Week 2 results are in the books!
Last week was all about the maturation of our motor neurons and astrocytes. This week the maturation process is complete, and some testing has been performed on our motor neurons with fascinating results. Let’s dive in.
Below are brightfield images of our healthy and ALS motor neurons 10 days post-seeding. Days 3, 5 and 7 were posted last week, so you may wish to look at and compare last week’s post to view the full maturation process.
Both motor neuron groups were expected to undergo complete differentiation 10 days post culturing. To assess whether this has occurred, immunofluorescence (IF) imaging was performed on both groups. The nuclei were stained blue while the neuronal differentiation marker Tuj1 (Beta III Tubulin) was stained red. Tuj1 is a tubulin known to be involved specifically during differentiation of neuronal cell types. The IF images below suggest that most of the motor neurons have been successfully differentiated.
Mutations in the gene encoding TAR DNA-binding protein 43 (TDP-43) are a well-recognised cause of ALS – both sporadic and familial. Wild type TDP-43 can also be found in neurotoxic oligomers and aggregates in both familial and sporadic ALS patients. eIF2α phosphorylation and TDP-43 mediated toxicity are strongly related to each other. It has been discovered that eIF2α inhibitors are able to mitigate the effects of TDP-43 toxicity. We want to see if our strategy for inhibiting eIF2α phosphorylation helps motor neurons resist the effects of TDP-43.
Graph 1 below illustrates the ratio of eIF2α phosphorylation for each motor neuron group tested. The ratio was determined by: phosphorylated eIF2α/total eIF2α.
The 4 groups are ALS motor neurons, healthy motor neurons, healthy motor neurons + solvent control and healthy motor neurons + compound (a known eIF2α kinase).
ALS motor neurons showed significantly higher eIF2α phosphorylation levels than healthy motor neurons, which is consistent with the literature. We also observed that healthy motor neurons treated with compound (known eIF2α kinase) showed significantly higher eIF2α phosphorylation levels than healthy motor neurons + solvent control.
And how are our astrocytes going?
Our astrocytes have finally reached passage 4! This is the minimum passage where they may be used for wet experimental work. The images below show our astrocytes at passage 3 and 4. Their distinct star-like shape with pronounced cell bodies and well-developed processes have become apparent. Last week we posted images of our astrocytes in passage 2. You may wish to glance back at last week’s post to view the full maturation process.
Limitations
As scientists we recognise the importance of identifying the limitations of our work. As such, we would like to share with you the limitations of this and all subsequent week’s results, and how we will be working around them.
To being with, the raw values obtained of eIF2α phosphorylation levels were a little bit low. To get around this, we will be testing a greater number of cells (increasing sample volume) which should provide us with a larger signal. This will hopefully provide us with more reliable results.
Additionally, we saw unexpectedly large variation of eIF2α phosphorylation levels with some wells having to be removed from the analysis as outliers. We believe this has to do with incomplete lysis reactions. To fix this, we have increased the time for lysis in the second run of testing.
Finally, the cell count of our IF imaging wasn’t as high as expected. Due to a shortage of our original media, we’ve had to use another type of media that has proven to be susceptible to depolymerisation when washing occurs for IF image preparation. The issue here is that due to this depolymerisation, our motor neurons aren’t adhering to the media as expected which is resulting in cell loss. We have identified PFA – a material of the cell washing procedure – as the culprit of depolymerisation. To improve IF images next week, we will be using a lower concentration PFA which will hopefully reduce polymerisation and therefore improve cell count and image quality.