Week 6

6 weeks down, 6 to go.

We are halfway through the 12-week challenge. It has been a rich and insightful journey so far. Let’s see what week 6 brings us.

As discussed, we have matured a fresh batch of astrocytes to repeat the BX559 experiment – determining the optimum dose and time for BX559 to mediate an inflammatory response. We have tested the 8- and 16-hour time points as we believed the 48- and 72-hour time points previously tested were too long for the incubation process.

Astrocytes:

Brightfield images of our astrocytes at passage 4 (Figure 1). As described in previous posts, the astrocytes are ready to be used once they reach passage 4.

Brightfield images of astrocytes at passage 4. Scale bar = 200 um.

However, before the experimental work can begin, we must confirm that differentiation has occurred successfully via immunofluorescence (IF) imaging (Figure 2). There are three stains in the IF images below:

1. Blue – Nuclei

2. Red – S100 calcium-binding protein B (S100b)

3. Green – N-Myc downstream-regulated gene 2 (NDRG2)

For descriptions of each of these markers, please look at the week 3s post.

IF images astrocytes. Scale bar = 50 um.

The IF images above tell us that differentiation has occurred successfully. We can now begin our BX559 experiment repeat. 8-hour and 16-hour response curves may be seen below.

8 hours:

The results are inconclusive. We do see a dose-dependent increase in the release of all 4 cytokines; however, there is no dosage that clearly induces a greater inflammatory response over the others. See 8-hour response graphs below:

Cytokine release in astrocytes 8 hours after introduction of BX559 at different concentrations.

16 hours:

Same as the 8-hour results. No dosage is clearly inducing a greater inflammatory response over the others. See 16-hour response graphs below:

Cytokine release in astrocytes 16 hours after introduction of BX559 at different concentrations.

After testing 9 different dosages at 8-, 16-, 24-, 48-, and 72-hour time points, the 12wdc team have concluded that the 24-hour time point (tested in week 4) at dose 0.8 or 1.6 is the optimum for BX559 to mediate an inflammatory response (see 24-hour response graphs below):

Cytokine release in astrocytes 24 hours after introduction of BX559 at different concentrations.

It is a bit strange that we aren’t getting any clear-cut results, but at least we can be sure that the astrocytes are secreting their inflammatory markers above background level in response to BX559. We will now proceed with the experiment and test whether our BX559 inhibitors can reduce the inflammatory response at 0.8 or 1.6ng/ml 24 hours post addition (next week).

Motor neuron maturation:

As mentioned last week, we have matured a new culture of motor neurons. Brightfield images of our motor neurons at days 3, 6, and 9 may be seen below:

Brightfield images of healthy motor neurons - days 3, 6, and 9. Scale bar = 200um

On day 9 post-seeding, IF imaging was performed to assess the expression of three specific neuronal markers.

1. Blue – Nuclei

2. Red – Beta III tubulin (S100b) – one of two structural components that form the neuronal microtubule network.

3. Green – Choline acetyltransferase (NDRG2) – An enzyme involved in synthesising acetylcholine, the neurotransmitter that regulates signal transduction at the neuromuscular junction.

Please see IF images below:

IF images of healthy motor neurons 9 days post-seeding. Scale bar = 200 um (zoom out) and 50 um (zoom in).

Motor neuron experiment details:

eIF2α phosphorylation induces an ALS phenotype. We will treat our mature, healthy motor neurons with a known eIF2α kinase (compound) and 4 potential compound inhibitors, to assess whether the potential compound inhibitors can mitigate the level of cell death associated with eIF2α phosphorylation. We will be adding a dye (CellTox Green Assay) that fluoresces when it binds to DNA. In healthy cells, this dye cannot permeate the cell membrane and bind to DNA in the nucleus, meaning that it will not fluoresce. However, during cell death, the cell membrane becomes disrupted, allowing the dye to enter the cells and give off a signal proportional to the amount of cell death. We will be taking fluorescence readings every 24h for a total of 96h to measure cell death in response to the drugs.

We will be comparing the results of the 4 potential compound inhibitors to the results of a known compound inhibitor. The comparison will show us how effective the potential compound inhibitors are at mitigating levels of cell death.

We will have the following setups:

Non-treated cells (to ensure the compound inhibitors aren't neurotoxic themselves):

1. Healthy ALS cells

2. Healthy ALS cells + known compound inhibitor

3. Healthy ALS cells + 1 of 4 potential compound inhibitors

Treated cells (to assess whether the compound inhibitors can mitigate cell death levels):

1. Healthy ALS cells

2. Healthy ALS cells + compound + known compound inhibitor

3. Healthy ALS cells + compound + 1 of 4 potential compound inhibitors

A lot is happening in the challenge, and we’re getting close to revealing key results. Keep up to date at 12wdc.com to make sure you don’t miss out!

#12wdc #ALS #MND #drugdiscovery #Ulysses