High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring.

Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-week-old male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippocampus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deficiency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These findings highlight the unintended negative consequences of supplemental folic acid.

Insight meditation and telomere biology: The effects of intensive retreat and the moderating role of personality.

A growing body of evidence suggests that meditation training may have a range of salubrious effects, including improved telomere regulation. Telomeres and the enzyme telomerase interact with a variety of molecular components to regulate cell-cycle signaling cascades, and are implicated in pathways linking psychological stress to disease. We investigated the effects of intensive meditation practice on these biomarkers by measuring changes in telomere length (TL), telomerase activity (TA), and telomere-related gene (TRG) expression during a 1-month residential Insight meditation retreat. Multilevel analyses revealed an apparent TL increase in the retreat group, compared to a group of experienced meditators, similarly comprised in age and gender, who were not on retreat. Moreover, personality traits predicted changes in TL, such that retreat participants highest in neuroticism and lowest in agreeableness demonstrated the greatest increases in TL. Changes observed in TRGs further suggest retreat-related improvements in telomere maintenance, including increases in Gar1 and HnRNPA1, which encode proteins that bind telomerase RNA and telomeric DNA. Although no group-level changes were observed in TA, retreat participants' TA levels at post-assessment were inversely related to several indices of retreat engagement and prior meditation experience. Neuroticism also predicted variation in TA across retreat. These findings suggest that meditation training in a retreat setting may have positive effects on telomere regulation, which are moderated by individual differences in personality and meditation experience. (ClinicalTrials.gov #NCT03056105).

APJ acts as a dual receptor in cardiac hypertrophy.

Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of ß-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.

Rcor2 underexpression in senescent mice: a target for inflammaging?

BACKGROUND: Aging is characterized by a low-grade systemic inflammation that contributes to the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). However, little knowledge is currently available on the molecular processes leading to chronic neuroinflammation. In this context, recent studies have described the role of chromatin regulators in inflammation and longevity including the REST corepressor (Rcor)-2 factor, which seems to be involved in an inflammatory suppressive program. METHODS: To assess the impact of Rcor2 in age-related inflammation, gene expression levels were quantified in different tissues and ages of the spontaneous senescence-accelerated P8 mouse (P8) using the SAMR1 mouse (R1) as a control. Specific siRNA transfection in P8 and R1 astrocyte cultures was used to determine Rcor2 involvement in the modulation of neuroinflammation. The effect of lipopolysaccharide (LPS) treatment on Rcor2 levels and neuroinflammation was analyzed both in vivo and in vitro. RESULTS: P8 mice presented a dramatic decrease in Rcor2 gene expression compared with R1 controls in splenocytes, an alteration also observed in the brain cortex, hippocampus and primary astrocytes of these mice. Rcor2 reduction in astrocytes was accompanied by an increased basal expression of the interleukin (Il)-6 gene. Strikingly, intraperitoneal LPS injection in R1 mice downregulated Rcor2 in the hippocampus, with a concomitant upregulation of tumor necrosis factor (Tnf-α), Il1-ß and Il6 genes. A negative correlation between Rcor2 and Il6 gene expression was also verified in LPS-treated C6 glioma cells. Knock down of Rcor2 by siRNA transfection (siRcor2) in R1 astrocytes upregulated Il6 gene expression while siRcor2 further increased Il6 expression in P8 astrocytes. Moreover, LPS activation provoked a further downregulation of Rcor2 and an amplified induction of Il6 in siRcor2-tranfected astrocytes. CONCLUSIONS: Data presented here show interplay between Rcor2 downregulation and increased inflammation and suggest that Rcor2 may be a key regulator of inflammaging.

Altered ß-adrenergic response in mice lacking myotonic dystrophy protein kinase.

The protein kinase product of the gene mutated in myotonic dystrophy 1 (DMPK) is reported to play a role in cardiac pathophysiology. To gain insight into the molecular mechanisms modulated by DMPK, we characterize the impact of DMPK ablation in the context of cardiac ß-adrenergic function. Our data demonstrate that DMPK knockout mice present altered ß-agonist-induced responses and suggest that this is due, at least in part, to a reduced density of ß(1)-adrenergic receptors in cardiac plasma membranes.

Epigenetic impact of a 1-week intensive multimodal group program for adolescents with multiple adverse childhood experiences.

Adverse childhood experiences (ACEs, i.e., abuse, neglect, household dysfunction) represent a potential risk factor for a wide range of long-lasting diseases and shorter life expectancy. We recently described a 1-week residential group program, based on mindfulness training, artistic expression and EMDR group therapy, that significantly reduced PTSD-related symptoms and increased attention/awareness-related outcomes in adolescent girls with multiple ACEs in a randomized controlled study. Since epigenetic mechanisms (i.e., DNA methylation) have been associated with the long-lasting effects of ACEs, the present report extends these prior findings by exploring genome-wide DNA methylation changes following the program. Saliva samples from all participants (n = 44) were collected and genomic DNA was extracted prior (T1) and following (T2) the intervention. Genome-wide DNA methylation analysis using the MethylationEPIC beadchip array (Illumina) revealed 49 differentially methylated loci (DML; p value < 0.001; methylation change > 10%) that were annotated to genes with roles in biological processes linked to early childhood adversity (i.e., neural, immune, and endocrine pathways, cancer and cardiovascular disease). DNA sequences flanking these DML showed significant enrichment of transcription factor binding sites involved in inflammation, cancer, cardiovascular disease, and brain development. Methylation changes in SIRT5 and TRAPPC2L genes showed associations with changes in trauma-related psychological measures. Results presented here suggest that this multimodal group program for adolescents with multiple victimization modulates the DNA methylome at sites of potential relevance for health and behavioral disorders associated with ACEs.

Changes in the expression of inflammatory and epigenetic-modulatory genes after an intensive meditation retreat.

Background: Meditation retreats are characterized by intensive or concentrated periods of meditation practice, commonly undertaken in a residential setting. Although research indicates that meditation training can positively influence physical and mental health outcomes, the biological consequences of meditation retreat interventions are relatively understudied. In this study, we examined the influence of a month-long, silent meditation retreat on the expression of genes involved in epigenetic modulation and immune processes. Method: We assessed gene expression changes in experienced meditators attending a month-long Insight meditation retreat (n = 28), as compared to a community control group (n = 34) of experienced practitioners living their everyday lives. Blood samples were collected on day two of the retreat (Time 1) and again 3 weeks later (Time 2). Control participants were also assessed across a 3-week interval, during which they maintained their regular daily routines. Results: As compared to controls, retreat participants showed differential changes in the expression of several genes involved in chromatin modulation and inflammation. The most substantive finding was downregulation of the TNF pathway in retreat participants, which was not observed in controls. Conclusions: These findings indicate that meditation retreat participation may influence some of the inflammatory mechanisms involved in the development of chronic diseases, and that this style of psychosocial intervention may have therapeutic potential, particularly in experienced practitioners.

Histone demethylase LSD1 regulates adipogenesis.

Epigenetic mechanisms, in particular the enzymatic modification of histones, are a crucial element of cell differentiation, a regulated process that allows a precursor cell basically to turn into a different cell type while maintaining the same genetic equipment. We have previously described that the promoters of adipogenic genes display significant levels of dimethylation at the Lys(4) of histone H3 (H3K4) in preadipocytes, where these genes are still silenced, thus maintaining the chromatin of the precursor cell in a receptive state. Here, we show that the expression of several histone demethylases and methyltransferases increases during adipogenesis, suggesting an important role for these proteins in this process. Knockdown of the H3K4/K9 demethylase LSD1 results in markedly decreased differentiation of 3T3-L1 preadipocytes. This outcome is associated with decreased H3K4 dimethylation and increased H3K9 dimethylation at the promoter of transcription factor cebpa, whose expression must be induced >200-fold upon stimulation of differentiation. Thus, our data suggest that LSD1 acts to maintain a permissive state of the chromatin in this promoter by opposing the action of a H3K9 methyltransferase. Knockdown of H3K9 methyltransferase SETDB1 produced the opposite results, by decreasing H3K9 dimethylation and increasing H3K4 dimethylation levels at the cebpa promoter and favoring differentiation. These findings indicate that the histone methylation status of adipogenic genes as well as the expression and function of the proteins involved in its maintenance play a crucial role in adipogenesis.

Mental health benefits of a 1-week intensive multimodal group program for adolescents with multiple adverse childhood experiences.

BACKGROUND: Adverse childhood experiences (ACEs) are associated with a wide range of diseases, unsafe behavior and shorter life expectancy. However, there is scarce evidence on effective interventions for children or adolescents who report multiple ACEs, including abuse, neglect and household dysfunction. OBJECTIVE: The aim of this study was to evaluate the mental health outcomes of a multimodal program designed for adolescents with multiple ACEs. PARTICIPANTS: Forty-four girls (aged 13-16 years, mean ACE score > 5) were randomized to an intervention group or a care-as-usual control group. METHODS: The intervention included mindfulness-based practices, expressive arts and EMDR (Eye Movement Desensitization and Reprocessing Integrative) group treatment. We used questionnaires for adolescents to assess trauma (SPRINT, CPSS) and attention/awareness-related outcomes (MAAS-A) at baseline (T1), post-intervention (T2) and two-months post-discharge (T3). RESULTS: Linear mixed effects model analyses showed significant Group by Time interactions on all the scales (F = 11.0, p = 0.015; F = 12.5 p < 0.001; and F = 6.4, p = 0.001, for SPRINT, CPSS and MAAS-A, respectively). After completing the program, the intervention group showed significant reduction in trauma-related outcomes (SPRINT, Δ%(T2-T1) = -73%, p < 0.001; CPSS, Δ%(T2-T1) = -26%, p < 0.001) while attention/awareness-related outcomes were improved by 57% (p < 0.001). These changes remained stable two months after discharge. SPRINT and CPSS scales were highly correlated (r = 0.833, p < 0.001) and outcomes from both trauma-related scales negatively correlated with mindfulness scores (MAAS-A/SPRINT, r = -0.515, p = 0.007; MAAS-A/CPSS, r = -0.553, p < 0.001). CONCLUSIONS: Results presented here support this multimodal group intervention as a feasible and promising program for reducing the psychological burden in adolescents with a history of multiple ACEs.

Microglial Hyperreactivity Evolved to Immunosuppression in the Hippocampus of a Mouse Model of Accelerated Aging and Alzheimer's Disease Traits.

Neuroinflammation is a risk factor for Alzheimer's disease (AD). We sought to study the glial derangement in AD using diverse experimental models and human brain tissue. Besides classical pro-inflammatory cytokines, we analyzed chitinase 3 like 1 (CHI3L1 or YKL40) and triggering receptor expressed on myeloid cells 2 (TREM2) that are increasingly being associated with astrogliosis and microgliosis in AD, respectively. The SAMP8 mouse model of accelerated aging and AD traits showed elevated pro-inflammatory cytokines and activated microglia phenotype. Furthermore, 6-month-old SAMP8 showed an exacerbated inflammatory response to peripheral lipopolysaccharide in the hippocampus and null responsiveness at the advanced age (for this strain) of 12 months. Gene expression of TREM2 was increased in the hippocampus of transgenic 5XFAD mice and in the cingulate cortex of autosomal dominant AD patients, and to a lesser extent in aged SAMP8 mice and sporadic early-onset AD patients. However, gene expression of CHI3L1 was increased in mice but not in human AD brain samples. The results support the relevance of microglia activation in the pathways leading to neurodegeneration and suggest diverse neuroinflammatory responses according to the AD process. Therefore, the SAMP8 mouse model with marked alterations in the dynamics of microglia activation and senescence may provide a complementary approach to transgenic mouse models for the study of the neuroinflammatory mechanisms underlying AD risk and progression.

