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Dementia Proteomics
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. (Nilsson Lab)ORCID iD: 0000-0002-3908-6476
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The term dementia encompass a number of conditions arising as a consequence of tissue degeneration in the brain. This degeneration is caused by molecular events occurring on a cellular level including inflammation, defective waste disposal and accumulation of insoluble proteins and peptides. Many of these molecular events are in turn also reflected in the composition of the cerebrospinal fluid (CSF) which circulates within and around the brain. This thesis summarise five studies conducted with the aim to explore and profile CSF proteins in the context of dementia and other neurodegenerative disorders. Protein profiles were obtained by so-called suspension bead arrays (SBAs), created by coupling antibodies to color-coded microspheres, allowing detection of more than 350 CSF proteins simultaneously. The majority of the explored proteins are referred to as brain-enriched, entailing that the corresponding genes are highly expressed in brain tissue in comparison to other tissues.

 

In Paper I, the SBA technology was utilised to profile about 280 proteins in CSF from several neurodegenerative disorders, i.e. Alzheimer’s disease (AD), dementia with Lewy Bodies and Parkinson’s disease. Distinct differences in the CSF proteome were identified depending on site of collection (ventricular or lumbar) and time point (post mortem or ante mortem). Disease-associated profiles for the two synaptic proteins neuromodulin (GAP43) and neurogranin (NRGN) could be confirmed, in which both proteins displayed higher levels in AD compared to controls. High levels of the two proteins were furthermore observed in patients at preclinical stages of AD in two independent cohorts. To verify the identified protein profiles, parallel reaction monitoring (PRM) assays were developed for 17 proteins in Paper II, including GAP43. Eight proteins displayed concordance to data generated with SBAs and among these were GAP43, cholecystokinin, neurofilament medium chain (NF-M), leucine-rich alpha-2-glycoprotein and vascular cell adhesion protein 1. 

 

In Paper III, the SBA technology was again applied to characterise early dementia-related changes in the CSF proteome by comparing samples from individuals with mild cognitive impairment (MCI), controls and AD patients in two independent cohorts. The MCI individuals were moreover stratified based on CSF concentration of the core AD biomarkers Aβ42 and tau. The six proteins amphiphysin, aquaporin 4, cAMP regulated phosphoprotein 21, β-synuclein, GAP43 and NF-M did all show significant differences between sample groups in both cohorts. Further exploration of how the pathological processes preceding dementia affect the CSF proteome, was done by analysis of 104 brain-enriched proteins in CSF from asymptomatic 70 year-olds in Paper IV. Protein profiles were correlated to Aβ42, t-tau and p-tau CSF concentration, revealing a large number of proteins displaying significant correlations to tau levels. Upon dividing the asymptomatic individuals based on Aβ42 CSF pathology, some proteins showed significantly different associations in the two groups. Most of these proteins yielding interesting profiles, were plasma membrane proteins or proteins connected to synaptic vesicle transport.

 

While AD is the most common form of dementia, accounting for more than 60 % of all cases worldwide, frontotemporal dementia (FTD) is the most frequently occurring form of young-onset dementia. In Paper V, CSF protein profiles were explored in the context of FTD. Patients with behavioural variant FTD and primary progressive aphasia, were compared to unaffected individuals with a high risk of developing FTD. Proteomic differences between patients with FTD and the unaffected individuals were observed already at a global level, and particularly for the six proteins NF-M, neurosecretory protein VGF, neuronal pentraxin receptor, prodynorphin, transmembrane protein 132D and tenascin-R.

 

The disease-associated profiles identified in the presented studies provide a basis for future research within dementia proteomics. Whether the proteins identified will have the possibility to aid in clinical diagnosis, prognosis or characterisation of dementia, remains to be evaluated. Given the fortunate situation, especially in Sweden, with access to large and well characterised CSF collections, there are ample opportunities for future proteomic studies to elucidate the true potential of these proteins.

