Transcriptomics analysis of human iPSC-derived dopaminergic neurons reveals a novel model for sporadic Parkinson's disease.

Parkinson's disease (PD) is a progressive, neurodegenerative disease affecting over 1% of the population beyond 65 years of age. Although some PD cases are inheritable, the majority of PD cases occur in a sporadic manner. Risk factors comprise next to heredity, age, and gender also exposure to neurotoxins from for instance pesticides and herbicides. As PD is characterized by a loss of dopaminergic neurons in the substantia nigra, it is nearly impossible to access and extract these cells from patients for investigating disease mechanisms. The emergence of induced pluripotent stem (iPSC) technology allows differentiating and growing human dopaminergic neurons, which can be used for in vitro disease modeling. Here, we differentiated human iPSCs into dopaminergic neurons, and subsequently exposed the cells to increasing concentrations of the neurotoxin MPP+. Temporal transcriptomics analysis revealed a strong time- and dose-dependent response with genes over-represented across pathways involved in PD etiology such as "Parkinson's Disease", "Dopaminergic signaling" and "calcium signaling". Moreover, we validated this disease model by showing robust overlap with a meta-analysis of transcriptomics data from substantia nigra from post-mortem PD patients. The overlap included genes linked to e.g. mitochondrial dysfunction, neuron differentiation, apoptosis and inflammation. Our data shows, that MPP+-induced, human iPSC-derived dopaminergic neurons present molecular perturbations as observed in the etiology of PD. Therefore we propose iPSC-derived dopaminergic neurons as a foundation for a novel sporadic PD model to study the pathomolecular mechanisms of PD, but also to screen for novel anti-PD drugs and to develop and test new treatment strategies.

Correction: MicroRNA regulation of persistent stress-enhanced memory.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

MicroRNA regulation of persistent stress-enhanced memory.

Disruption of persistent, stress-associated memories is relevant for treating posttraumatic stress disorder (PTSD) and related syndromes, which develop in a subset of individuals following a traumatic event. We previously developed a stress-enhanced fear learning (SEFL) paradigm in inbred mice that produces PTSD-like characteristics in a subset of mice, including persistently enhanced memory and heightened cFos in the basolateral amygdala complex (BLC) with retrieval of the remote (30-day-old) stress memory. Here, the contribution of BLC microRNAs (miRNAs) to stress-enhanced memory was investigated because of the molecular complexity they achieve through their ability to regulate multiple targets simultaneously. We performed small-RNA sequencing (smRNA-Seq) and quantitative proteomics on BLC tissue collected from mice 1 month after SEFL and identified persistently changed microRNAs, including mir-135b-5p, and proteins associated with PTSD-like heightened fear expression. Viral-mediated overexpression of mir-135b-5p in the BLC of stress-resilient animals enhanced remote fear memory expression and promoted spontaneous renewal 14 days after extinction. Conversely, inhibition of BLC mir-135b-5p in stress-susceptible animals had the opposite effect, promoting a resilient-like phenotype. mir-135b-5p is highly conserved across mammals and was detected in post mortem human amygdala, as well as human serum samples. The mir-135b passenger strand, mir-135b-3p, was significantly elevated in serum from PTSD military veterans, relative to combat-exposed control subjects. Thus, miR-135b-5p may be an important therapeutic target for dampening persistent, stress-enhanced memory and its passenger strand a potential biomarker for responsivity to a mir-135-based therapeutic.

Evolving DNA methylation and gene expression markers of B-cell chronic lymphocytic leukemia are present in pre-diagnostic blood samples more than 10 years prior to diagnosis.

