal Tau in PSP (34,35) Normal A 42 in PSP (41,42) Normal GFAP in PSP (12) CBD versus controls - Normal NFH in CBD (16) A. Increased Tau in CBD (34, 36) B. Normal Tau in CBD (35) - - MSA-P contra MSA-C No difference in NFL between MSA-P and MSA-C (11,12,15) A. No difference in NFH between MSA-C and MSA-P (15,16); B. Increased NFH in MSA-C compared wih MSA-P but not statistically verified (16). No difference in Tau between MSA-P and MSA-C (13,15) No difference in A 42 between MSA-P and MSA-C (41,42) No difference in GFAP between MSA- P and MSA-C (12,13) PD contra MSA, MSA- P, PSP, CBD Higher NFL in MSA, MSA-P and PSP than in PD (1,11,12) A. Higher NFH in MSA, MSA-P and PSP than in PD and CBD. No difference in NFH between PD and CBD (1,16); B. No difference in NFH between PD and MSA-P but not statistically verified (16). Higher Tau in MSA and MSA-P than in PD (1,12) A. Decreased A 42 in MSA but not in PD and PSP (41); B. No difference in A 42 between PD, MSA and PSP (42). No difference in GFAP between PD, MSA and PSP (12,13) MSA contra PSP No difference in NFL between MSA and PSP (11,12) No difference in NFH between MSA and PSP (16) Higher Tau in MSA than in PSP (indirect conclusion, no study) No difference in A 42 between MSA and PSP (42) No difference in GFAP between MSA and PSP (12) CBD contra PSP - Higher NFH in PSP than in CBD (16) A. Higher Tau in CBD than in PSP (34); B. No difference in Tau between CBD and PSP (35) - - MSA contra CBD - Higher NFH in MSA than in CBD (16) Contradictory results (indirect conclusion, no studies) - - Parkinson s disease (PD), multiple system atrophy (MSA), multiple system atrophy with predominantly parkinsonism (MSA-P), multiple system atrophy with predominantly cerebellar symptom corticobasal degeneration (CBD) Radu Constantinescu, in manuscript

Frontallobsdemens 25 3 12 2 25 1 T-tau pg/ml 15 1 P-tau pg/ml 2 15 1 AE42 pg/ml 8 6 4 5 5 2 AD MCI FTD AD MCI FTD AD MCI FTD 2OVVRQ HW DO &OLQ &KHP 2WKHU FDQGLGDWH PDUNHUV VXFK DV 6E DQG QHXURILODPHQW KDYH IDLOHG WR UHDFK KLJK HQRXJK VHQVLWLYLW\ DQG VSHFLILFLW\ UHTXLUHG IRU XVH DV GLDJQRVWLF )7' WHVWV

Search for new CSF protein markers using SELDI-TOF CSF is applied to protein chips and protein binding is analyzed by MS Different types of chips bind different types of proteins Hydrophobic Anionic Cationic Metal Ion Normal Phase

Search for new CSF protein markers using SELDI-TOF 6 5 1 15 2 About 2 protein peaks can be identified and analyzed Differences in protein peaks between controls and FTD are quantified 4 2 6 4 2 6 4 2 7.5 5 2.5 15 1 5 15 1 5 Control cont. CM1 SPA FTD FTD CM1 SPA cont. Control Q1 SPA FTD FTD Q1 SPA cont. Control IMAC3 SPA FTD FTD IMAC3 SPA CM1 Q1 IMAC C3 1 7.5 5 2.5 7.5 5 2.5 cont. Control H5 SPA FTD FTD H5 SPA H5 5 1 15 2

SEARCH FOR BIOMARKERS FOR FRONTOTEMPORAL DEMENTIA USING SELDI Study design: CSF samples from 16 FTD cases 12 healthy controls Protein chips CM1 weak cation exchange Q1 strong anion exchange IMAC3 immobilized metal affinity capture H5 reversed phase / hydrophobic interaction Matrix CHCA = α-cyano-4-hydroxy cinnamic acid Small proteins < 15 kda SPA = sinapinic acid Large proteins > 1 kda Results: About 2 peaks detected, possible to quantify Primary analysis: 42 peaks with p<.1 Exclusion of peaks with low signal-to-noise ratio differentially charged peaks 1 peaks for PLS-DA analysis

SEARCH FOR BIOMARKERS FOR FRONTOTEMPORAL DEMENTIA USING SELDI =HWWHUEHUJ HW DO ([S 1HXURO

Examples of individual peaks in FTD and controls 6, M 3689 4, M 564 5, 4, 3, 3, 2, 2, 1, 1,,, FTD Controls FTD Controls 3, M 13766 4, M 13887 3, 2, 2, 1, 1,, FTD Controls, FTD Controls =HWWHUEHUJ HW DO ([S 1HXURO

Sex Age T-tau P-tau AE42 M 3689 M 564 M 526 M 6251 M 13766 M 13887 M 1254 M 58932 M 6247 M 422.1. -.1 Quantitative differences between FTD and controls Variable =HWWHUEHUJ HW DO ([S 1HXURO CoeffCS[1](Diagnosis)

PLS-DA loadings for all variables.4.3.2.1. -.1 -.2 -.3 -.4 -.5 3 Control M 13887 M 1254 M 6247 M 526 M 13766 AE42 P-tau M 3689 M 564 FTD M 6251 M 58932 T-tau M 422 -.3 -.2 -.1..1.2.3 Loadings for: 1 SELDI-TOF peaks 3 CSF markers (T-tau, P-tau Aβ42) 2 1-1 -2-3 -7-6 -5 FTD-2 C-9 C-3 FTD-12 FTD-11 C-8 FTD-14 FTD-16 FTD-5 FTD-9 FTD-1 FTD-4 FTD-13 FTD-15 FTD-1 FTD-7 FTD-8 FTD-6 C-2 C-5 C-11 C-7 C-6 C-1 C-4 C-1 C-12 FTD-3-4 -3-2 -1 1 2 3 4 5 6 7 =HWWHUEHUJ HW DO ([S 1HXURO

