Thesis title: DNA methylation-dependent miR-29a regulation contributes to the amyloidogenic processing.
Abstract
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the aggregation of two proteins in the brain, β-amyloid peptide (Aβ) and hypermethylated tau protein, leading to the formation of extracellular neuritic plaques and intracellular neurofibrillary tangles, respectively. AD patients further exhibit memory decline, disorientation, and inability to perform normal routine activities. Epidemiological studies estimate that Alzheimer’s dementia affects 24 million individuals aged 65 years and older, with projections indicating a quadrupling of this figure by 2050. For many years, the extensive research effort has focused on LOAD due to the incidence and substantial social impact arising from the care costs associated with affected patients.
Care provision encompasses support for communication, coordination among caregivers and family members, aiming to ensure patient security and manage their health. This condition is distressing for both patients and their families. Consequently, the absence of a functional and effective therapy emphasizes the necessity to acquire further knowledge about the underlying mechanisms, identifying potential targets for selective prevention or treatment.
Alzheimer’s disease manifests in two forms. Late-onset Alzheimer's Disease (LOAD) is the most clinically and socio-economically relevant, accounting for over 90% of cases. Unlike the early-onset form, where specific genes are identified to increase susceptibility, LOAD has a minimal genetic component (ApoE) and is highly multifactorial. The mutations in the genes associated with hereditary AD, such as Presenilin 1 (PSEN1), Presenilin 2 (PSEN2), and Amyloid Precursor Protein (AβPP), are apparently not involved in LOAD.
PSEN1, the catalytic subunit of the γ-secretase complex, participates in intramembrane cleavage of different proteins, including the β-cleaved-APP peptide resulting by the BACE1 (β-secretase 1) mediated-cleavage of APP. This process generates the β-amyloid peptide (Aβ), possibly leading to the aggregation observed in AD patients. APP, a critical membrane protein primarily expressed at synapses, plays a role in their formation, neuronal plasticity, and iron export.
Several studies have been conducted regarding the epigenetic mechanisms involved in AD pathogenesis. In our laboratory, we had studied the effects of DNA methylation as expression regulator of several genes contributing to the amyloid processing. We investigated on DNA methylation by modulating the biochemical pathway called ‘one carbon metabolism’, responsible for the endogenous production of the methyl groups, through the deficiency of B-group vitamins (B12, B6, Folate) and/or the supplementation of the methylating agent, S-adenosylmethionine (SAM). Alterations in this pathway as well as hyperhomocysteinemia have been associated to dementia and AD development.
In this framework, we demonstrated that PSEN1 gene expression is directly mediated by the DNA-methylation status of its promoter. Interestingly, BACE1 expression is modulated by one-carbon metabolism alterations but the methylation state of its promoter remains unchanged, suggesting the presence of a mediator responsible for its regulation induced by one-carbon metabolism alterations.
microRNAs are small, non-coding RNAs that exerts their role by directing posttranscriptional repression of protein-coding genes they pair to. In order to evaluate the possible effects of one-carbon metabolism alterations on miRNAs expression, I performed microRNAs profiling studies on SK-N-BE neuroblastoma cell line in hypermethylating conditions (SAM supplementation) compared to the control. This assay allowed to select a subgroup of microRNAs potentially modulated by DNA methylation. I focused on miR-29a-3p because of its DNA-methylation mediated regulation, its involvement in AD and because it is known to target BACE1, being a promising candidate as mediator of one-carbon metabolism-associated BACE1 regulation.
miR-29a modulation was confirmed by qRT-PCR both in in vitro and in vivo models, in hyper- and hypo- methylating conditions, showing an expression profile counterintuitive respect to possible direct DNA-methylation mediated regulation (i.e., overexpression in hypermethylating conditions and vice versa).
Moreover, I have observed miR-29a down-regulation in post-mortem human brain tissues from AD patients in comparison with age-matched healthy subjects, supporting the idea of its protective role in AD.
To better understand the link between miR-29a and DNA-methylation, I investigated on the methylation pattern of the genetic locus encoding for this miRNA. DNA methylation of the miR-29a locus gene sequence increases with SAM supplementation and decreases with B-vitamins deficiency. I used miRNA-specific mimic and inhibitor to perform gain- and loss- of function assays on human neuroblastoma SK-N-BE cell lines, to clarify the effects of miR-29a on its targets. I confirmed that among other genes, miR-29a targets BACE1 mRNA, repressing it when up-regulated. Therefore, it has a protective role in AD and could correspond to the mediator responsible for the regulation of BACE1 expression. Through a collaboration with the University of Naples, the study on the miR-29a allowed to generate a biosensor to detect its levels in the blood as a possible starting point to propose this microRNA as an AD biomarker.
Since the accumulation of Aβ is dependent on its processing, transportation and cleavage, different mechanisms and cell types inside the nervous system may contribute to AD pathogenesis. As a side branch of the present project and through the collaboration with the Université d’Artois, in Lens, France, I started to study the effects of the ‘one carbon metabolism’ modulation, induced by SAM supplementation, on the blood-brain-barrier (BBB). Maintaining BBB integrity is crucial for the highly relevant central nervous system (CNS) environment homeostasis. BBB breakdown can trigger neurodegenerative pathways. In neurodegenerative disorders like AD, BBB tightness is also related to epigenetic modifications, notably DNA methylation.
During a 3-month permanence in Lens I investigated the effects of SAM supplementation on a human in vitro BBB model, consisting of pericytes and endothelial cells, the main cellular component of the barrier.
These preliminary results showed that SAM has no negative on the BBB integrity and, on the contrary, SAM treatment is associated to permeability decrease (an index of increased BBB efficiency) after 48 hours. To confirm these observations, I assessed SAM effects on tight junction (TJs) proteins (C15 and ZO-1) by qRT-PCR and by immunoblotting, thus confirming a possible protective role of SAM through the up-regulation of TJs genes expression. DNA methylation and miR-29a gene expression analyses are ongoing on the human in vitro BBB model.
Both the studies on miR-29a and on BBB can support the development of a novel diagnostic and therapeutic approach.