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Identification of autophagy-associated blood biomarkers for Parkinson’s disease

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dc.contributor.advisor장, 재락-
dc.contributor.author강, 주희-
dc.date.accessioned2021-11-10T05:53:04Z-
dc.date.available2021-11-10T05:53:04Z-
dc.date.issued2021-
dc.identifier.urihttp://repository.ajou.ac.kr/handle/201003/19871-
dc.description.abstractThe current diagnosis of Parkinson’s disease (PD) mostly relies on clinical rating scales related to motor dysfunction. Since symptoms of PD begins to appear only after a loss of significant numbers of nerve cells, it is necessary to find biomarkers for early diagnosis of PD. In this study, Idiopathic PD patients and age-matched patients with essential tremor (ET) according to the UK Brain Bank Criteria were consecutively enrolled to identify peripheral blood biomarkers for PD. Clinical data were obtained by clinical survey and assessment. We used albumin- and IgG-depleted plasma samples of 20 PD patients and 20 controls with ET, and peripheral blood mononuclear cell (PBMC) samples of 37 PD patients and 40 controls with ET. We performed immunoblot analysis of autophagy-lysosome pathway (ALP)-associated proteins and compared the levels of proteins to those of the control group. We found that the levels of BCL2-associated athanogene 2 (BAG2) and cathepsin D were significantly decreased in PD patient plasma. We also analyzed the correlation between the levels of candidate proteins and clinical characteristics and the correlation among candidate proteins. The levels of plasma BAG2 were significantly correlated with Cross-Culture Smell Identification Test (CC-SIT) score, which indicates the olfactory function of the patients. Finally, we validated our biomarker models using receiver operating characteristic (ROC) curve analysis. The result showed that our plasma biomarker model with BAG2 and cathepsin D showed 87.5% diagnostic accuracy (AUC=0.875, p<0.0001) in distinguishing PD patients from control subjects. We also found that the levels of lysosomal-associated membrane protein 1 (LAMP1) were increased and levels of optineurin (OPTN) were decreased in PBMCs derived from PD patients. Autophagic flux of the PBMCs was correlated with early and delayed phases of metaiodobenzylguanidine (MIBG) uptake. Autophagic flux was increased and LAMP1 levels were decreased in PD patient PBMCs with a cutoff value less than 1.6, which indicates cardiac MIBG uptake reduction. Our PBMC biomarker model with LAMP1 and OPTN showed 92.4% diagnostic accuracy (AUC=0.924, p<0.0001) in distinguishing PD patients from control subjects. Taken together, we showed that levels of ALP-associated proteins were altered both in plasma and PBMCs of PD patients. In addition, this study suggests BAG2 and cathepsin D as plasma biomarker candidates and OPTN and LAMP1 as PBMC biomarker candidates for early PD diagnosis.-
dc.description.abstract파킨슨병은 현재 주로 임상평가척도에 근거해서 진단된다. 파킨슨병의 운동 이상 증상은 신경세포의 상당수가 소실된 후에야 나타나기 때문에 파킨슨병의 조기 진단을 위한 바이오마커를 찾는 것이 필요하다. 이 연구에서 우리는 혈액 바이오마커 동정을 통해 파킨슨병을 조기에 진단할 수 있는 방법을 찾고자 했다. 우리는 UK Brain Bank Criteria에 따라 진단된 특발성 파킨슨병 환자군 및 연령대가 일치하는 비교군을 모집했고, 임상 조사 및 평가를 통해 임상 데이터를 확보했다. 채혈을 통해 확보한 환자 혈액 자원 중, 20명의 파킨슨병 환자군과 20명의 본 태성 떨림을 가진 대조군의 혈장을 분석했다. 단백질 면역 블랏 방법을 이용한 오토파지-라이소좀 기전 연관 단백질에 대한 정량 분석 결과, BAG2와 cathepsin D가 PD 환자 혈장에서 유의미한 수준으로 감소해 있음을 확인했다. 오토파지-라이소좀 연관 단백질들의 양적 정보와 각 환자의 임상적 특성 사이의 상관 관계 분석 결과, 혈장 내 BAG2의 양이 환자의 후각능력과 높은 연관성이 있다는 것을 확인했다. 끝으로 ROC curve 분석 및 로지스틱 회귀분석을 통해 BAG2와 cathepsin D 혈장 바이오마커 모델이 87.5%의 정확도로 대조군과 파킨슨병 환자들을 구분할 수 있다는 것을 확인했다. 이어서 37명의 파킨슨병 환자군과 40명의 대조군의 PBMC를 분석한 결과, 파킨슨병 환자들의 PBMC에서 LAMP1의 양은 증가, OPTN의 양은 감소되어 있음을 확인했고, PBMC의 오토파지 활성도 및 LAMP1의 양이 MIBG uptake 정도와 상관성이 높음을 확인했다. ROC curve 분석 및 로지스틱 회귀분석을 통해 LAMP1과 OPTN PBMC 바이오마커 모델이 92.4%의 정확도로 대조군과 파킨슨병 환자들을 구분할 수 있다는 것을 확인했다. 종합하면, 본 연구를 통해 우리는 파킨슨병 환자의 혈액을 이용한 오토파지-라이소좀 기전 연관 단백질의 양적 변화 분석을 통해 파킨슨병 진단을 위한 혈장 및 PBMC 바이오마커 후보를 동정했다.-
dc.description.tableofcontentsⅠ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 4
Subjects and study approval 4
Plasma sample preparation 4
Purification of PBMCs and preparation of the cell lysates 5
Antibody validation 5
Immunoblot analysis 5
Flow cytometry of lysosomal pH 6
Statistical analysis 6
Ⅲ. RESULTS 7
A. Analysis of ALP-associated protein levels in the plasma of PD patients 7
1. Demographic and clinical characteristics of subjects for plasma analysis 7
2. Preparation of patient plasma samples for SDS-PAGE 7
3. Screening of biomarker candidate proteins associated with the autophagy-lysosome system in plasma samples 7
4. BAG2 levels are decreased in the plasma of patients with PD 8
5. Cathepsin D levels are decreased in the plasma of patients with PD 14
6. Association of plasma BAG2 and cathepsin D levels with clinical characteristics 17
7. The BAG2 and cathepsin D levels of plasma as potential biomarkers of PD 17
B. Analysis of ALP-associated protein levels and cellular activities of PD patient PBMCs 22
1. Demographic and clinical characteristics of subjects for PBMC analysis 22
2. The intracellular protein levels of LAMP1 and OPTN are altered in the PBMCs of PD patients 22
3. Autophagic activity analysis in the PBMCs of control and PD patients 22
4. Association of PBMC LAMP1 levels and autophagic flux with cardiac MIBG uptake in PD patients 23
5. Correlation analysis among the ALP-associated markers in PBMCs 33
6. The intracellular protein levels of LAMP1, OPTN and ATG5 as potential biomarkers of PD 33
Ⅳ. DISCUSSION 39
Ⅴ. CONCLUSION 43
REFERENCE 44
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dc.language.isoen-
dc.titleIdentification of autophagy-associated blood biomarkers for Parkinson’s disease-
dc.typeThesis-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000031081-
dc.description.degreeMaster-
dc.contributor.department대학원 의생명과학과-
dc.contributor.affiliatedAuthor강, 주희-
dc.date.awarded2021-
dc.type.localTheses-
dc.citation.date2021-
dc.embargo.liftdate9999-12-31-
dc.embargo.terms9999-12-31-
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Theses > Graduate School of Biomedical Sciences > Master
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