We're getting ready for more experiments!
As mentioned last week, we did not achieve the results we were expecting for our astrocyte experiment. We are unable to determine the optimal dose and time for BX559 to induce an inflammatory response in our astrocytes. We believe that the time points we tested – 48hr and 72h – was too long of a period to allow the astrocytes to sit with BX559, resulting in an inconclusive dose response.
This is science. You do not always get the results you want. Sometimes experiments need to be repeated and tweaked to achieve the desired results. You must be willing to deal with the curveballs. The 12wdc team strives for excellence and so this week, we have begun a repeat of this experiment.
Again, we must mature our astrocytes. They were thawed in T25 flasks in astrocyte medium. They will undergo a maturation process until they reach their mature structure (passage 4), with media changing every 2-3 days. Below are the brightfield images of our astrocytes at passage 2 and 3.
Upcoming motor neuron experiment:
As discussed in week 2, a proportion of ALS cases are associated with TDP-43 mutations. eIF2α phosphorylation and TDP-43 mediated toxicity are strongly related to each other. Therefore, high eIF2α phosphorylation levels may result in cell death and induce an ALS phenotype. It has been discovered that eIF2α inhibitors are able to mitigate the effects of TDP-43 toxicity.
In week 2, we tested the effective eIF2α phosphorylation levels of 4 different motor neuron groups.
ALS motor neurons
Healthy motor neurons
Healthy motor neurons + solvent control
Healthy motor neurons + compound (a known eIF2α kinase)
We did this to confirm that healthy motor neurons + compound (a known eIF2α kinase) had the highest eIF2α phosphorylation levels. We did in fact find this (refer to graph 1 in week 2).
Now that we have confirmed that this is the best model to proceed with, we will mature another culture of healthy motor neurons, treat them with the compound as well as 4 different compound inhibitors that GenieUs identified in their in silico screening. We hope to see that some, if not all of the compound inhibitors are able to reduce eIF2α phosphorylation levels and hence, reduce cell death. This would suggest that the inhibitors may be able to protect motor neurons and reverse the ALS disease phenotype.
Again, our motor neurons will undergo a maturation process for 10-14 days, with media changing every 2-3 days. They are currently at day 1 after thawing. Brightfield images of the maturation process will be available next week.