Myotonic dystrophy protein kinase (DMPK) and its role in the pathogenesis of myotonic dystrophy 1.

Myotonic dystrophy 1 (DM1) is an autosomal, dominant inherited, neuromuscular disorder. The DM1 mutation consists in the expansion of an unstable CTG-repeat in the 3'-untranslated region of a gene encoding DMPK (myotonic dystrophy protein kinase). Clinical expression of DM1 is variable, presenting a progressive muscular dystrophy that affects distal muscles more than proximal and is associated with the inability to relax muscles appropriately (myotonia), cataracts, cardiac arrhythmia, testicular atrophy and insulin resistance. DMPK is a Ser/Thr protein kinase homologous to the p21-activated kinases MRCK and ROCK/rho-kinase/ROK. The most abundant isoform of DMPK is an 80 kDa protein mainly expressed in smooth, skeletal and cardiac muscles. Decreased DMPK protein levels may contribute to the pathology of DM1, as revealed by gene target studies. Here we review current understanding of the structural, functional and pathophysiological characteristics of DMPK.

Epigenetics and meditation.

In the last decade, epigenetics has taken center stage to explain the relationships between stress exposure, health and behavior. Acquired or inherited epigenetic changes modulate gene expression states without modifying the DNA sequence itself, they can be long-lasting, yet, they are potentially reversible. Several studies have explored whether meditation-based interventions can influence gene expression profiles towards healthier directions, identifying candidate genes and biological pathways that seem to be sensitive to contemplative practices. However, to date, the clinical implications of these molecular outcomes and their potential long-lasting epigenetic bases remain mostly unknown. The present article addresses these topics from a broad perspective and analyzes future research questions and perspectives at the crossroads of contemplative sciences and epigenetics.

Early Manifestations of Brain Aging in Mice Due to Low Dietary Folate and Mild MTHFR Deficiency.

Folate is an important B vitamin required for methylation reactions, nucleotide and neurotransmitter synthesis, and maintenance of homocysteine at nontoxic levels. Its metabolism is tightly linked to that of choline, a precursor to acetylcholine and membrane phospholipids. Low folate intake and genetic variants in folate metabolism, such as the methylenetetrahydrofolate reductase (MTHFR) 677 C>T polymorphism, have been suggested to impact brain function and increase the risk for cognitive decline and late-onset Alzheimer's disease. Our study aimed to assess the impact of genetic and nutritional disturbances in folate metabolism, and their potential interaction, on features of cognitive decline and brain biochemistry in a mouse model. Wild-type and Mthfr+/- mice, a model for the MTHFR 677 C>T polymorphism, were fed control or folate-deficient diets from weaning until 8 and 10 months of age. We observed short-term memory impairment measured by the novel object paradigm, altered transcriptional levels of synaptic markers and epigenetic enzymes, as well as impaired choline metabolism due to the Mthfr+/- genotype in cortex or hippocampus. We also detected changes in mRNA levels of Presenillin-1, neurotrophic factors, one-carbon metabolic and epigenetic enzymes, as well as reduced levels of S-adenosylmethionine and acetylcholine, due to the folate-deficient diet. These findings shed further insights into the mechanisms by which genetic and dietary folate metabolic disturbances increase the risk for cognitive decline and suggest that these mechanisms are distinct.

Role of Resveratrol and Selenium on Oxidative Stress and Expression of Antioxidant and Anti-Aging Genes in Immortalized Lymphocytes from Alzheimer's Disease Patients.

