WNK3 Maintains the GABAergic Inhibitory Tone, Synaptic Excitation and Neuronal Excitability via Regulation of KCC2 Cotransporter in Mature Neurons

10.3389/fnmol.2021.762142Frontiers in Molecular Neuroscience1476214

An optogenetic approach for assessing formation of neuronal connections in a co-culture system

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.Published versio

Choline Ameliorates Disease Phenotypes in Human iPSC Models of Rett Syndrome

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene account for approximately 95 % of all RTT cases. To model RTT in vitro, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of two RTT patients with different mutations (MECP2R306C and MECP21155Δ32) in their MECP2 gene. We found that these iPSCs were capable of differentiating into functional neurons. Compared to control neurons, the RTT iPSC-derived cells had reduced soma size and a decreased amount of synaptic input, evident both as fewer Synapsin 1-positive puncta and a lower frequency of spontaneous excitatory postsynaptic currents. Supplementation of the culture media with choline rescued all of these defects. Choline supplementation may act through changes in the expression of choline acetyltransferase, an important enzyme in cholinergic signaling, and also through alterations in the lipid metabolite profiles of the RTT neurons. Our study elucidates the possible mechanistic pathways for the effect of choline on human RTT cell models, thereby illustrating the potential for using choline as a nutraceutical to treat RTT.NRF (Natl Research Foundation, S’pore)NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore)Accepted versio

Taurine induces proliferation of neural stem cells and synapse development in the developing mouse brain

10.1371/journal.pone.0042935PLoS ONE78

Extending neurites sense the depth of the underlying topography during neuronal differentiation and contact guidance

The topography of the extracellular microenvironment influences cell morphology, provides conduct guidance and directs cell differentiation. Aspect ratio and dimension of topography have been shown to affect cell behaviours, but the ability and mechanism of depth-sensing is not clearly understood. We showed that murine neural progenitor cells (mNPCs) can sense the depth of the micro-gratings. Neurite elongation, alignment and neuronal differentiation were observed to increase with grating depth. We proposed a mechanism for depth-sensing by growing neurites: filopodial adhesion in the growth cones favour elongation but the bending rigidity of the neurite cytoskeleton resists it. Thus, perpendicular extension on deeper grooves is unfavourable as neurites need to bend over a larger angle. A quantitative model was developed and its prediction of neurite growth on gratings fit well with the experimental data. The results indicated that mNPC fate can be directed by appropriately designed patterned surfaces

Human rett-derived neuronal progenitor cells in 3D graphene scaffold as an in vitro platform to study the effect of electrical stimulation on neuronal differentiation

Studies of electrical stimulation therapies for the treatment of neurological disorders, such as deep brain stimulation, have almost exclusively been performed using animal-models. However, because animal-models can only approximate human brain disorders, these studies should be supplemented with an in vitro human cell-culture based model to substantiate the results of animal-based studies and further investigate therapeutic benefit in humans. This study presents a novel approach to analyze the effect of electrical stimulation on the neurogenesis of patient-induced pluripotent stem cell (iPSC) derived neural progenitor cell (NPC) lines, in vitro using a 3D graphene scaffold system. The iPSC-derived hNPCs used to demonstrate the system were collected from patients with Rett syndrome, a debilitating neurodevelopmental disorder. The graphene scaffold readily supported both the wild-type and Rett NPCs. Electrical stimulation parameters were optimized to accommodate both wild-type and Rett cells. Increased cell maturation and improvements in cell morphology of the Rett cells was observed after electrical stimulation. The results of the pilot study of electrical stimulation to enhance Rett NPCs neurogenesis were promising and support further investigation of the therapy. Overall, this system provides a valuable tool to study electrical stimulation as a potential therapy for neurological disorders using patient-specific cells.NRF (Natl Research Foundation, S’pore)Accepted versio