Week 7

The motor neuron results are in!

Illustration by Henris Kas with permission

Last week we discussed an upcoming motor neuron experiment. We treated our mature, healthy motor neurons with 4 potential compound inhibitors (with and without a known EIF2α kinase (compound)), to assess whether the compound inhibitors can mitigate the level of cell death associated with EIF2α phosphorylation. We have compared these results against mature, healthy motor neurons + a known compound inhibitor (K-control) to evaluate the efficacy of the 4 potential compound inhibitors.

Instead of just adding a CellTox Green assay (measuring cell death), we decided to add a CellTiter-Glo assay at the final time point (96h) to measure cell viability.

For privacy reasons, the 4 potential compound inhibitors will be labelled:

• X

• Y

• Z

• W

We found a cell viability value (using CellTiter-Glo) and a cell toxicity value for each of the following treatments:

1. Motor neurons

2. Motor neurons + a known EIF2a kinase + known compound inhibitor (K-control line)

3. Motor neurons + a known EIF2a kinase + 1 of the 4 compound inhibitors

Treatment 1s signal is represented by numeral 100 on the y-axis

Treatment 2s signal is represented by the K-control line

Treatment 3s signal is represented by the coloured dots

Unfortunately, when the cells were treated with the known EIF2α kinase + 1 of 4 compound inhibitors for up to 96h, we were unable to obtain a CellTiter Glo signal. We believe that the kinase is causing a significant impact on either cell health, or on the normal cell metabolism, and by the end of the 96h, the cells are completely depleted of ATP. As a consequence, we are unable to comment on whether the compounds are neuroprotective.

Cell toxicity graphs for each potential compound inhibitor (X, Y, Z and W). K-control line = known compound inhibitor.

Cell viability graphs for each potential compound inhibitor (X, Y, Z and W). K-control line = known compound inhibitor.

We can deduce from both graphs that compounds X, Y and Z are well tolerated over the dose range. Compound W seems to induce cell death/reduce cell viability in a dose-dependent manner. Therefore, compound W is not an appropriate drug to continue further testing with as it is contributing to neurotoxicity.

We are now going to culture a fresh batch of healthy motor neurons and measure total EIF2α phosphorylation levels after the addition of a known EIF2α kinase and compounds X, Y, and Z. Below are brightfield images of our healthy motor neurons at days 3 and 6.

Brightfield images of astrocytes at days 3 and 6. Scale bar = 200 um.

Upcoming astrocyte experiment:

As discussed last week, we repeated our astrocyte experiment to determine the optimal dose and time for BX559 to induce an inflammatory response in our astrocytes. After testing multiple different doses and timepoints, we concluded that 24 hours at dosage point 0.8 or 1.6ng/ml was the optimum. Now, we are going to mature a fresh batch of astrocytes, induce an inflammatory response, and test the effects of the BX559 inhibitors that GenieUs identified in their in-silico screening on the inflammatory response. Brightfield images of our astrocytes at passage 2 may be seen below. As usual, we will wait until they reach passage 4 before we use them for the experimental work.

Brightfield images of astrocytes at passage 2. Scale bar = 200 um.

#12wdc #ALS #MND #drugdiscovery #Ulysses