Background: Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects a significant proportion of the aging population. Its initial form is known as mild cognitive impairment (MCI), which progresses with variable speed to AD dementia. Biomarkers could provide better and earlier insight into the molecular-genetic mechanisms encompassing course of the disease.
Aim: In our study, we aimed to evaluate the association of polymorphisms in genes involved in important metabolic pathways associated with AD and MCI susceptibility and to assess the associations between genetic variability and biochemical biomarkers from the cerebrospinal fluid (CSF). We also evaluated the associations of miRNAs expression in peripheral blood, CSF and extracellular vesicles (EVs) with AD susceptibility and CSF biomarkers.
Methods: In our study, we included a total of 207 subjects with cognitive decline: 82 AD patients, 63 patients with MCI and 62 control subjects with no diagnosed neurodegenerative diseases. In AD and MCI patients, we also obtained data on CSF biomarker levels. Using bioinformatic analysis, we identified genes and miRNAs that could serve as biomarkers of AD or MCI. Polymorphisms in selected genes associated with AD risk (APOE, TOMM40, ACE, SORCS, BCHE, IL6R), inflammatory pathways (IL1B, IL6, MRNA146A, TNF , CARD8, NLRP3, GSTP1, NOS1), oxidative stress (CAT, GPX1, SOD2, KEAP1, NFE2L2) and incretin receptors (GLP1R, GIPR) were analysed using competitive allele-specific PCR. In AD and MCI patients, the expression of target miRNAs (hsa-miR-146a-5p, hsa-miR-451a, hsa-miR-30c-5p, hsa-miR-375-3p, hsa-miR-107, hsa-miR-193b-3p and hsa-miR-29c) in blood plasma, CSF samples and in EVs, isolated from both fluids, was evaluated using quantitative PCR.
Results: TNF rs1800629 (p=0.017) was associated with lower risk, while GLP1R rs10305420 was associated with higher risk for developing dementia (p<0.001). Lower AD risk was observed for IL1B rs16944 (p=0.039) and TNF rs1800629 (p=0.025), while GLP1R rs10305420 was associated with increased risk (p=0.003). Carriers of polymorphic NFE2L2 rs35652124 (p=0.021), TOMM40 rs2075650 (p=0.025) and TOMM40 rs157581 (p=0.001) alleles were more frequent in AD compared to MCI.
Genetic variability was associated with various CSF biomarkers. IL1B rs16944 was associated with higher Aβ42 (p=0.020 in cognitive decline and p=0.038 in AD), while TOMM40 rs157581 (p=0.033) and NFE2L2 rs35652124 (p=0.031) were associated with lower Aβ42. MIR146A rs2910164 (p=0.043), GLP1R rs10305420 (p=0.041) and GLP1R rs6923761 (p=0.050) were associated with an increased Aβ42/40 ratio. TOMM40 rs157581 was associated with increased total tau (p=0.032) and p-tau181 (p=0.034) in cognitive decline. In AD, GLP1R rs6923761 was associated with lower p-tau181 (p=0.022), SORCS1 rs1358030 with lower total tau (p=0.019), while SORCS1 rs1416406 was associated with lower total tau (p=0.013) and p-tau181 (p=0.036). Carriers of polymorphic CAT rs1001179 (p=0.022), KEAP1 rs1048290 (p=0.019) and KEAP1 rs9676881 (p=0.019) alleles as well as carriers of at least one polymorphic GSTP1 rs1138272 allele (p=0.005) achieved higher cognitive test results. In contrast, the polymorphic NFE2L2 rs35652124 (p=0.030) and BCHE rs1803274 alleles (p=0.029) were associated with lower values on the cognitive test.
We observed no differences in target miRNAs expression between AD and MCI patients. In EVs from plasma, we observed lower expression of miR-146a-5p, miR-451a, miR-375-3p and higher expression of miR-107, compared to total plasma (all p<0.001). In cognitive decline, we also observed associations between miR-30c-5p expression in blood plasma and higher total tau levels (p=0.035), and between miR-375-3p expression and total tau (p=0.027) and p-tau181 (p=0.018) levels in blood plasma EVs. In AD, higher expression of miR-146a-5p from blood plasma was associated with lower Aβ42/40 ratio (p=0.027), while higher expression of miR-107 in EVs from blood plasma was associated with higher p-tau181 (p=0.040). Additionally, higher expression of miR-146a-5p in CSF was associated with higher p-tau181 (p=0.042), higher expression of miR-107 with higher total tau (p=0.025), higher miR-29c expression was associated with lower Aβ42/40 ratio (p=0.023), while in EVs isolated from CSF, higher expression of miR-451a was associated with higher level of Aβ42 (p=0.042). In cognititve decline, we also observed a correlation between higher expression of miR-146a-5p in the CSF of and lower values on the cognitive test (p=0.039).
Conclusion: In our study, we confirmed that some of the studied genes, associated with AD risk, could serve as biomarkers of MCI and AD. We also observed the associations of their genetic variability with the individual CSF biomarker levels. The hypothesis that miRNAs can serve as biological markers of AD and MCI was not entirely confirmed, as we did not observe differences in expression between AD and MCI. However, the expression of target miRNAs was associated with CSF biomarkers. We also partially confirmed the hypothesis that miRNAs in EVs are a better biomarker of MCI and AD than cell-free miRNAs. We have shown the significant impact of molecular mechanisms, especially inflammation and oxidative stress, on the occurrence of cognitive decline, therefore our results represent a basis for further studies searching for biomarkers for less invasive diagnosis of AD and MCI.
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