BACKGROUND: B-cell chronic lymphocytic leukemia (CLL) is a common type of adult leukemia. It often follows an indolent course and is preceded by monoclonal B-cell lymphocytosis, an asymptomatic condition, however it is not known what causes subjects with this condition to progress to CLL. Hence the discovery of prediagnostic markers has the potential to improve the identification of subjects likely to develop CLL and may also provide insights into the pathogenesis of the disease of potential clinical relevance. RESULTS: We employed peripheral blood buffy coats of 347 apparently healthy subjects, of whom 28 were diagnosed with CLL 2.0-15.7 years after enrollment, to derive for the first time genome-wide DNA methylation, as well as gene and miRNA expression, profiles associated with the risk of future disease. After adjustment for white blood cell composition, we identified 722 differentially methylated CpG sites and 15 differentially expressed genes (Bonferroni-corrected p < 0.05) as well as 2 miRNAs (FDR < 0.05) which were associated with the risk of future CLL. The majority of these signals have also been observed in clinical CLL, suggesting the presence in prediagnostic blood of CLL-like cells. Future CLL cases who, at enrollment, had a relatively low B-cell fraction (<10%), and were therefore less likely to have been suffering from undiagnosed CLL or a precursor condition, showed profiles involving smaller numbers of the same differential signals with intensities, after adjusting for B-cell content, generally smaller than those observed in the full set of cases. A similar picture was obtained when the differential profiles of cases with time-to-diagnosis above the overall median period of 7.4 years were compared with those with shorted time-to-disease. Differentially methylated genes of major functional significance include numerous genes that encode for transcription factors, especially members of the homeobox family, while differentially expressed genes include, among others, multiple genes related to WNT signaling as well as the miRNAs miR-150-5p and miR-155-5p. CONCLUSIONS: Our findings demonstrate the presence in prediagnostic blood of future CLL patients, more than 10 years before diagnosis, of CLL-like cells which evolve as preclinical disease progresses, and point to early molecular alterations with a pathogenetic potential.

Over-expression of Arabidopsis AtCHR23 chromatin remodeling ATPase results in increased variability of growth and gene expression.

BACKGROUND: Plants are sessile organisms that deal with their -sometimes adverse- environment in well-regulated ways. Chromatin remodeling involving SWI/SNF2-type ATPases is thought to be an important epigenetic mechanism for the regulation of gene expression in different developmental programs and for integrating these programs with the response to environmental signals. In this study, we report on the role of chromatin remodeling in Arabidopsis with respect to the variability of growth and gene expression in relationship to environmental conditions. RESULTS: Already modest (2-fold) over-expression of the AtCHR23 ATPase gene in Arabidopsis results in overall reduced growth compared to the wild-type. Detailed analyses show that in the root, the reduction of growth is due to reduced cell elongation. The reduced-growth phenotype requires sufficient light and is magnified by applying deliberate abiotic (salt, osmotic) stress. In contrast, the knockout mutation of AtCHR23 does not lead to such visible phenotypic effects. In addition, we show that over-expression of AtCHR23 increases the variability of growth in populations of genetically identical plants. These data indicate that accurate and controlled expression of AtCHR23 contributes to the stability or robustness of growth. Detailed RNAseq analyses demonstrate that upon AtCHR23 over-expression also the variation of gene expression is increased in a subset of genes that associate with environmental stress. The larger variation of gene expression is confirmed in individual plants with the help of independent qRT-PCR analysis. CONCLUSIONS: Over-expression of AtCHR23 gives Arabidopsis a phenotype that is markedly different from the growth arrest phenotype observed upon over-expression of AtCHR12, the paralog of AtCHR23, in response to abiotic stress. This demonstrates functional sub-specialization of highly similar ATPases in Arabidopsis. Over-expression of AtCHR23 increases the variability of growth among genetically identical individuals in a way that is consistent with increased variability of expression of a distinct subset of genes that associate with environmental stress. We propose that ATCHR23-mediated chromatin remodeling is a potential component of a buffer system in plants that protects against environmentally-induced phenotypic and transcriptional variation.

Molecular insights into PCB neurotoxicity: Comparing transcriptomic responses across dopaminergic neurons, population blood cells, and Parkinson's disease pathology.

Parkinson's disease (PD) is a complex neurodegenerative disorder influenced by genetic factors and environmental exposures. Polychlorinated biphenyls (PCBs), a group of synthetic organic compounds, have been identified as potential environmental risk factors for neurodegenerative diseases, including PD. We explored PCB-induced neurotoxicity mechanisms using iPSC-derived dopaminergic neurons and assessed their transcriptomic responses to varying PCB concentrations (0.01 µM, 0.5 µM, and 10 µM). Specifically, we focused on PCB-180, a congener known for its accumulation in human brains. The exposure durations were 24 h and 74 h, allowing us to capture both short-term and more prolonged effects on gene expression patterns. We observed that PCB exposure led to the suppression of oxidative phosphorylation, synaptic function, and neurotransmitter release, implicating these pathways in PCB-induced neurotoxicity. In our comparative analysis, we noted similarities in PCB-induced changes with other PD-related compounds like MPP+ and rotenone. Our findings also aligned with gene expression changes in human blood derived from a population exposed to PCBs, highlighting broader inflammatory responses. Additionally, molecular patterns seen in iPSC-derived neurons were confirmed in postmortem PD brain tissues, validating our in vitro results. In conclusion, our study offers novel insights into the multifaceted impacts of PCB-induced perturbations on various cellular contexts relevant to PD. The use of iPSC-derived dopaminergic neurons allowed us to decipher intricate transcriptomic alterations, bridging the gap between in vitro and in vivo findings. This work underscores the potential role of PCB exposure in neurodegenerative diseases like PD, emphasizing the need to consider both systemic and cell specific effects.

Beyond CSF and Neuroimaging Assessment: Evaluating Plasma miR-145-5p as a Potential Biomarker for Mild Cognitive Impairment and Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia. New strategies for the early detection of MCI and sporadic AD are crucial for developing effective treatment options. Current techniques used for diagnosis of AD are invasive and/or expensive, so they are not suitable for population screening. Cerebrospinal fluid (CSF) biomarkers such as amyloid ß1-42 (Aß1-42), total tau (T-tau), and phosphorylated tau181 (P-tau181) levels are core biomarkers for early diagnosis of AD. Several studies have proposed the use of blood-circulating microRNAs (miRNAs) as potential novel early biomarkers for AD. We therefore applied a novel approach to identify blood-circulating miRNAs associated with CSF biomarkers and explored the potential of these miRNAs as biomarkers of AD. In total, 112 subjects consisting of 28 dementia due to AD cases, 63 MCI due to AD cases, and 21 cognitively healthy controls were included. We identified seven Aß1-42-associated plasma miRNAs, six P-tau181-associated plasma miRNAs, and nine Aß1-42-associated serum miRNAs. These miRNAs were involved in AD-relevant biological processes, such as PI3K/AKT signaling. Based on this signaling pathway, we constructed an miRNA-gene target network, wherein miR-145-5p has been identified as a hub. Furthermore, we showed that miR-145-5p performs best in the prediction of both AD and MCI. Moreover, miR-145-5p also improved the prediction performance of the mini-mental state examination (MMSE) score. The performance of this miRNA was validated using different datasets including an RT-qPCR dataset from plasma samples of 23 MCI cases and 30 age-matched controls. These findings indicate that blood-circulating miRNAs that are associated with CSF biomarkers levels and specifically plasma miR-145-5p alone or combined with the MMSE score can potentially be used as noninvasive biomarkers for AD or MCI screening in the general population, although studies in other AD cohorts are necessary for further validation.

Integrating SNPs-based genetic risk factor with blood epigenomic response of differentially arsenic-exposed rural subjects reveals disease-associated signaling pathways.

Arsenic (As) contamination in groundwater is responsible for numerous adverse health outcomes among millions of people. Epigenetic alterations are among the most widely studied mechanisms of As toxicity. To understand how As exposure alters gene expression through epigenetic modifications, a systematic genome-wide study was designed to address the impact of multiple important single nucleotide polymorphisms (SNPs) related to As exposure on the methylome of drinking water As-exposed rural subjects from Pakistan. Urinary As levels were used to stratify subjects into low, medium and high exposure groups. Genome-wide DNA methylation was investigated using MeDIP in combination with NimbleGen 2.1 M Deluxe Promotor arrays. Transcriptome levels were measured using Agilent 8 × 60 K expression arrays. Genotyping of selected SNPs (As3MT, DNMT1a, ERCC2, EGFR and MTHFR) was measured and an integrated genetic risk factor for each respondent was calculated by assigning a specific value to the measured genotypes based on known risk allele numbers. To select a representative model related to As exposure we compared 9 linear mixed models comprising of model 1 (including the genetic risk factor), model 2 (without the genetic risk factor) and models with individual SNPs incorporated into the methylome data. Pathway analysis was performed using ConsensusPathDB. Model 1 comprising the integrated genetic risk factor disclosed biochemical pathways including muscle contraction, cardio-vascular diseases, ATR signaling, GPCR signaling, methionine metabolism and chromatin modification in association with hypo- and hyper-methylated gene targets. A unique pathway (direct P53 effector) was found associated with the individual DNMT1a polymorphism due to hyper-methylation of CSE1L and TRRAP. Most importantly, we provide here the first evidence of As-associated DNA methylation in relation with gene expression of ATR, ATF7IP, TPM3, UBE2J2. We report the first evidence that integrating SNPs data with methylome data generates a more representative epigenome profile and discloses a better insight in disease risks of As-exposed individuals.

