Biology subjects

Castellani, C. A.

Publications and source records attributed to Castellani, C. A..

10 recordsLinked to original sources

bioRxiv2025

Transcriptomic analysis of cells following decreased mitochondrial DNA-copy number reveals compensatory mechanisms in mitochondrial DNA replication and cellular energetics

Mitochondrial DNA copy number (mtDNA-CN) is a metric of mitochondrial function that has been associated with a variety of diseases including cardiovascular disease and all-cause mortality. To investigate genes and pathways affected by mtDNA-CN variation, we perturbed HEK 293T cells with ethidium bromide to deplete mtDNA. Using RNASeq and methylation microarrays, we evaluated transcriptomic and methylomic changes in treated cell lines. We observed an 8-fold decrease in mtDNA-CN and compensatory shifts in mitochondrial transcription to support mtDNA replication. Nuclear transcriptomic and methylomic analysis highlighted changes in metabolic pathways, including oxidative phosphorylation and canonical glycolysis. Longitudinal analyses revealed that the identified genes and pathways have different response timing, with nuclear response lagging behind mitochondrial response. These findings further elucidate the mechanisms behind mtDNA maintenance and responses to cellular energetics as well as mitochondrial-nuclear crosstalk dynamics.

genetics↗

bioRxiv2025

The effect of fatty acid binding protein-3 exposure on endothelial transcriptomics

BackgroundFatty acid binding protein-3 (FABP3) is released in circulation following myocardial infarction and an increased level of circulatory FABP3 is also reported in peripheral artery disease patients exposing endothelial cells to higher level of FABP3. Recently, loss of endothelial FABP3 was shown to protect endothelial cells against inflammation-induced endothelial dysfunction, however, the effect of FABP3-exposure on endothelial cells is unknown. Accordingly, to study the effect of FABP3-exposure on endothelial cells we performed transcriptomic profiling following recombinant human FABP3 (rhFABP3) treatment to endothelial cells. MethodCultured human endothelial cells were treated with either vehicle or rhFABP3 (50 ng/ml, 6h) and then RNA-sequencing was performed. Gene expression analysis followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to identify differentially expressed genes and affected cellular functions and pathways. ResultsDifferential gene expression analysis revealed kinesin family member 26b (KIF26B) as the most upregulated and survival of motor neuron 2 (SMN2) as the most downregulated genes in rhFABP3-treated in comparison to vehicle-treated endothelial cells. Most of the differentially expressed genes were associated with endothelial cell motility, immune response, and angiogenesis. GO and KEGG analyses indicated that rhFABP3 exposure impacts several crucial pathways, prominently "Regulation of leukocyte mediated cytotoxicity" and "Natural killer cell mediated cytotoxicity", suggesting its involvement in endothelial cell physiology and response mechanisms to cardiovascular stress. ConclusionThis is the first study evaluating rhFABP3-induced transcriptomics in human endothelial cells. Our data reveal novel genes and pathways affected by FABP3-exposure to endothelial cells. Further research is necessary to validate these findings and fully understand FABP3s role in endothelial biology and in cardiovascular diseases like myocardial infarction and peripheral artery disease.

biochemistry↗

bioRxiv2024

EpigeneticAgePipeline: an R package for comprehensive assessment of epigenetic age metrics from methylation microarrays

Epigenetic age is a biological age estimate based on nuclear DNA methylation patterns. Epigenetic clocks measure biological age by analyzing predictable changes in DNA methylation sites associated with aging. This study introduces EpigeneticAgePipeline, an R package that streamlines the estimation of epigenetic age metrics including Horvath, Horvath skin and blood, Hannum, PhenoAge/Levine, GrimAge (V1, and V2), and DunedinPACE plus additional acceleration metrics based on all other clocks. Quality control includes detection p-value filtering (sample- and probe-level), bead-count thresholds, and Illumina quality control intensity checks. EpigeneticAgePipeline supports Illumina Infinium methylation microarrays (HumanMethylation27, HumanMethylation450, HumanMethylationEPIC/EPICv2, and Human Methylation Screening Array). It offers functionalities including data preprocessing, normalization, cell count imputation, residual generation accounting for principal components and batch effects, and extensive visualizations for improved interpretability. Validation was performed using GEO dataset GSE237561, confirming the accuracy of the pipeline. EpigeneticAgePipeline provides an integrated workflow from raw data to advanced statistical analyses and visualizations, improving usability over existing tools. In addition to the traditional clocks mentioned, the package also integrates a set of additional epigenetic age clocks (PedBE, Wu, TL, BLUP, and EN). Future updates will include emerging epigenetic age measures to maintain relevance in this evolving field.

