Mesenchymal Stem Cells from Familial Alzheimer's Patients Express MicroRNA Differently

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the predominant form of dementia globally. No reliable diagnostic, predictive techniques, or curative interventions are available. MicroRNAs (miRNAs) are vital to controlling gene expression, making them valuable biomarkers for diagnosis and prognosis. This study examines the transcriptome of olfactory ecto-mesenchymal stem cells (MSCs) derived from individuals with the PSEN1(A431E) mutation (Jalisco mutation). The aim is to determine whether this mutation affects the transcriptome and expression profile of miRNAs and their target genes at different stages of asymptomatic, presymptomatic, and symptomatic conditions. Expression microarrays compare the MSCs from mutation carriers with those from healthy donors. The results indicate a distinct variation in the expression of miRNAs and mRNAs among different symptomatologic groups and between individuals with the mutation. Using bioinformatics tools allows us to identify target genes for miRNAs, which in turn affect various biological processes and pathways. These include the cell cycle, senescence, transcription, and pathways involved in regulating the pluripotency of stem cells. These processes are closely linked to inter- and intracellular communication, vital for cellular functioning. These findings can enhance our comprehension and monitoring of the disease's physiological processes, identify new disorder indicators, and develop innovative treatments and diagnostic tools for preventing or treating AD.

Keywords: A431E; Alzheimer’s disease; Jalisco mutation; PSEN1; biomarkers; epigenetics; familial Alzheimer’s disease; mesenchymal stem cells; miRNA; olfactory; transcriptome.

Figure 1

Family pedigree of the study subjects with PSEN1(A431E) mutation. Modified from [34].

Figure 2

Evaluation of the integrity of the total RNA samples. Yellow letters correspond to the PSEN1(A431E) mutation carriers (up) and green letters correspond to the healthy controls. The electropherograms display the various species of RNAs present in the total RNA extractions, ranging from ribosomal RNAs to small RNAs less than 200 nucleotides in length. Additionally, the gel-type image highlights the prominent ribosomal RNA bands. We did not detect any genomic DNA (gDNA).

Figure 3

Selection and types of transcripts by disease stage. (A) The selection criteria were employed using the data obtained from the microarrays to determine the number of transcripts that would be examined. (B–E) display the categorization of transcripts and the corresponding distribution percentages according to groups. S represents the symptomatic stage, while P represents the presymptomatic stage. A3 refers to the asymptomatic stage in individuals who are 30 years old, while A2 refers to the asymptomatic stage in individuals who are 20 years old. lncRNA stands for long non-coding RNA, snoRNA stands for small nucleolar RNA, snRNA stands for small nuclear RNA, and vtRNA refers to vault RNA. * Deployed probesets were obtained by searching functional annotations of uncharacterized probesets in the DAVID, NCBI, and Biotools databases to access more results.

Figure 4

miRNAs expression profiles by the groups. The miRNAs with upregulated expression are depicted in red, while those with downregulated expression are in blue. (A) Individuals exhibiting symptoms. (B) Individuals in their 40s who have not yet developed disease symptoms may do so soon. (C) Individuals in their 30s who do not show any symptoms of the disease. (D) Individuals in their 20s who do not show any symptoms of the disease.

Figure 5

Putative and validated target interactions and genes for the altered miRNAs by group. (A) The table displays the putative and validated target interactions categorized by the group in each database utilized. The putative interactions were obtained from mirDB, DIANA-microT, and Target Scan, while the validated interactions were sourced from TarBase and miRTarBase. (B) Venn diagram illustrating the top 10 genes that experience higher regulatory pressure from miRNAs. In green the symptomatic stage, in yellow the presymptomatic stage and in purple and pink the asymptomatic A3 and A2 stages respectively (C) Table displaying the distinct target genes that were putative and validated for each stage. * Number of targets shared between mirDB, DIANA-microT, and Target Scan databases.

Figure 6

Putative and validated target genes that coincide with the altered transcripts in MSCs of PSEN1(A431E) mutation carriers. (A) Symptomatic S, (B) Presymptomatic P, (C) Asymptomatic A3, (D) Asymptomatic A2.

Figure 7

Enriched pathways in putative, validated, and altered mRNAs in PSEN1(A431E) mutation carriers by stage. (A) 50s Symptomatic S, (B) 40s Presymptomatic P, (C) 30s Asymptomatic A3, (D) 20s Asymptomatic A2. Outside numbers indicate the pathways resulted with the targets in each database and the numbers inside the shared pathways between databases.

Figure 8

Transcriptomic profile of MSCs derived from PSEN1(A431E) mutation carriers and controls. (A) Principal component analysis (PCA) of miRNA expression profiles in MSCs carrying the PSEN1(A431E) mutation. Each point represents a sample, and colors correspond to age groups. The yellow ellipsoid represents cells from individuals with the PSEN1(A431E) mutation, and the green one represents cells from control individuals. (B) Heat map of the expression profile of coding and non-coding RNAs differentially expressed in MSCs from healthy individuals and individuals affected by the A431E mutation. Each gene is represented in rows, and each sample is displayed in columns. Average linkage calculated the distance between the two groups. Genes without differences in expression have zero value and are represented in black. Genes with increased expression are depicted in red, while those with reduced expression are shown in blue. The data were standardized so that the average of each transcript was zero and the standard deviation was 1, ensuring all transcripts had the same weight. Graphs were generated with Partek v6.6beta software accessed on 9 April 2019).

Figure 9

miRNA profile of PSEN1(A431E) mutation carriers. (A) Venn diagram of miRNAs altered by symptomatologic stage. In green the symptomatic stage, in yellow the presymptomatic stage and in purple and pink the asymptomatic A3 and A2 stages respectively. MiRNAs shared in more than 1 mutation carrier are in bold and correspond to the 54 miRNAs in the orange circle of Venn diagram in figure (B), blue circle shows miRNAs with a FC ≥ 1.5 or ≤−1.5. This FC was obtained with the average differential expression of PSEN1(A431E) mutation carriers versus the average of MSCs without the mutation. The 29 resulting miRNAs are shown in the graphic, where green corresponds to the symptomatic stage, yellow to presymptomatic, and purple and pink to the asymptomatic stages A3 and A2, respectively. (C) Top miRNAs by FC (FC ≥ 2, ≤−2). * Correspond to the 29 shared miRNAs, and miRNAs with red letters are upregulated and green downregulated.

Figure 10

Gene ontology annotation and signaling pathways enriched with the targets of miRNAs altered in MSCs from PSEN1(A431E) mutation carriers. (A) Biological process (BP), (B) cellular component (CC), (C) molecular function (MF), (D) KEGG-enriched signaling pathways (KESP), (E) shared signaling pathways between symptomatologic stages.

Figure 11

miRNAs altered in MSCs from PSEN1(A431E) mutation carriers linked to AD previously reported. Tables with the miRNAs previously reported are linked to the AD number of references found in the PUBMED search. (A) Symptomatic S (green), (B) Presymptomatic P (yellow), (C) Asymptomatic stages A3 (purple), and A2 (pink). (D) miRNAs changed in more than 1 disease condition. ↑: miRNA upregulated and ↓: downregulated. Only the miRNAs with more than four references are shown.

Figure 12

Diagram showing the steps involved in labeling, amplifying, fragmentation, labelling and hybridizing the microarrays.