Week 8

EIF2α phosphorylation results are in!

As discussed last week, we treated our healthy motor neurons with 4 compound inhibitors (X, Y, Z, W) (with and without a known EIF2α kinase (compound)) and assessed cell viability and cell toxicity levels.

This week, we tested total EIF2α phosphorylation levels after the addition of 1 of 4 compound inhibitors (with and without a known EIF2α kinase).

Below are brightfield images of our healthy motor neurons at day 10.

Brightfield image of healthy motor neurons at day 10. Scale bar = 200 um.

As usual, on day 10 post seeding, motor neurons are assessed for the expression of two specific neuronal markers.

1. Blue – Nuclei

2. Red – Beta III tubulin (S100b)

3. Choline acetyltransferase (NDRG2)

For descriptions of each of these markers, please refer to week 6s post.

Immunofluorescence images of healthy motor neurons 10 days post-seeding. Scale bar = 200 um (zoom out) and 50 um (zoom in).

Results: Total EIF2a phosphorylation levels

In the data below, the graphs on left indicate the total EIF2a phosphorylation levels of:

· Healthy motor neurons + 1 of 4 compound inhibitors

The K control line represents the value obtained when the cells were treated with a known compound inhibitor

The graphs on the right indicate the total EIF2a phosphorylation levels of:

· Healthy motor neurons + 1 of 4 compound inhibitors + compound

The K control line represents the value obtained when the cells were treated with a known compound inhibitor

Total EIF2a phosphorylation levels. (left) healthy motor neurons + 1 of 4 compound inhibitors. (right) healthy motor neurons + 1 of 4 compound inhibitors + compound

What can we conclude from the data?

• The compound significantly induced EIF2α phosphorylation levels.

• Our assumptions last week of compound W contributing to neurotoxicity may be confirmed.

• We can see compounds X, Y, and Z causing slight reductions in EIF2α phosphorylation levels, in particular in the presence of the compound, however, the effect is not dramatic.

• It would be interesting to test increasing concentrations (up to 30-50 μm) to see if we can increase the neuroprotective effects of compounds X, Y, and Z (without causing neurotoxicity).

• It would be interesting to test longer incubation times (48-72 h), provided we can overcome the known compound-related long-term toxicity. (e.g., washing out the compound after 24 h).

We are now maturing another batch of astrocytes and motor neurons for next week’s experiment. We will be treating our astrocytes with BX559 to induce an inflammatory response. We will know an inflammatory response has been induced when the supernatant contains cytokines – TNFa, IL-1B, IL-8 and IL-6.

We are then going to test the effects of 4 different BX559 inhibitors that GenieUs identified in their in-silico screening.

Then, we will treat mature, healthy motor neurons with the astrocyte conditioned media to induce an inflammatory response. The motor neurons will be treated with the 4 different BX559 inhibitors and the effects will be assessed.

Below are brightfield images of the astrocytes at passage 3 and the motor neurons at day 3 and 6.

Brightfield images of astrocytes at passage 3. Scale bar = 200 um.

Brightfield images of healthy motor neurons - days 3 and 6. Scale bar = 200um

Stay tuned as we are approaching the final stretch of the 12-week challenge. More fascinating results coming soon!

#12wdc #ALS #MND #drugdiscovery #Ulysses