Sustained synchronized neuronal network activity in a human astrocyte co-culture system

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer's disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal) function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases

Molecular characterisation of the N-methyl-D-aspartate receptor expressed in mammalian cells

L-Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system, mediating its effects via interaction with glutamate receptors. Several pharmacological subclasses of the glutamate receptor have been identified, including the N-methyl-D-aspartate (NMDA) receptor. Molecular cloning has recently identified five genes encoding the NMDA receptor subunits, NMDAR1 and NMDAR2A-D. In this thesis, work is described in which conditions for the optimal transient expression of homo- and heteromeric NMDA receptors in mammalian cells were established, thus providing a model system for structure-function studies of this important brain protein. Thus, the cDNAs encoding the NMDAR1-1a and NMDAR2A subunits were subcloned into the mammalian expression vector, pCIS. The resultant constructs were transfected alone or in combination in human embryonic kidney (HEK) 293 cells by the calcium phosphate method. Co-transfection studies resulted in cell death, which was prevented by inclusion of NMDA receptor antagonists in the cell culture medium post-transfection. A study was made of the efficacy for the prevention against cell death for a series of different NMDA receptor antagonists, which included DL-2-amino-5 phosphonovalerate (AP5), 5,7-dichlorokynurenic acid (DKA) and Mg2+. The percentage cell death was quantified by either Trypan Blue exclusion or the CytoTox 96TM assay system. The combination of AP5 and DKA gave optimal protection with no significant difference between cotransfected and control samples. Site-directed in vitro mutagenesis was used to generate point mutations of the NMDAR1-1a subunit. Co-transfection studies with the wild-type and mutant NMDA receptor cDNAs were carried out and the effect of the mutations on cell viability quantified. Most notably, it was found that the mutation (N598Q), previously shown to reduce the Ca2+ permeability of cloned receptors, significantly reduced cell toxicity, thus providing evidence for the role of Ca2+ in NMDA-receptor mediated cytotoxic mechanisms

135PAM1 shifts the concentration response curves of Relaxin-3_NH2 and R3/I5_NH2.

<p>HEK-293 cells coexpressing RXFP3 and G_qI5 were incubated with fixed concentrations of 135PAM1 (0, 0.2, 2 and 20 µM) 10 min before the addition of increasing concentrations of Relaxin-3_NH2 (A), R3I5_NH2 (B), Relaxin-3_OH (C) or R3I5_OH (D).</p

135PAM1 lacks affinity at the orthosteric binding site of RXFP3 receptor.

<p>135PAM1 did not displace [125I] R3/I5_NH2 at concentrations of up to 20 µM, but instead increased total binding. R3/I5_NH2 displaced the tracer with a pIC₅₀ of 8.76 (8.91 to 8.61).</p

Structure of 135PAM1 (3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea).

<p>Structure of 135PAM1 (3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea).</p

Using Human iPSC-Derived Neurons to Model TAU Aggregation

<div><p>Alzheimer’s disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.</p></div

K18 seeding induces TAU aggregation and hyperphosphorylation in human TAU-P301L neurons.

<p>Optimal timelines are shown for AAV transduction, 96w final plating, K18 seeding and final assay. <b>(A, B)</b> AlphaLISA data show that K18 seeding induces an increase in both hTAU10 (P<0,001; A) and AT8 (P<0,001; B) TAU aggregation assays. <b>(C, D)</b> AlphaLISA results demonstrate around 2-fold increase in TAU phosphorylation (AT8/hTAU10; P<0,001; C) while total TAU levels remain unchanged (HT7/hTAU10; P = NS; D). <b>(E)</b> CellTiter-Glo® results showing that general cell health is unaffected after K18 addition (P = NS). For all assays in <b>(A-E)</b>: *** P<0,001; 1-way ANOVA with Tukey’s post hoc; n≥3 independent experiments. <b>(F)</b> Representative blot of Native PAGE followed by Western blot showing two monomeric HT7-positive TAU bands (around 66kDa) in all conditions. Notably, non-migrated HT7-positive TAU proteins (>1236kDa) in K18-seeded samples suggest the presence of TAU aggregates. <b>(G, H)</b> Representative Western blots after Sarkosyl extraction showing soluble (S) and insoluble (IS) fractions after blotting with antibodies against total TAU (HT7; G) and hyperphosphorylated TAU (AT8; H). Aggregates are only present in the insoluble pellet after addition of 6nM or 50nM of K18 fibrils. Note the presence of monomeric 3R and 4R TAU protein in the soluble fraction. <b>(I)</b> Immunostaining for AT8 and neuronal HuC/D after 1%Triton/PFA fixation, to remove monomeric TAU, reveals AT8-positive neurons after K18 seeding. Scale bar = 25μm.</p