bioinformatics↗

bioRxiv2024

Mitochondrial DNA copy number reduction via in vitro TFAM knockout remodels the nuclear epigenome and transcriptome

Mitochondrial DNA copy number (mtDNA-CN) is associated with several age-related chronic diseases and is a predictor of all-cause mortality. Here, we examine site-specific differential nuclear DNA (nDNA) methylation and differential gene expression resulting from in vitro reduction of mtDNA-CN to uncover shared genes and biological pathways mediating the effect of mtDNA-CN on disease. Epigenome and transcriptome profiles were generated for three independent human embryonic kidney (HEK293T) cell lines harbouring a mitochondrial transcription factor A (TFAM) heterozygous knockout generated via CRISPR-Cas9, and matched control lines. We identified 4,242 differentially methylated sites, 228 differentially methylated regions, and 179 differentially expressed genes associated with mtDNA-CN. Integrated analysis uncovered 381 Gene-CpG pairs. GABAA receptor genes and related pathways, the neuroactive ligand receptor interaction pathway, ABCD1/2 gene activity, and cell signalling processes were overrepresented, providing insight into the underlying biological mechanisms facilitating these associations. We also report evidence implicating chromatin state regulatory mechanisms as modulators of mtDNA-CN effect on gene expression. We demonstrate that mitochondrial DNA variation signals to the nuclear DNA epigenome and transcriptome and may lead to nuclear remodelling relevant to development, aging, and complex disease.

genomics↗

bioRxiv2021

Genome-wide analysis of mitochondrial DNA copy number reveals multiple loci implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation

Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses were used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 x 10-15) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 x 10-8) and mtDNA replication (p = 1.2 x 10-7). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 x 10-4).

genetics↗

bioRxiv2020

Blood-derived mitochondrial DNA copy number is associated with gene expression across multiple tissues and is predictive for incident neurodegenerative disease

BackgroundMitochondrial DNA copy number (mtDNA-CN) can be used as a proxy for mitochondrial function and is associated with a number of aging-related diseases. However, it is unclear how mtDNA-CN measured in blood can reflect risk for diseases that primarily manifest in other tissues. Using the Genotype-Tissue Expression Project, we interrogated the relationships between mtDNA-CN measured in whole blood and gene expression from whole blood as well as 47 additional tissues. ResultsWe evaluated associations between blood-derived mtDNA-CN and gene expression in whole blood for 418 individuals, correcting for known confounders and surrogate variables derived from RNA-sequencing. Using a permutation-derived cutoff (p<2.70e-6), mtDNA-CN was significantly associated with expression for 721 genes in whole blood, including nuclear genes that are required for mitochondrial DNA replication. Significantly enriched pathways included splicing (p=1.03e-8) and ubiquitin-mediated proteolysis (p=2.4e-10). Genes with target sequences for the mitochondrial transcription factor NRF1 were also enriched (p=1.76e-35). In non-blood tissues, there were more significantly associated genes than expected in 30 out of 47 tested tissues, suggesting that global gene expression in those tissues is correlated with mtDNA-CN. Pathways that were associated in multiple tissues included RNA-binding, catalysis, and neurodegenerative disease. We evaluated the association between mtDNA-CN and incident neurodegenerative disease in an independent dataset, the UK Biobank, using a Cox proportional-hazards model. Higher mtDNA-CN was significantly associated with lower risk for incident neurodegenerative disease (HR=0.73, 95% CI= 0.66;0.90). ConclusionsThe observation that mtDNA-CN measured in whole blood is associated with gene expression in other tissues suggests that blood-derived mtDNA-CN can reflect metabolic health across multiple tissues. Key pathways in maintaining cellular homeostasis, including splicing, RNA binding, and catalytic genes were significantly associated with mtDNA-CN, reinforcing the importance of mitochondria in aging-related disease. As a specific example, genes involved in neurodegenerative disease were significantly enriched in multiple tissues. This finding, validated in a large independent cohort study showing an inverse association between mtDNA-CN and neurodegenerative disease, solidifies the link between blood-derived mtDNA-CN, altered gene expression in both blood and non-blood tissues, and aging-related disease.

