Unraveling pathogenic proteins and pathways in Alzheimer disease : a focus on proteomics

Alzheimer disease (AD) is a multifactorial and complex neurodegenerative disorder. To date, different mechanisms, such as impairment of synaptic, mitochondrial and autophagic function, neuroinflammation and many more, are found to disrupt cellular homeostasis in AD brains. Despite the increased knowledge, it is still difficult to pinpoint which of these mechanisms is the main culprit driving the pathologic cascade, especially in the form of late- onset, sporadic AD, accounting for more than 95% of all patients. In this thesis, we used human-based or translational approaches to investigate which pathological alterations indeed occur in AD brains. In Paper I, we investigated an amyloid precursor protein (APP)-derived band with a molecular weight of 20 kDa, most likely corresponding to the APP C-terminal fragment (CTF) called CTF-η, and showed that it is expressed at low levels in the human brain. However, we also noted that several antibodies directed to APP or other proteins also detects a presumably non-specific band of a similar size. In Paper II, IV and V, we explored changes in the proteome of postmortem AD brains and CSF of AD patients and App knock-in mice. In Paper II, our aim was to identify proteins and pathways that could underlie synaptic dysfunction, a pathogenic event that happens early in disease progression. We thus explored the proteome of the outer molecular layer (OML) of the dentate gyrus using mass spectrometry (MS). This region is relatively cell-free and highly enriched in synaptic connections, and more importantly receives the main input of the hippocampus called the perforant path, which is highly affected in AD pathogenesis. Our comprehensive data analysis indicates that the OML indeed exhibits presynaptic changes, which is in line with previously published reports, whereas postsynaptic density proteins were not altered. To follow-up on the hypothesis of presynaptic impairment in AD OML, using immunofluorescence, we measured the staining densities of five presynaptic proteins in sub- regions of the hippocampus in Paper III. Similarly, we found decreased staining densities of complexin-1, syntaxin-1a, synaptotagmin-1 and synaptogyrin-1 in AD OML. However, the analysis of other hippocampal sub-regions showed no significant alterations in these presynaptic proteins, except syntaxin-1a, which showed increased staining densities in AD. Although other molecular layers of hippocampus also receive the perforant path input (as well as other important inputs), it was intriguing to find that presynaptic impairment was restricted to the OML. Together, Paper II and III point out to presynaptic failure in AD hippocampus. To further compare our proteomic findings with the published ones, in which proteome of AD-affected brain regions (e.g. temporal and frontal cortices) was analyzed, and to identify commonalities and discrepancies between the studies, in Paper IV, we performed a meta- analysis of labeled (11753 proteins and 168 cases) and label-free (4292 proteins and 632 cases) data. We found approximately 500 significantly altered proteins that were associated with pathways such as synaptic signaling, neuron and axon development, neurogenesis, cellular respiration and catabolic process, some of which are previously reported to be involved in AD pathogenesis. Additionally, seven novel proteins were found to be consistently altered in AD. In Paper V, we studied the CSF proteome of App knock-in mice and identified alterations in several blood-brain barrier and extracellular matrix proteins, for example decorin. Furthermore, in order to explore translational changes between mouse and human CSF, we compared our findings from Paper V with the CSF proteome of human patients, reflecting different stages in AD continuum (i.e normal cognition, mild cognitive impairment and AD dementia), from a recently published study. Interestingly, decorin was significantly upregulated both in the AppNL-F/NL-F mice and in the subjects with normal cognition and Aβ- positive and tau-negative CSF levels. Additionally, this study revealed alterations in proteins that were shared in all groups and extensively associated with pathways such as cell adhesion, neurogenesis, cholesterol and lipid metabolism and acute inflammatory response. In summary, this thesis has contributed with new knowledge on potential presynaptic failure in AD hippocampus and expanded our understanding of altered pathways that could be involved in AD pathogenesis. Future studies on this work may facilitate the development of new CSF biomarkers and therapeutic strategies for AD

Evaluation of Incidence and Clinical Features of Antibody-Associated Autoimmune Encephalitis Mimicking Dementia