Principles for identification of SELDI-TOF peaks AD AD AD AD On-chip tryptic digestion Normal SELDI-TOF Normal Normal #97 RT:11.3 AV:1 NL:5.56E6 F: + c d Full ms2 146.7@35. [ 275.-2.] 1 Peptide mapping + PCI1-MS/MS sequencing 129.4 ProteinChip to monitor purification Relative Abundance 8 6 4 2 362.4 45.4 77.7 918.4 78.8 836.6 673.7 746.5 111. 54.4 631.5 5 4 6 8 1 13.3 m/z SDS-PAGE 21 kda 14 Tryptic digestion Micro-fractionation using matched Biosepra columns ATL-1 Normal 6

1 3689.3523 GRPEAQPPPLSSEHKEPVAGDAVPGPKDGSAPEVRGA b 21 3671.3984 Marker 3689: Neurosecretory protein VGF % 218.3813 2181.3784 b 28 2845.8345 2846.8481 3669.3933 y y 158.42 16 843.557 9 2182.3916 2844.8389 3653.3872 y 4 42.315 227.1445 639.4114 159.164 b 844.5549 13 b 1489.9984 1741.125 16 y 1947.2386 21 151.15 845.5497 1148.7816 1612.529 2183.3865 b 25 2847.8293 2827.864 2826.8455 2342.4832 254.5845 3651.4136 3627.3757 2 4 6 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 HH 1 275.1337 y 4 439.24 y 4 457.295 FQDKQYSSHHTAE 14.5 Marker 6251: Chromogranin B % 226.1255 165.1167 12.998 HT 239.1251 b 2 276.1421 QDK-28 344.1585 b 3 458.1992 y 5 576.2619 y 5 b 5 647.3194 594.27 b 5 y 7 b 6 y 931.3945 8 y 768.3528 732.3419 6 81.3927 124.4319 984.5195 a 9 193.5117 176.5146 b 9 1121.5272 1187.55 a1 123.5865 b 1122.5173 1258.5677 1 1259.646 y 1 b 12 1578.7126 1577.7317 156.6779 1331.615 1431.6563 1542.6946 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 m/z

FTD versus controls VGF Truncated cystatin C chromogranin B transthyretin S-cysteinylated transthyretin Sensitivitet 94% Specificitet 83% =HWWHUEHUJ HW DO ([S 1HXURO

AD mot kontroller och FTD helt oberoende studie p-value p- value M/Z ID AD vs Healthy Level in AD control AD vs FTD 243.77 Integral membrane protein 2B CT.39 Up n.s. 3379.265.39 Down.1 3495.122.36 Up.7 399.32 Chromogranin A peptide (Vasostatin II).11 Down.26 481.349 VGF peptide.2 Down n.s. 519.393.118 Down n.s. 5749.135 <.1 Down.11 6437.276 Apo C-1 (2 aa deleted from NT) <.1 Down n.s. 7229.774.9 Down.7 8937.64 C3a des-arg.4 Up n.s. 12282.91.23 Down n.s. 12524.14 Cystatin C -8aa from NT <.1 Up <.1 13347.95 Cystatin C <.1 Down <.1 14559.94 Pancreatic ribonuclease <.1 Up n.s. 14944.76 <.1 Down n.s. Suboptimala sensitivitets- och specificitetssiffror för att skilja FTD från AD Simonsen et al., Dem Ger Cogn Dis (27) 24:434-44

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Vad är på gång, november 27??? 7DX LVRIRUPP QVWHU Borroni et al., Neurobiol Aging 27, IP-WB Kvoten mellan Tau 33 kda och Tau 55 kda kraftigt sänkt i PSP jämfört med - kontroller - AD - PD - CBD - LBD D6\QXFOHLQ Brit Mollenhauer et al., ELISA (submitted) Och: 7RNXGD HW DO %%5& LQ SUHVV /lww VlQNW DV\QXFOHLQNRQFHQWUDWLRQ L &6) L 3' D6\QXFOHLQDJJUHJDW ELISA från Biosource Funkar ej för CSF 7'3 Virginia Lee och John Trojanowski Den gemensamma länken mellan ALS och FTD?

TDP-43 = transactive response (TAR)-DNA-binding protein 43 verkar vara patogenetiskt involverat i både sporadisk ALS och i olika FTD-former. Normalt intranukleärt men vid dessa tillstånd extranukleära inklusionskroppar. FTD (hjärna) ALS (ryggmärg)

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Tack för uppmärksamheten! Dept of Neurochemistry and Psychiatry Sahlgrenska Academy, Göteborg University Kaj Blennow Erik Portelius Ulf Andreasson Ann Brinkmalm Gunnar Brinkmalm Anders Wallin Clinical Memory Research Unit, Department of Clinical Sciences Malmö, Lund University, Sweden; Lennart Minthon Elisabet Londos Oskar Hansson Chiphergen Anja Hviid Simonsen Vladimir N Podust Excellent technical assistance Åsa Källén, Shirley Fridlund, Mona Thunell, Sara Hullberg, Monica Christiansson Research Support The Swedish Research Council, the Swedish Council for Working Life and Social Research, the Göteborg Medical Society, the Swedish Medical Society, the Swedish Society for Medical Research, Pfizer inc. s grant for clinical research in neuroscience, Stiftelsen Gamla Tjänarinnor, Alzheimerfonden