Oxidative damage is involved in the pathophysiology of age-related ailments, including Alzheimer's disease (AD). Studies have shown that the brain tissue and also lymphocytes from AD patients present increased oxidative stress compared to healthy controls (HCs). Here, we use lymphoblastoid cell lines (LCLs) from AD patients and HCs to investigate the role of resveratrol (RV) and selenium (Se) in the reduction of reactive oxygen species (ROS) generated after an oxidative injury. We also studied whether these compounds elicited expression changes in genes involved in the antioxidant cell response and other aging-related mechanisms. AD LCLs showed higher ROS levels than those from HCs in response to H2O2 and FeSO4 oxidative insults. RV triggered a protective response against ROS under control and oxidizing conditions, whereas Se exerted antioxidant effects only in AD LCLs under oxidizing conditions. RV increased the expression of genes encoding known antioxidants (catalase, copper chaperone for superoxide dismutase 1, glutathione S-transferase zeta 1) and anti-aging factors (sirtuin 1 and sirtuin 3) in both AD and HC LCLs. Our findings support RV as a candidate for inducing resilience and protection against AD, and reinforce the value of LCLs as a feasible peripheral cell model for understanding the protective mechanisms of nutraceuticals against oxidative stress in aging and AD.

Temporal Integrative Analysis of mRNA and microRNAs Expression Profiles and Epigenetic Alterations in Female SAMP8, a Model of Age-Related Cognitive Decline.

A growing body of research shows that epigenetic mechanisms are critically involved in normal and pathological aging. The Senescence-Accelerated Mouse Prone 8 (SAMP8) can be considered a useful tool to better understand the dynamics of the global epigenetic landscape during the aging process since its phenotype is not fully explained by genetic factors. Here we investigated dysfunctional age-related transcriptional profiles and epigenetic programming enzymes in the hippocampus of 2- and 9-month-old SAMP8 female mice using the Senescent-Accelerated Resistant 1 (SAMR1) mouse strain as control. SAMP8 mice presented 1,062 genes dysregulated at 2 months of age, and 1,033 genes at 9 months, with 92 genes concurrently dysregulated at both ages compared to age-matched SAMR1. SAMP8 mice showed a significant decrease in global DNA methylation (5-mC) at 2 months while hydroxymethylation (5-hmC) levels were increased in SAMP8 mice at 2 and 9 months of age compared to SAMR1. These changes were accompanied by changes in the expression of several enzymes that regulate 5-mC and methylcytosine oxidation. Acetylated H3 and H4 histone levels were significantly diminished in SAMP8 mice at 2-month-old compared to SAMR1 and altered Histone DeACetylase (HDACs) profiles were detected in both young and old SAMP8 mice. We analyzed 84 different mouse miRNAs known to be altered in neurological diseases or involved in neuronal development. Compared with SAMR1, SAMP8 mice showed 28 and 17 miRNAs differentially expressed at 2 and 9 months of age, respectively; 6 of these miRNAs overlapped at both ages. We used several bioinformatic approaches to integrate our data in mRNA:miRNA regulatory networks and functional predictions for young and aged animals. In sum, our study reveals interplay between epigenetic mechanisms and gene networks that seems to be relevant for the progression toward a pathological aging and provides several potential markers and therapeutic candidates for Alzheimer's Disease (AD) and age-related cognitive impairment.

Omega-3 supplements reduce self-reported physical aggression in healthy adults.

There is emerging evidence that Omega-3 polyunsaturated fatty acids (PUFA) supplements can decrease aggression. However, experimental studies with adults from non-specific populations are scarce. We hypothesized that Omega-3 supplements would decrease self-reported aggression among non-clinical participants. In a double-blind randomized trial, two groups of participants (N = 194) aged 18-45 from the general population followed a 6-weeks treatment with 638mg docosahexaenoic acid (DHA) and 772mg eicosapentaenoic acid (EPA) per day or the equivalent quantity of copra oil (placebo). Self-reported aggressiveness was measured at baseline and after the 6-week treatment period. Findings showed that Omega-3 supplements significantly decreased self-reported aggressiveness at the end of the 6-week period (d = 0.31). In conclusion, this experiment indicates that Omega-3 administration has beneficial effects in reducing aggression among the general population.

Epigenetic clock analysis in long-term meditators.