Background. Anti-neuronal autoimmunity may cause cognitive impairment that meets the criteria for dementia. Objective. Our aim was to detect the incidence and clinical features of autoimmune encephalitis imitating clinical findings of primary dementia disorders and to delineate the validity of anti-neuronal antibody screening in dementia patients. Methods. Fifty consecutive patients fulfilling the clinical criteria for primary dementia, 130 control patients, and 50 healthy controls were included. Their sera were investigated for several ion channel and glutamic acid decarboxylase (GAD) antibodies by a cell-based assay, radioimmunoassay, and ELISA, as required. Results. Sixteen patients satisfying dementia criteria had atypical findings or findings suggestive of autoimmune encephalitis. N-methyl-D-aspartate receptor (NMDAR) antibody was detected in a patient with dementia, Parkinsonism, and REM sleep behavior disorder (RBD) fulfilling the criteria for dementia with Lewy bodies (DLB). One control patient with bipolar disease displayed low anti-GAD antibody levels. Conclusions. Our study showed for the first time the presence of parkinsonism and RBD in an anti-NMDAR encephalitis patient mimicking DLB. Although autoimmune encephalitis patients may occasionally present with cognitive decline, most dementia patients do not exhibit anti-neuronal antibodies, suggesting that routine analysis of these antibodies in dementia is not mandatory, even though they display atypical features

Mitochondrial carrier homolog 1 (Mtch1) antibodies in neuro-Behcet's disease

WOS: 000326258200019PubMed ID: 24035008Efforts for the identification of diagnostic autoantibodies for neuro-Behcet's disease (NBD) have failed. Screening of NBD patients' sera with protein macroarray identified mitochondrial carrier homolog 1 (Mtch1), an apoptosis-related protein, as a potential autoantigen. ELISA studies showed serum Mtch1 antibodies in 68 of 144 BD patients with or without neurological involvement and in 4 of 168 controls corresponding to a sensitivity of 47.2% and specificity of 97.6%. Mtch1 antibody positive NBD patients had more attacks, increased disability and lower serum nucleosome levels. Mtch1 antibody might be involved in pathogenic mechanisms of NBD rather than being a coincidental byproduct of autoinflammation. (C) 2013 Elsevier B.V. All rights reserved

Identification of a Novel BRCA2 and CHEK2 A-C-G-C Haplotype in Turkish Patients Affected with Breast Cancer

Background: Many breast cancers are caused by certain rare and familial mutations in the high or moderate penetrance genes BRCA1, BRCA2 and CHEK2. The aim of this study was to examine the allele and genotype frequencies of seven mutations in BRCA1, BRCA2 and CHEK2 genes in breast cancer patients and to investigate their isolated and combined associations with breast cancer risk. Methods: We genotyped seven mutations in BRCA1, BRCA2 and CHEK2 genes and then analyzed single variations and haplotype associations in 106 breast cancer patients and 80 healthy controls. Results: We found significant associations in the analyses of CHEK2-1100delC (p=0.001) and BRCA1-5382insC (p=0.021) mutations in breast cancer patients compared to controls. The highest risk was observed among breast cancer patients carrying both CHEK2-1100delC and BRCA2-Met784Val mutations (OR=0.093; 95% CI 0.021-0.423; p=0.001). We identified one previously undescribed BRCA2 and a CHEK2 four-marker haplotype of A-C-G-C which was overrepresented (chi(2)=7.655; p=0.0057) in the patient group compared to controls. Conclusion: In this study, we identified a previously undescribed BRCA2 and CHEK2 A-C-G-C haplotype in association with the breast cancer in our population. Our results further suggest that the CHEK2-1100delC mutation in combination with BRCA2-Met784Val may lead to an unexpected high risk which needs to be confirmed in larger cohorts in order to better understand their role in the development and prognosis of breast cancer

Identification of a Novel BRCA2 and CHEK2 A-C-G-C Haplotype in Turkish Patients Affected with Breast Cancer

Background: Many breast cancers are caused by certain rare and familial mutations in the high or moderate penetrance genes BRCA1, BRCA2 and CHEK2. The aim of this study was to examine the allele and genotype frequencies of seven mutations in BRCA1, BRCA2 and CHEK2 genes in breast cancer patients and to investigate their isolated and combined associations with breast cancer risk. Methods: We genotyped seven mutations in BRCA1, BRCA2 and CHEK2 genes and then analyzed single variations and haplotype associations in 106 breast cancer patients and 80 healthy controls. Results: We found significant associations in the analyses of CHEK2-1100delC (p=0.001) and BRCA1-5382insC (p=0.021) mutations in breast cancer patients compared to controls. The highest risk was observed among breast cancer patients carrying both CHEK2-1100delC and BRCA2-Met784Val mutations (OR=0.093; 95% CI 0.021-0.423; p=0.001). We identified one previously undescribed BRCA2 and a CHEK2 four-marker haplotype of A-C-G-C which was overrepresented (chi(2)=7.655; p=0.0057) in the patient group compared to controls. Conclusion: In this study, we identified a previously undescribed BRCA2 and CHEK2 A-C-G-C haplotype in association with the breast cancer in our population. Our results further suggest that the CHEK2-1100delC mutation in combination with BRCA2-Met784Val may lead to an unexpected high risk which needs to be confirmed in larger cohorts in order to better understand their role in the development and prognosis of breast cancer