On this page, I have divided up the sections into:
1) Current gene trapping research projects in the lab
2) Advice on what you need to do to carry out a gene trap screen
3) Technical resources
How does the mood stabiliser Lithium work in the treatment of bipolar disorder - and why do some patients fail to respond?
Today, lithium salts are part of a class of medications
known as mood stabilisers and are routinely prescribed to those diagnosed with
bipolar disorder. It can be highly effective, and is clearly responsible for
reducing risk of suicide. However, it has a narrow therapeutic index - meaning
the clinically useful dose is very close to the potentially toxic dose. Lithium
toxicity primarily affects kidney function and so newly-prescribed patients are
closely monitored for circulating levels of lithium to avoid this issue. A
second issue with lithium treatment is that 20-30% of patients show no
beneficial response – and many more show only moderate response. The likely
explanation is that human genetic variation results in a spectrum of response
to the drug. Gene-drug interactions are formally studied by the science of
pharmacogenetics or pharmacogenomics (depending on scale of study). It’s
tempting to speculate that the considerable genetic complexity of bipolar
disorder is also directly or indirectly linked to the genetic complexity of
lithium response.
The pharmacogenomic description of lithium response forms
the first, and most comprehensive, application of gene trapping that has been
attempted in the lab. In simple terms, we wish to identify those genes which
influence the way that laboratory cells respond to lithium – as a model for the
varying patient response to treatment. There’s another potential dividend to
this work: lithium is perhaps the most ‘off-target’ medication in existence.
Typically, drugs are designed and screened to act on a very specific ‘target’
(for example a particular enzyme or receptor). That way you can modulate the
biological deficit in a disorder with a fine precision – avoiding any collateral
damage that might result if other biological processes were affected. If the
intention is to create drugs that are laser-guided missiles, lithium can be
thought of as carpet bombing. Numerous studies have claimed to identify the targets
of lithium: GSK3beta, phosphomonoesterases, and inositol monophosphatase are generally
accepted to be among them. Less is known about which are therapeutically most relevant
to mood stabilisation in bipolar disorder.
Our first gene trap experiment on lithium response was published in
2013. In it we made use of the fact that lithium slows down and stops the
growth (meaning proliferation/division, not size) of a laboratory cell line
called SH-SY5Y that has properties that make it an OK model of neurons (there’s
a lot of subtext in that word, ‘OK’!). We treated a library of gene trapped
SH-SY5Y cells and essentially asked which mutant cells ‘keep calm and carry on
dividing’ as if lithium isn’t there. The majority of cells stopped dividing and
eventually died. However, peppered around the culture dishes were the odd
surviving cell. Over time, these cells divided to form patches of genetically
(mutationally) identical daughter cells – we call those patches ‘colonies’ and
we can physically pick them up and grow into large numbers of identical
(clonal) cells for molecular analysis. That analysis aims to identify the genes
disrupted in lithium-resistant colonies. This we did, and identified a number
of genes, one of which, MED10, could
be justifiably placed within the GSK3beta pathway. With hindsight, this was a
good ‘proof of principle’ experiment, but perhaps lacked the scale to shed much
new light on lithium action. The Mark II version of this experiment has now
been completed and I am now just trying to finish off validating the results
with the aim of submitting it for publication in the New Year. This version
features a much more sophisticated and quantitative output, and a much larger
scale of analysis. The work has been carried out in conjunction with David Sims
at CGAT at the University of Oxford. I am very excited about the findings, but that
enthusiasm has to be matched by that of the reviewers of the eventual paper! Watch
this space.
One last point on this approach. There is a fundamental
assumption made in this work – that the growth rate of SH-SY5Y cells in
laboratory culture has a mechanistic bearing on the alteration of mood in a
living human brain. I would argue that this FUNCTIONAL experiment is isolating genuine
effects of lithium on cellular protein function, and that the complexity of
expressed proteins in these cells is of the same order as that in the brain. I
would also go so far as to state that I believe that many of the pathways that
result in growth in the laboratory cells will also have pleiotropic action
(same gene/protein- different role) in our brain neurons that do not divide.
Again, the paper will reveal whether this is the case.
Which human genes encode proteins that are used by bacteria to invade our cells?
Which genes are responsible for tumours becoming resistant to chemotherapy agents such as cisplatin?