In this paper, we examined whether meditation practice influences the epigenetic clock, a strong and reproducible biomarker of biological aging, which is accelerated by cumulative lifetime stress and with age-related chronic diseases. Using the Illumina 450K array platform, we analyzed the DNA methylome from blood cells of long-term meditators and meditation-naïve controls to estimate their Intrinsic Epigenetic Age Acceleration (IEAA), using Horvath's calculator. IEAA was similar in both groups. However, controls showed a different IEAA trajectory with aging than meditators: older controls (age≥52) had significantly higher IEAAs compared with younger controls (age <52), while meditators were protected from this epigenetic aging effect. Notably, in the meditation group, we found a significant negative correlation between IEAA and the number of years of regular meditation practice. From our results, we hypothesize that the cumulative effects of a regular meditation practice may, in the long-term, help to slow the epigenetic clock and could represent a useful preventive strategy for age-related chronic diseases. Longitudinal randomized controlled trials in larger cohorts are warranted to confirm and further characterize these findings.

SIRT1 Overexpression in Mouse Hippocampus Induces Cognitive Enhancement Through Proteostatic and Neurotrophic Mechanisms.

SIRT1 induces cell survival and has shown neuroprotection against amyloid and tau pathologies in Alzheimer's disease (AD). However, protective effects against memory loss or the enhancement of cognitive functions have not yet been proven. We aimed to investigate the benefits induced by SIRT1 overexpression in the hippocampus of the AD mouse model 3xTg-AD and in control non-transgenic mice. A lentiviral vector encoding mouse SIRT1 or GFP, selectively transducing neurons, was injected into the dorsal CA1 hippocampal area of 4-month-old mice. Six-month overexpression of SIRT1 fully preserved learning and memory in 10-month-old 3xTg-AD mice. Remarkably, SIRT1 also induced cognitive enhancement in healthy non-transgenic mice. Neuron cultures of 3xTg-AD mice, which show traits of AD-like pathology, and neuron cultures from non-transgenic mice were also transduced with lentiviral vectors to analyze beneficial SIRT1 mechanisms. We uncovered novel pathways of SIRT1 neuroprotection through enhancement of cell proteostatic mechanisms and activation of neurotrophic factors not previously reported such as GDNF, present in both AD-like and healthy neurons. Therefore, SIRT1 may increase neuron function and resilience against AD.

Plasma miR-34a-5p and miR-545-3p as Early Biomarkers of Alzheimer's Disease: Potential and Limitations.

Plasma microRNAs (miRNAs) have been proposed as potential biomarkers in Alzheimer's disease (AD). Here, we explored their use as early sensors of the preclinical phase of the disease, when brain pathology is being developed and no cognitive loss is detected. For this purpose, we analyzed a set of ten mature plasma miRNAs in symptomatic patients with AD from a cohort that also included healthy controls (HC) and patients with preclinical Alzheimer's disease (PAD) (cohort 1). Plasmas from subjects with Parkinson's disease (PD) were used to control for disease specificity. We found that miR-15b-5p, miR-34a-5p, miR-142-3p, and miR-545-3p levels significantly distinguished AD from PD and HC subjects. We next examined the expression of these four miRNAs in plasma from subjects with PAD. Among these, miR-34a-5p and miR-545-3p presented good diagnostic accuracy to distinguish both AD and PAD from HC subjects, according to the receiver operating characteristic (ROC) curve analysis. Both miRNAs also demonstrated a significant positive correlation with Aß1-42 levels in cerebrospinal fluid (CSF). Taking into account the clinical potential of these findings, we decided to validate the diagnostic accuracy of miR-34a-5p and miR-545-3p in plasma samples from an independent cohort (cohort 2), in which we did not observe the alterations described by us and others in AD and PAD samples. Although miR-34a-5p and miR-545-3p might be promising early biomarker candidates for AD, our study highlights possible sources of variability in miRNA analysis across hospitals, which currently prevents their use as reliable clinical tools.