Week 10

Only 2 weeks remain!

We are nearing the end of the 12-week challenge. We hope that you guys are enjoying the project as much as we are. The work has taught us some valuable lessons, especially on how to handle the curveballs that cell/molecular biological studies can throw at any given time. Besides executing the project to a high level, we aimed to give the public real-life insight into the workings of a scientific study whilst making it interactive and easy to understand.


Without further ado, let’s get into the Week 10 update. Last week, it was all about maturing for the upcoming experiment. This week, the experiment is complete. Let’s have a quick refresher. The experiment was split up into 2 parts.


Part 1: Astrocyte treatment

  • Treat astrocytes with BX559 (+/- BX559), or BX559 co-dosed with 4 different BX559 inhibitors (X, Y, Z, and W) that GenieUs identified in their in-silico screening.

  • After 24h, astrocyte supernatant tested for TNFα, IL-1β, IL-8, and IL-6 to assess levels of inflammation.


Part 2: Motor neuron treatment

  • Healthy motor neurons were treated with astrocyte conditioned media (+/- BX559, or BX559 co-dosed with 4 different BX559 inhibitors (X, Y, Z, and W)).

  • Cell viability was monitored after 72h.


Results:

  • Part 1

The astrocytes were treated with BX559 at a concentration of 1ng/ml. We observed no significant difference in inflammatory responses between the BX559 treated and untreated astrocytes. The graphs below show the raw values of BX559 induced astrocyte inflammation.

Graphs illustrating inflammation of BX559-treated astrocytes vs non-treated astrocytes

We ran controls to ensure our techniques were correct and that there was no issue with the astrocytes themselves. The control group were healthy astrocytes treated with lipopolysaccharide (LPS). LPS is the major component of the outer membrane of gram-negative bacteria. It is known to be toxic and has shown an ability to induce a very strong inflammatory response.


We did observe a high inflammatory response in the astrocytes after the addition of LPS – indicated by the release of the 4 cytokines. Additionally, the raw values were much higher than we observed for BX559 suggesting that either BX559 is incapable of producing a strong inflammatory response, or BX559 is unable to penetrate the cells to produce an inflammatory response. The graphs below show raw values of LPS induced astrocyte inflammation.


Graphs illustrating inflammation of LPS-treated astrocytes vs non-treated astrocytes
  • Part 2

Healthy motor neurons were treated with the astrocyte conditioned media (after introduction of BX559) and 4 BX559 inhibitors (X, Y, Z, and W) where the viability of the motor neurons was assessed. However, we just established that BX559 did not produce a significant inflammatory response in our astrocytes and thus, we expected to observe no significant effects on cell viability when the conditioned media was combined with the healthy motor neurons.


The graphs below illustrate cell viability values that have been expressed as a percentage compared to non-treated motor neurons (no BX559, no compounds), represented by the dashed line (100% viability). The dots represent the viability of motor neurons in the presence of astrocyte conditioned media.


Motor neuron viability graphs

As expected, there is no significant difference in cell viability between non-treated motor neurons and treated motor neurons. We can see that all the tested compounds retain cell viability, like non-treated cells.


We believe that the experiment requires optimisation to get the most out of the BX559 treatment. We need to answer questions such as:

- Is BX559 being delivered not the cells?

- Do we need to conjugate it to another molecule to increase uptake?

- Should we use a transfection delivery method?

- Is this the best model to perform the experiment?


These are important questions to answer but unfortunately, we do not have enough time (or cells) to do this as part of 12wdc. We encourage further research to investigate this.

Finally, we are maturing our final batches of healthy and ALS motor neurons to repeat and extend some of the work done in previous weeks. More details will be provided next week. Below are brightfield images of healthy and ALS motor neurons at day 3. As usual, motor neurons will undergo a maturation process for 10 days, with media changing every 2-3 days.


Brightfield image of healthy and ALS motor neurons at day 3. Scale bar = 200 um.

Only 2 weeks remain in the 12-week challenge. We want to thank everyone for their support throughout it. We plan on finishing the final weeks strong and providing an encouraging and exciting wrap up at the end.


Stay tuned at 12wdc.com for further updates.


#12wdc #ALS #MND #drugdiscovery #Ulysses



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