Abstract [sv]

Demens är ett samlingsnamn som inbegriper en rad symptom orsakade av skador på hjärnvävnaden, så kallad neurodegeneration. Dessa skador kan uppstå på grund av exempelvis inflammation eller som en följd av rubbningar i molekylära processer i hjärnans celler. Gemensamt för dessa cellulära störningar är att de kan orsaka extracellulär ansamling av svårlösliga proteiner och peptider. Proteiner och andra molekyler som uppkommer under cellulära obalans, kan detekteras i den vätska som omger hjärnan – cerebrospinalvätskan (CSF). Den här avhandlingen innefattar fem studier med syfte att undersöka och profilera proteiner i CSF från individer med demens eller andra neurodegenerativa sjukdomar. Genom att använda en särskild typ av antikroppsarrayer (SBA, suspension bead array) i vilka antikropparna är kovalent bundna till mikroskopiska färg-kodade kulor, kan fler än 350 CSF-proteiner analyseras parallellt i ett och samma prov. Majoriteten av de proteiner som analyserats i denna avhandling definieras som hjärn-specifika då generna som kodar för dem uttrycks i högre grad i hjärnvävnad jämfört med andra vävnader.

 

Artikel I användes SBA-teknologin för att analysera 280 hjärn-specifika proteiner i CSF från individer med Alzheimers sjukdom (AD), Lewykroppsdemens och Parkinsons sjukdom. Tydliga skillnader i CSF-proteomet kunde identifieras mellan individer beroende på var (lumbalt eller ventrikulärt) och vid vilken tidpunkt (post mortem eller ante mortem) provtagningen skett. För de två proteinerna neuromodulin (GAP43) och neurogranin (NRGN) verifierades tidigare observerade profiler, vilka visat förhöjda proteinnivåer i patienter med AD jämfört med kontrollindivider. Vidare observerades dessa höga proteinnivåer även i prekliniska AD-patienter i två oberoende patientkohorter. För att verifiera de påvisade proteinprofilerna utvecklades i Artikel II ett masspektrometriflöde (PRM, parallel reaction monitoring) för 17 av de analyserade proteinerna, däribland GAP43. Med detta flöde konstaterades starka korrelationer mellan data genererat med SBA-teknologi och PRM för åtta proteiner, bland andra GAP43, cholecystokinin, neurofilament medium (NF-M), leucine-rich alpha-2-glycoprotein och vascular cell adhesion protein 1.

 

Artikel III användes återigen SBA-teknologin, nu för att jämföra individer med mild kognitiv svikt (MCI), AD och kontrollindivider med avsikten att karaktärisera tidiga demensrelaterade förändringar i CSF-proteomet. Individerna med MCI stratifierades därtill med avseende på nivåer av de etablerade biomarkörena för AD, Aβ42 och tau. Signifikanta skillnader mellan de undersökta grupperna kunde identifieras för de sex proteinerna amphiphysin, aquaporin 4, cAMP regulated phosphoprotein 21, β-synuclein, GAP43 och NF-M. För att ytterligare undersöka hur de patofysiologiska processerna som föregår demenssjukdomar påverkar CSF-proteomet, analyserades även 104 hjärn-specifika proteiner i CSF från asymtomatiska 70-åringar i Artikel IV. Ett stort antal av de analyserade proteinerna visade signifikanta korrelationer med koncentrationerna av AD-biomarkörerna Aβ42, total-tau och phospho-tau. De asymtomatiska individerna delades därefter upp i två grupper utifrån de uppmätta Aβ42 nivåerna och flera proteiner uppvisade signifikant skilda associationer till de tre biomarkörerna. Bland dessa proteiner identifierades två proteingrupper, membranproteiner och proteiner involverade i transport av synaptiska vesiklar.

 

Artikel V undersöktes CSF-proteomet i relation till frontallobsdemens (FTD), vilken är den vanligast förekommande demensformen hos individer under 65 års ålder. Patienter med beteendevarianten av FTD och primär progressiv afasi jämfördes i denna studie med symptomfria individer som hade hög risk att själva utveckla FTD. Skillnader mellan patienter med FTD och symptomfria individer kunde observeras på en global proteinnivå, och framförallt för de proteinerna NF-M, neurosecretory protein VGF, neuronal pentraxin receptor, prodynorphin, transmembrane protein 132D and tenascin-R.