genetics↗

bioRxiv2019

Mitochondrial DNA Copy Number and Incident Atrial Fibrillation

BackgroundMechanistic studies suggests that mitochondria DNA (mtDNA) dysfunction may be associated with increased risk of atrial fibrillation (AF). The association between mtDNA copy number (mtDNA-CN) and incident AF in the general population, however, remains unknown. MethodsWe conducted prospective analyses of 19,709 participants from the Atherosclerosis Risk in Communities Study (ARIC), the Multi-Ethnic Study of Atherosclerosis (MESA) and the Cardiovascular Health Study (CHS). mtDNA-CN from peripheral blood was calculated from probe intensities on the Affymetrix Genome-Wide Human single nucleotide polymorphisms (SNP) Array 6.0 in ARIC and MESA, and from multiplexed real time quantitative polymerase chain reaction (qPCR) in CHS. Incident AF cases were identified through electrocardiograms, review of hospital discharge codes, Medicare claims, and death certificates. ResultsThe median follow-up time was 21.4 years in ARIC, 12.9 years in MESA and 11.0 years in CHS, during which 4,021 participants developed incident atrial fibrillation (1,761 in ARIC, 790 in MESA, and 1,470 in CHS). The fully-adjusted pooled hazard ratio for incident atrial fibrillation comparing the 1st to the 5th quintile of mitochondria DNA copy number was 1.13 (1.01, 1.27). The fully-adjusted pooled hazard ratio comparing the 10th vs the 90th percentile of mitochondria DNA copy number was 1.13 (1.04, 1.24). Dose-response spline analysis also showed an inverse association between mitochondria DNA copy number and hazard for atrial fibrillation for all three cohorts. These associations were consistent across subgroups. ConclusionsMitochondria DNA copy number was inversely associated with the risk of AF independent of traditional cardiovascular risk factors. These findings implicate mitochondria DNA copy number as a novel risk factor for atrial fibrillation. Further research is warranted to understand the underlying mechanisms and to evaluate the role of mitochondria DNA copy number in the management of atrial fibrillation risk.

genetics↗

bioRxiv2019

Hippocampal transcriptome analysis following maternal separation implicates altered RNA processing in a mouse model of fetal alcohol spectrum disorder

Fetal alcohol spectrum disorders (FASD) are common, seen in 1-5% of the population in the United States and Canada. Children diagnosed with FASD are not likely to remain with their biological parents, facing early maternal separation and foster placements throughout childhood. We have modeled FASD in mice via prenatal alcohol exposure and further induce early life stress through maternal separation. We report an association between adult hippocampal gene expression and prenatal ethanol exposure followed by postnatal separation stress that is related to behavioral changes. Clustering of expression profiles through weighted gene co-expression network analysis (WGCNA) identifies a set of transcripts, module 19, associated with anxiety-like behavior (r = 0.79, p = 0.002) as well as treatment group (r = 0.68, p = 0.015). Genes in this module are overrepresented by genes involved in transcriptional regulation and other pathways related to neurodevelopment. Interestingly, one member of this module, Polr2a, polymerase (RNA) II (DNA directed) polypeptide A, is downregulated by the combination of prenatal ethanol and postnatal stress in an RNA-Seq experiment and qPCR validation (q = 2e-12, p = 0.004, respectively). Together, transcriptional control in the hippocampus is implicated as a potential underlying mechanism leading to anxiety-like behavior via environmental insults. Further research is required to elucidate the mechanism involved and use this insight towards early diagnosis and amelioration strategies involving children born with FASD.\n\nSUMMARY STATEMENTMouse hippocampal gene expression alterations following prenatal alcohol exposure and maternal separation are associated with behavioral deficits. Transcriptomic analysis implicates systems defect involving RNA processing, specifically including downregulation of Polr2a.

