We are a quarter of the way through!
We are a quarter of the way through our 12-week ALS challenge! We just want to remind everyone the purpose of this project – to potentially find a new ALS treatment in 12 weeks – and so far, the results have been fantastic. We hope you guys are enjoying the progress as much as we are.
Let’s get into Week 3's update.
Last week we performed immunofluorescence (IF) imaging on our mature motor neurons to illustrate motor neuron differentiation. This week, we did the same for our astrocytes. In the IF images below, we have 3 stains:
Blue – Nuclei
Red – S100 calcium-binding protein B (S100b)
Green – N-Myc downstream-regulated gene 2 (NDRG2)
S100b is a multifunctional protein that is abundantly expressed in astrocytes and has been implicated in the regulation of several intracellular activities such as proliferation and differentiation.
NDRG2 – a well-studied tumour suppressor gene – is principally expressed in astrocytes in mammalian brains. It has been shown to be a multifunctional protein associated with cell proliferation, differentiation, transmembrane transport, and stress response.
Now that our astrocytes have differentiated, we can begin experimental work.
BX559 is a potent activator of the glial cells of the brain. When BX559 was administered to rats spinal cords, glial activation and proliferation occurred rapidly alongside increased secretion of inflammatory cytokines and the loss of motor neurons. In this sense, BX559 treatment induces a distinct ALS phenotype. This is the premise for our upcoming astrocyte experiment.
We are testing the impact of BX559 on astrocyte mediated cytokine release. We want to determine the optimum dose and time for BX559 mediated inflammatory secretion in astrocytes by testing the supernatant for 4 cytokines – TNFa, IL-1b, IL-8 and IL-6.
We added BX559 at 9 different doses and tested the media 24hr, 48hr and 72hr post addition. If BX559 generates an inflammatory reaction, the cytokines above should be released into the media by the astrocytes.
The analysis is still in progress. Next week we will reveal the optimal time and dosage. These will be the conditions for all future 12wdc astrocyte experiments. Stay tuned to find out!