 

Om de proteiner som identifierats i denna avhandling kan användas för att förbättra demensdiagnostik och prognostisering, eller för ytterligare karakterisering av demenssjukdomar återstår att se. De observerade proteinprofilerna har emellertid bidragit till att utöka kunskapen om hur CSF-proteomet påverkas i, och under utvecklingen av, våra två vanligaste demenssjukdomar. Denna kunskap kan förhoppningsvis ligga till grund för framtida forskning inom demensproteomik.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2020. , p. 89
Series
TRITA-CBH-FOU ; 2020:17
Keywords [en]
Dementia, Proteomics, Alzheimer's disease, Frontotemporal dementia, Antibody array, Suspension bead array, Cerebrospinal fluid, Protein profiling, Biomarker discovery
National Category
Medical Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-273596ISBN: 978-91-7873-544-0 (print)OAI: oai:DiVA.org:kth-273596DiVA, id: diva2:1431298
Public defence
2020-06-12, https://kth-se.zoom.us/j/62560073937, 09:00 (English)
Opponent
Supervisors
Note

QC 2020-05-20

Available from: 2020-05-20 Created: 2020-05-19 Last updated: 2020-05-20Bibliographically approved
List of papers
1. CSF profiling of the human brain enriched proteome reveals associations of neuromodulin and neurogranin to Alzheimer's disease
Open this publication in new window or tab >>CSF profiling of the human brain enriched proteome reveals associations of neuromodulin and neurogranin to Alzheimer's disease
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2016 (English)In: PROTEOMICS - Clinical Applications, ISSN 1862-8346, E-ISSN 1862-8354, Vol. 10, no 12, p. 1242-1253Article in journal (Refereed) Published
Abstract [en]

Purpose: This study is part of a larger effort aiming to expand the knowledge of brain-enriched proteins in human cerebrospinal fluid (CSF) and to provide novel insight into the relation between such proteins and different neurodegenerative diseases. Experimental design: Here 280 brain-enriched proteins in CSF from patients with Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are profiled. In total, 441 human samples of ventricular CSF collected post mortem and lumbar CSF collected ante mortem are analyzed using 376 antibodies in a suspension bead array setup, utilizing a direct labelling approach. Results: Among several proteins displaying differentiated profiles between sample groups, we focus here on two synaptic proteins, neuromodulin (GAP43) and neurogranin (NRGN). They are both found at elevated levels in CSF from AD patients in two independent cohorts, providing disease-associated profiles in addition to verifying and strengthening previously observed patterns. Increased levels are also observed for patients for whom the AD diagnosis was not established at the time of sampling. Conclusions and clinical relevance: These findings indicate that analyzing the brain-enriched proteins in CSF is of particular interest to increase the understanding of the CSF proteome and its relation to neurodegenerative disorders. In addition, this study lends support to the notion that measurements of these synaptic proteins could potentially be of great relevance in future diagnostic tests for AD.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2016
Keywords
Alzheimer's disease, Cerebrospinal fluid, Neurogranin, Neuromodulin, Neuroproteomics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-199507 (URN)10.1002/prca.201500150 (DOI)000389221400009 ()27604409 (PubMedID)2-s2.0-84990913359 (Scopus ID)
Note

QC 20170117

Available from: 2017-01-17 Created: 2017-01-09 Last updated: 2020-05-20Bibliographically approved
2. Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease
Open this publication in new window or tab >>Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease
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2019 (English)In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 494, p. 79-93Article in journal (Refereed) Published
Abstract [en]

Detailed knowledge of protein changes in cerebrospinal fluid (CSF) across healthy and diseased individuals would provide a better understanding of the onset and progression of neurodegenerative disorders. In this study, we selected 20 brain-enriched proteins previously identified in CSF by antibody suspension bead arrays (SBA) to be potentially biomarkers for Alzheimer's disease (AD) and verified these using an orthogonal approach. We examined the same set of 94 CSF samples from patients affected by AD (including preclinical and prodromal), mild cognitive impairment (MCI), non-AD dementia and healthy individuals, which had previously been analyzed by SBA. Twenty-eight parallel reaction monitoring (PRM) assays were developed and 13 of them could be validated for protein quantification. Antibody profiles were verified by PRM. For seven proteins, the antibody profiles were highly correlated with the PRM results (r > 0.7) and GAP43, VCAM1 and PSAP were identified as potential markers of preclinical AD. In conclusion, we demonstrate the usefulness of targeted mass spectrometry as a tool for the orthogonal verification of antibody profiling data, suggesting that these complementary methods can be successfully applied for comprehensive exploration of CSF protein levels in neurodegenerative disorders.

Place, publisher, year, edition, pages
Elsevier B.V., 2019
Keywords
AD, Alzheimer's disease, Biomarkers, Cerebrospinal fluid, Parallel reaction monitoring (PRM), Suspension bead array (SBA), alpha 1 aantitrypsin, alpha 1 antichymotrypsin, apolipoprotein, biological marker, cathepsin D, cholecystokinin, creatine kinase B type, dickkopf related protein 3, fibrinogen alpha, fructose bisphosphate aldolase C, glucose regulated protein 94, inter alpha trypsin inhibitor heavy chain H1, leucine rich alpha 2 glycoprotein, neurobeachin, neurofilament medium polypeptide, neuromodulin, plasminogen, prosaposin, protein S100B, SPARC like protein 1, unclassified drug, vascular cell adhesion protein 1, adult, aged, Alzheimer disease, Article, clinical article, cohort analysis, controlled study, correlational study, disease course, female, human, male, mass spectrometry, middle aged, mild cognitive impairment, multiple reaction monitoring, priority journal, protein blood level, protein cerebrospinal fluid level, protein microarray, suspension bead array, very elderly
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-252444 (URN)10.1016/j.cca.2019.03.243 (DOI)000470950400013 ()30858094 (PubMedID)2-s2.0-85063002689 (Scopus ID)
Note

QC 20190715

Available from: 2019-07-15 Created: 2019-07-15 Last updated: 2020-05-20Bibliographically approved
3. Multi-cohort protein profiling reveals higher levels of six brain-enriched proteins in Alzheimer’s disease patients
Open this publication in new window or tab >>Multi-cohort protein profiling reveals higher levels of six brain-enriched proteins in Alzheimer’s disease patients
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology
Research subject
Biotechnology; Biotechnology
Identifiers
urn:nbn:se:kth:diva-273620 (URN)
Available from: 2020-05-20 Created: 2020-05-20 Last updated: 2020-05-20
4. Novel CSF protein pattern correlates with biomarker evidence of tau and amyloid β pathology in asymptomatic 70-year-olds
Open this publication in new window or tab >>Novel CSF protein pattern correlates with biomarker evidence of tau and amyloid β pathology in asymptomatic 70-year-olds
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology
Research subject
Biotechnology; Biotechnology
Identifiers
urn:nbn:se:kth:diva-273621 (URN)
Available from: 2020-05-20 Created: 2020-05-20 Last updated: 2020-05-20
5. Altered levels of CSF proteins in patients with FTD, presymptomatic mutation carriers and non-carriers
Open this publication in new window or tab >>Altered levels of CSF proteins in patients with FTD, presymptomatic mutation carriers and non-carriers
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(English)In: Translational Neurodegeneration, ISSN 2047-9158Article in journal (Refereed) Accepted
Abstract [en]

Background. The clinical presentations of frontotemporal dementia (FTD) are diverse and overlap with other neurological disorders. There are, as of today, no biomarkers in clinical practice for diagnosing the disorders. Here, we aimed to find protein markers in cerebrospinal fluid (CSF) from patients with FTD, presymptomatic mutation carriers and non-carriers.

Methods. Antibody suspension bead arrays were used to analyse 328 proteins in CSF from patients with behavioural variant FTD (bvFTD, n=16) and progressive primary aphasia (PPA, n=13), as well as presymptomatic mutation carriers (PMC, n=16) and non-carriers (NC, n=8). A total of 492 antibodies were used to measure protein levels by direct labelling of the CSF samples. The findings were further examined in an independent cohort including 13 FTD patients, 79 patients with Alzheimer’s disease and 18 healthy controls.

Results. We found significantly altered protein levels in CSF from FTD patients compared to unaffected individuals (PMC and NC) for 26 proteins. The analysis show patterns of separation between unaffected individuals and FTD patients, especially for those with a clinical diagnosis of bvFTD. The most statistically significant differences in protein levels were found for VGF, TN-R, NPTXR, TMEM132D, PDYN and NF-M. Patients with FTD were found to have higher levels of TN-R and NF-M, and lower levels of VGF, NPTXR, TMEM132D and PDYN, compared to unaffected individuals. The main findings were reproduced in the independent cohort.

Conclusion. In this pilot study, we show a separation of FTD patients from unaffected individuals based on protein levels in CSF. Further investigation is required to explore the CSF profiles in larger cohorts, but the results presented here has the potential to enable future clinical utilization of these potential biomarkers within FTD.

Keywords
Frontotemporal dementia, cerebrospinal fluid, biomarkers, proteomics, antibody suspension bead array
National Category
Neurosciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-273182 (URN)
Note

QC 20200511

Available from: 2020-05-11 Created: 2020-05-11 Last updated: 2020-05-20Bibliographically approved

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