DNA Methyltransferase 1 (DNMT1) Shapes Neuronal Activity of Human iPSC-Derived Glutamatergic Cortical Neurons

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.

A mitochondria cluster at the proximal axon initial segment controls axodendritic TAU trafficking in rodent primary and human iPSC-derived neurons

“…We thus tested whether calcium homeostasis is perturbed in our paradigms (rather low and non-cell-toxic concentrations of respiratory chain inhibitors) sufficient to induce TAU missorting. We consequently investigated cytosolic calcium levels via live-cell-imaging with a calcium-sensitive dye, Fluo-4, similar as before [ 22 ]. In agreement with previous experiments showing that mitochondrial impairment leads to increased cytosolic calcium levels (as calcium import into mitochondria requires a proper proton gradient, for review see [ 33 ]) and our initial findings that TAU missorting in response to mitochondrial impairment occurs within hours (Figs.…”

“…After four days, the same amount of neuronal maintenance medium (Neurobasal media (TFS), 1 × Antibiotic-/Antimycotic solution (TFS), 1 × NS21 (Panbiotech) and AraC (Sigma-Aldrich)) was added and cells were kept in culture as long as indicated. Human WTC11 iPSCs carrying a doxycycline-inducible Neurogenin2 (Ngn2) transgene were differentiated into neurons as previously described and characterized [ 22 , 23 ].…”

The TAU isoform 1N4R restores vulnerability of MAPT knockout human iPSC-derived neurons to Amyloid beta-induced neuronal dysfunction

“…To monitor MAPT mRNA expression, iPSCs were differentiated into glutamatergic neurons (iNeurons) for up to 14 days, using doxycycline-induced Ngn2 expression as described before 31,32 . MAPT transcripts were ampli ed by qRT-PCR (Fig.…”

Maternal immune activation primes deficiencies in adult hippocampal neurogenesis

“…Importantly, reservations do exist as to whether hiPSCs are a suitable model to assess epigenetic effects due to major epigenetic remodelling involved in their generation ( Perrera and Martello, 2019 ). During differentiation, hiPSC-derived cells do undergo epigenetic remodelling ( Bachmann et al, 2021 , Su et al, 2021 ), and genome-wide DNA methylation patterns and gene expression patterns are preserved between neurons, derived from hiPSCs and human embryonic stem cells ( de Boni, 2018 ). This provides confidence that hiPSC-derived cells would undergo MIA-associated epigenetic changes in similar manner as seen in in vivo models, thus making them a potentially useful model to investigate this.…”