Abstract
Epigenetic mechanisms are emerging key players for the regulation of brain function, synaptic activity, and the formation of neuronal engrams in health and disease. As one important epigenetic mechanism of transcriptional control, DNA methylation was reported to distinctively modulate synaptic activity in excitatory and inhibitory cortical neurons in mice. Since DNA methylation signatures are responsive to neuronal activity, DNA methylation seems to contribute to the neuron’s capacity to adapt to and integrate changing activity patterns, being crucial for the plasticity and functionality of neuronal circuits. Since most studies addressing the role of DNA methylation in the regulation of synaptic function were conducted in mice or murine neurons, we here asked whether this functional implication applies to human neurons as well. To this end, we performed calcium imaging in human induced pluripotent stem cell (iPSC)-derived excitatory cortical neurons forming synaptic contacts and neuronal networks in vitro. Treatment with DNMT1 siRNA that diminishs the expression of the DNA (cytosine-5)-methyltransferase 1 (DNMT1) was conducted to investigate the functional relevance of DNMT1 as one of the main enzymes executing DNA methylations in the context of neuronal activity modulation. We observed a lowered proportion of actively firing neurons upon DNMT1-knockdown in these iPSC-derived excitatory neurons, pointing to a correlation of DNMT1-activity and synaptic transmission. Thus, our experiments suggest that DNMT1 decreases synaptic activity of human glutamatergic neurons and underline the relevance of epigenetic regulation of synaptic function also in human excitatory neurons.
Highlights
All learned behavior and the retrieval of memories, cognition, sensory perception, and motor control depend on the formation of stable synaptic connections to form sustained neuronal ensembles
To approach the functional implication of DNA (cytosine-5)-methyltransferase 1 (DNMT1) in activity regulation of human cortical neurons, we made use of human induced pluripotent stem cells and their potential to differentiate into functional neurons
After having tested different available neuronal differentiation protocols, we settled for a coculture system with murine astrocytes (Figure 1b) that resulted in a higher percentage of neurons showing reliable neuronal activity as assessed by calcium imaging (Supplementary Files A1 and A2)
Summary
All learned behavior and the retrieval of memories, cognition, sensory perception, and motor control depend on the formation of stable synaptic connections to form sustained neuronal ensembles. Since neurons are required to develop distinct gene expression profiles to enforce their role in neuronal circuits and memory engrams [2,3], epigenetic mechanisms recently emerged as new examination targets in the regulation of synaptic function and plasticity Their dynamic nature and responsiveness to altered neuronal activity [4,5,6] could explain the translation of external stimuli, e.g., as presented during learning, into changed gene expression that underlies structural and functional synaptic adaptations. While enzymes of the DNA methyltransferase (DNMT) family catalyze cytosine methylation, active demethylation is executed via oxidation by ten-eleven translocation (TET) proteins and subsequent iterative oxidation and base excision repair [9,10,11] These mechanisms enable dynamic reconfigurations of DNA methylation signatures, observed during and being proposed to be critical for neuronal differentiation and maturation [12,13,14,15]
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