genomics↗

bioRxiv2019

Mitochondrial DNA Copy Number (mtDNA-CN) Can Influence Mortality and Cardiovascular Disease via Methylation of Nuclear DNA CpGs

BackgroundMitochondrial DNA copy number (mtDNA-CN) has been associated with a variety of aging-related diseases, including all-cause mortality. However, the mechanism by which mtDNA-CN influences disease is not currently understood. One such mechanism may be through regulation of nuclear gene expression via the modification of nuclear DNA (nDNA) methylation. MethodsTo investigate this hypothesis, we assessed the relationship between mtDNA-CN and nDNA methylation in 2,507 African American (AA) and European American (EA) participants from the Atherosclerosis Risk in Communities (ARIC) study. To validate our findings we assayed an additional 2,528 participants from the Cardiovascular Health Study (CHS) (N=533) and Framingham Heart Study (FHS) (N=1,995). We further assessed the effect of experimental modification of mtDNA-CN through knockout of TFAM, a regulator of mtDNA replication, via CRISPR-Cas9. ResultsThirty-four independent CpGs were associated with mtDNA-CN at genome-wide significance (P<5x10-8). Meta-analysis across all cohorts identified six mtDNA-CN associated CpGs at genome-wide significance (P<5x10-8). Additionally, over half of these CpGs were associated with phenotypes known to be associated with mtDNA-CN, including coronary heart disease, cardiovascular disease, and mortality. Experimental modification of mtDNA-CN demonstrated that modulation of mtDNA-CN directly drives changes in nDNA methylation and gene expression of specific CpGs and nearby transcripts. Strikingly, the neuroactive ligand receptor interaction KEGG pathway was found to be highly overrepresented in the ARIC cohort (P= 5.24x10-12), as well as the TFAM knockout methylation (P=4.41x10-4) and expression (P=4.30x10-4) studies. ConclusionsThese results demonstrate that changes in mtDNA-CN influence nDNA methylation at specific loci and result in differential expression of specific genes that may impact human health and disease via altered cell signaling.

genomics↗

bioRxiv2019

Evaluation of mitochondrial DNA copy number estimation techniques

Mitochondrial DNA copy number (mtDNA-CN), a measure of the number of mitochondrial genomes per cell, is a minimally invasive proxy measure for mitochondrial function and has been associated with several aging-related diseases. Although quantitative real-time PCR (qPCR) is the current gold standard method for measuring mtDNA-CN, mtDNA-CN can also be measured from genotyping microarray probe intensities and DNA sequencing read counts. To conduct a comprehensive examination on the performance of these methods, we use known mtDNA-CN correlates (age, sex, white blood cell count, Duffy locus genotype, incident cardiovascular disease) to evaluate mtDNA-CN calculated from qPCR, two microarray platforms, as well as whole genome (WGS) and whole exome sequence (WES) data across 1,085 participants from the Atherosclerosis Risk in Communities (ARIC) study and 3,489 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). We observe mtDNA-CN derived from WGS data is significantly more associated with known correlates compared to all other methods (p < 0.001). Additionally, mtDNA-CN measured from WGS is on average more significantly associated with traits by 5.6 orders of magnitude and has effect size estimates 5.8 times more extreme than the current gold standard of qPCR. We further investigated the role of DNA extraction method on mtDNA-CN estimate reproducibility and found mtDNA-CN estimated from cell lysate is significantly less variable than traditional phenol-chloroform-isoamyl alcohol (p = 5.44x10-4) and silica-based column selection (p = 2.82x10-7). In conclusion, we recommend the field moves towards more accurate methods for mtDNA-CN, as well as re-analyze trait associations as more WGS data becomes available from larger initiatives such as TOPMed.

genetics↗