Journal Article

Neuronal methylome reveals CREB-associated neuro-axonal impairment in multiple sclerosis

Kramarova, Tatiana, Adzemovic, Milena Z., Tegnér, Jesper, Piket, Eliane, Needhamsen, Maria, Jagodic, Maja, Ewing, Ewoud, Gomez-Cabrero, David, Piehl, Fredrik, Kular, Lara, Beck, Stephan, Brundin, Lou

2019

Overview

Background Due to limited access to brain tissue, the precise mechanisms underlying neuro-axonal dysfunction in neurological disorders such as multiple sclerosis (MS) are largely unknown. In that context, profiling DNA methylation, which is a stable and cell type-specific regulatory epigenetic mark of genome activity, offers a unique opportunity to characterize the molecular mechanisms underpinning brain pathology in situ. We examined DNA methylation patterns of neuronal nuclei isolated from post-mortem brain tissue to infer processes that occur in neurons of MS patients. Results We isolated subcortical neuronal nuclei from post-mortem white matter tissue of MS patients and non-neurological controls using flow cytometry. We examined bulk DNA methylation changes (total n  = 29) and further disentangled true DNA methylation (5mC) from neuron-specific DNA hydroxymethylation (5hmC) ( n  = 17), using Illumina Infinium 450K arrays. We performed neuronal sub-type deconvolution using glutamate and GABA methylation profiles to further reduce neuronal sample heterogeneity. In total, we identified 2811 and 1534 significant (genome-wide adjusted P value < 0.05) differentially methylated and hydroxymethylated positions between MS patients and controls. We found striking hypo-5mC and hyper-5hmC changes occurring mainly within gene bodies, which correlated with reduced transcriptional activity, assessed using published RNAseq data from bulk brain tissue of MS patients and controls. Pathway analyses of the two cohorts implicated dysregulation of genes involved in axonal guidance and synaptic plasticity, with meta-analysis confirming CREB signalling as the most highly enriched pathway underlying these processes. We functionally investigated DNA methylation changes of CREB signalling-related genes by immunohistofluoresence of phosphorylated CREB in neurons from brain sections of a subcohort of MS patients and controls ( n  = 15). Notably, DNA methylation changes associated with a reduction of CREB activity in white matter neurons of MS patients compared to controls. Conclusions Our data demonstrate that investigating 5mC and 5hmC modifications separately allows the discovery of a substantial fraction of changes occurring in neurons, which can escape traditional bisulfite-based DNA methylation analysis. Collectively, our findings indicate that neurons of MS patients acquire sustained hypo-5mC and hyper-5hmC, which may impair CREB-mediated neuro-axonal integrity, in turn relating to clinical symptoms.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: 5-Methylcytosine - analogs & derivatives / 5-Methylcytosine - metabolism / Adult / Aged / Aged, 80 and over / Analysis / Autopsy / Axon guidance / Axonal guidance / Axonal plasticity / Bioinformatics / Biomedical and Life Sciences / Biomedicine / Biotechnology industries / Bisulfite / Cadaver / Case-Control Studies / Criminal investigation / Cyclic AMP response element-binding protein / Cyclic AMP Response Element-Binding Protein - metabolism / Deoxyribonucleic acid / DNA / DNA fingerprinting / DNA hydroxymethylation / DNA Methylation / Down-Regulation / Epigenesis, Genetic / Epigenetic inheritance / Epigenetics / Female / Flow cytometry / GABA / Gene Function / Genes / Genetic research / Genomes / Genomics / Glutamate / High-Throughput Nucleotide Sequencing / Human Genetics / Humans / Male / Medical research / Methylation / Middle Aged / Molecular modelling / Multiple sclerosis / Multiple Sclerosis - genetics / Multiple Sclerosis - metabolism / Neurological diseases / Neurology and Psychiatry Epigenetics / Neurons / Neurons - chemistry / Neurons - cytology / Pathology / Phosphorylation / RNA sequencing / Sequence Analysis, DNA / Signal Transduction / Substantia alba / Sulfites / Synaptic plasticity / Transcription / Transcription (Genetics) / White Matter - chemistry / White Matter - cytology / γ-Aminobutyric acid