Lead-exposure associated miRNAs in humans and Alzheimer's disease as potential biomarkers of the disease and disease processes.

Alzheimer's disease (AD) is a neurodegenerative disease that eventually affects memory and behavior. The identification of biomarkers based on risk factors for AD provides insight into the disease since the exact cause of AD remains unknown. Several studies have proposed microRNAs (miRNAs) in blood as potential biomarkers for AD. Exposure to heavy metals is a potential risk factor for onset and development of AD. Blood cells of subjects that are exposed to lead detected in the circulatory system, potentially reflect molecular responses to this exposure that are similar to the response of neurons. In this study we analyzed blood cell-derived miRNAs derived from a general population as proxies of potentially AD-related mechanisms triggered by lead exposure. Subsequently, we analyzed these mechanisms in the brain tissue of AD subjects and controls. A total of four miRNAs were identified as lead exposure-associated with hsa-miR-3651, hsa-miR-150-5p and hsa-miR-664b-3p being negatively and hsa-miR-627 positively associated. In human brain derived from AD and AD control subjects all four miRNAs were detected. Moreover, two miRNAs (miR-3651, miR-664b-3p) showed significant differential expression in AD brains versus controls, in accordance with the change direction of lead exposure. The miRNAs' gene targets were validated for expression in the human brain and were found enriched in AD-relevant pathways such as axon guidance. Moreover, we identified several AD relevant transcription factors such as CREB1 associated with the identified miRNAs. These findings suggest that the identified miRNAs are involved in the development of AD and might be useful in the development of new, less invasive biomarkers for monitoring of novel therapies or of processes involved in AD development.

Small RNA Sequencing of Aqueous Humor and Plasma in Patients With Primary Open-Angle Glaucoma.

Purpose: Identify differentially expressed microRNAs (miRNAs) in aqueous humor (AH) and blood of primary open-angle glaucoma (POAG) patients by using small RNA sequencing. These may provide insight into POAG pathophysiology or serve as diagnostic biomarker. Methods: AH and plasma of nine POAG patients and 10 cataract control patients were small RNA sequenced on Illumina NovaSeq 6000. Identification of gene transcripts targeted by differentially expressed miRNAs was done with miRWalk and MirPath. These targets were used for pathway analysis and Gene Ontology enrichment. Diagnostic potential was evaluated by receiver operating characteristics analysis. Results: We identified 715 miRNAs in plasma and 62 miRNAs in AH. Plasma miRNA profile did not differ between POAG and control. In contrast, in AH, seven miRNAs were differentially expressed. Hsa-miR-30a-3p, hsa-miR-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were upregulated, whereas hsa-miR-92a-3p, hsa-miR-451a, and hsa-miR-486-5p were downregulated in POAG. Compared to previous studies, hsa-mir-143-3p, hsa-miR-211-5p, and hsa-miR-221-3p were reported previously, strengthening their involvement in POAG whereas hsa-miR-30a-3p, hsa-miR-92a-3p, and hsa-miR-486-5p are implicated in POAG for the first time. Identified gene transcripts were involved in several pathways, some implicated in glaucoma before (e.g., TGF-ß and neurotrophin signaling), whereas others are new (e.g., prolactin and apelin signaling). In respect to diagnostics, AH concentration of hsa-mir-143-3p had an area under the curve (AUC) of 0.889. Combined with hsa-miR-221-3p, AUC improved to 0.96. Conclusions: Small RNA sequencing identified seven differentially expressed miRNAs in AH of POAG patients. The differentially expressed miRNAs may be useful as POAG biomarkers or could become targets for new therapeutic strategies.