Progress report from the research team
Principal Investigator, Prof. Alan Mackay-Sim provided this report:
Science is never predictable and the last 3 months in the lab have been slower than hoped, for both technical and biological reasons.
The technical problems have been with the half-million dollar microscope system used for imaging living cells. The problem has taken a while to diagnose due to the complexity of the instrument but it is now repaired and working properly, however time has been lost. Other technical issues arose with the purchase of new reagents that do not behave as the old ones did. This has taken some trouble shooting and re-optimisation by Fan.
The SPAST (SPG4) mutated mice are now through all the regulatory, supply and quarantine hurdles and are now being housed in facilities at Griffith University, having previously been housed at the University of Queensland for quarantine.
Our thanks to Dr Andy Grierson and his team at the University of Sheffield who supplied the breeding pairs and to Emily Duggan at the University of Queensland who arranged their importation and quarantine for us.
Initially one mouse quickly became pregnant but the first litter was lost early on, possibly due to the stresses of travel on the parents. Since then all have behaved well with the first successful litter now a month-old with others following. Fan is genotyping them to identify the next generation of mutated SPAST mice.
A paper based on Gautam’s research work was submitted to a journal for review and, although rejected, received some useful feedback from the referees. We are taking their advice and will do another couple of experiments that will help pin down how peroxisome transport deficits in SPAST cells can lead to cell death.
Gautam’s work on establishing induced pluripotent stem cells to differentiate into neurons with the SPAST mutation is continuing. Earlier this year, Gautam generated induced pluripotent stem cells (iPSCs) from an HSPer with a SPAST mutation and from two healthy people as controls.
Over the last few months he has been testing batches of iPSCs to make sure that the required genetic engineering was successful. These quality control experiments are essential for reliable science and include measures such as chromosome stability, differentiation potential and pluripotency status. Uniform populations of iPSCs are notoriously hard to establish due to the variability across the cell population following the genetic engineering. For any results from subsequent downstream testing to be reliable, the cell population being tested must reach a high standard of uniformity in order to make meaningful comparisons of the data produced.
Gautam is now working on differentiating the iPSCs into neurons. This involves growing them for several months in a complex mix of proteins, growth factors, ions and sugars. These cell colonies are fragile; they must be fed with fresh growth medium every few days and demand closely controlled environmental conditions. The purpose is to mimic the growth conditions in the developing human embryo, aiming to turn the iPSCs into cortical neurons, which is the type of nerve cell most affected in people with HSP. The procedure is long and complicated, fraught with difficulty and randomness, still part “art” as much as “science”. When all this comes together he will have human neurons that will be used to further test his earlier findings of the effects of SPAST mutations on peroxisome trafficking and the efficacy of drug treatment candidate, epothilone D.