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

Identification of Z-Tyr-Ala-CHN 2, a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN 2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN 2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN 2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication

Plasmids for heavy metal resistance in Alcaligenes eutrophus CH34: Mechanisms and applications

Alcaligenes eutrophus CH34 is the main representative of a group of strongly related strains (mostly facultative chemolithotrophs) that are well adapted to environments containing high levels of heavy metals. It harbors the megaplasmids pMOL28 and pMOL30 which carry resistance determinants to Co2+, Ni2+, CrO42-, Hg2+, Tl+, Cd2+, Cu2+ and Zn2+. Among the best characterized determinants are the cnr operon (resistance to Co, Ni) an pMOL28 and the czc operon on pMOL30 (resistance to Co, Cd and Zn). Although the two systems reveal a significant degree of amino acid similarity in the structural genes, the regulation of the operons is different. The resistance mechanism in both cases is based on efflux. The efflux mechanism leads to a pH increase outside of the cytoplasmic membrane. Metals are sequestered from the external medium through the bioprecipitation of metal carbonates formed in the saturated zone around the cell. This latter phenomenon can be exploited in bioreactors designed to remove metals from effluents. The bacteria are immobilized on composite membranes in a continuous tubular membrane reactor (CTMR). The effluent continuously circulates through the intertubular space, while the external surface of the tubes is in contact with the growth medium. Metal crystals are eventually removed by the effluent stream and collected on a glass bead column. The system has been applied to effluents containing Cd, Zn, Co, Ni and Cu. By introducing catabolic plasmids involved in the aerobic degradation of PCBs and 2,4-D into metal-resistant A. eutrophus strains, the application range was widened to include effluents polluted with both organic and inorganic substances. Biosensors have been developed which are based on the fusion of genes induced by metals to a reporter system, the lux operon of Vibrio fischeri. Bacterial luciferases produce light through the oxidation of fatty aldehydes. The gene fusions are useful both for the study of regulatory genes and for the determination of heavy metal concentrations in the environment

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

Model validation: autophagy inducers reduce TAU hyperphosphorylation and aggregation in TAU-P301L neurons.

<p><b>(A)</b> CellTiter-Glo® data showing that rapamycin is not toxic (P = NS). <b>(B, C)</b> Rapamycin dose-dependently reduces hTAU10 (P<0,001; B) and AT8 aggregated TAU measured with AlphaLISA (P = 0,025 at 10 nM and P<0.001 at 1 μM; C) and compared to DMSO. <b>(D, E)</b> Also general TAU phosphorylation is reduced (AT8/hTAU10; P<0,001; D) to a similar extent as the reduction in total TAU (HT7/hTAU10; P<0,001; E). <b>(F)</b> CellTiter-Glo® results show that trehalose is highly toxic at 250 mM (P<0,001). <b>(G, H)</b> AlphaLISA results reveal that trehalose significantly reduces hTAU10 (P<0,001) and AT8 TAU aggregation levels <i>versus</i> control (P = 0,006 at 31,5 mM and P = 0,014 at 125 mM). <b>(I, J)</b> Only at 125 mM of trehalose, both phosphorylated TAU (P<0,001, I) and total TAU levels (P<0,001, J) are decreased. ***P<0,001; **P<0,01; *P<0,05; ^#P<0,001 due to toxicity; 1-way ANOVA with Dunnett’s post hoc; n≥3 independent experiments</p

Optimization of dynamic range of hTAU10 aggregation AlphaLISA.

<p><b>(A)</b> K18 sonication significantly improves seeding potency (P<0.001, n≥3 independent experiments). Seeds were added at week 1. In all further experiments, sonicated K18 is used. <b>(B)</b> K18 seeding does not induce aggregation in control (no virus) and WT virus (AAV WT TAU 2N4R) transduced neurons (P = NS; n≥3 independent experiments). <b>(C)</b> Weekly repeated K18 seeding of K18 significantly increases the dynamic range (P<0.001 for both 1xF <i>versus</i> 2xF (wk 1+2) and 1xF <i>versus</i> 3xF (wk1+wk2+wk3); n≥3 independent experiments). <b>(D)</b> Finally, also the timing of seeding has an effect on the aggregation potency. Addition of K18 at week 2 (wk2) significantly increases TAU aggregation compared to week 1 (P<0.001, n≥3 independent experiments) while addition of fibrils at week 3 shows significantly less aggregation (P<0.001; n≥3 independent experiments) probably due to the shorter (1 week) K18 incubation period before AlphaLISA. ***P<0,001; 2-way-ANOVA with Dunnett’s post hoc.</p

AlphaLISA optimizations on human brain extracts for total TAU, TAU aggregation and phosphorylation.

<p><b>(A, B)</b> AlphaLISA on 2 different AD brain extracts show high hTAU10 (A) and AT8 (B) TAU aggregation signals compared to control brain samples. <b>(C, D)</b> AT8/hTAU10 (C) AlphaLISA on these AD brain extracts reveals high levels of phosphorylated TAU compared to control brain samples while both AD and control brain extracts display high HT7/hTAU10 (D) levels. Decreasing signals with increasing dilutions suggest no hooking of the samples. Representative curve of 1 experiment with 2 technical replicates is shown as RFU (relative fluorescence units) ± SD. <b>(E, F)</b> Western Blot on soluble (S) and insoluble (IS) fractions of control and AD brain extracts after Sarkosyl extraction shows HT7-positive (E) and AT8-positive (F) bands only in the Sarkosyl insoluble pellets of both AD patients, confirming the presence of TAU aggregates. M represents Magic Marker (band sizes) and T represents TAU ladder with all 6 TAU isoforms. All experiments have been confirmed at least twice.</p

Identification of Z-Tyr-Ala-CHN 2, a Cathepsin L Inhibitor with Broad-Spectrum Cell-Specific Activity against Coronaviruses, including SARS-CoV-2.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN 2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN 2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN 2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication