Scepticism

The first thing to clear up for anyone reading this, scepticism is not the same as pessimism.

Pessimism is to see the worst in the world and life, scepticism is to doubt the truth of something, to question the validity of something. Scepticism literally originates from the Greek for 'To examine'. Pessimism is a negative attitude, scepticism is a healthy questioning attitude. I want to avoid this post being like verbal diarrhoea so I will do my best to structure it coherently.

Scepticism; becoming more important in modern living?

Believing everything one reads is unfortunately something many people do, it becomes more of a problem because public health is always a hot topic with most people showing interest. Though healthcare and human science (my research) is a very specialist research area that takes a lot of understanding to formulate an opinion based on fact. Therefore when a non-expert reads an article on a 'new' discovery they almost have to assume it is real or that the person writing the article has employed scepticism for you. Sadly this is not the case now, some articles are written by people with some understanding but not the critical analysis skills to employ scepticism or someone with very little understanding on the subject. More dangerous reasons include that the person relaying the information is not allowed the time to do the necessary research to trust the information and the need to sell more of their product or in the case of news sites on the internet and social media, wanting to get as many views as possible to make more money from ads through site traffic, this is the 'click bait' phenomenon. The 'click bait' approach seems to have shifted the balance from delivering factually accurate news that has had scepticism employed for the consumer, to a focus on entertainment. This has led to reporting of information that is not verified and may require lots more knowledge on the subject than the lay person has to make an informed judgement. This essentially forces the lay person to believe what they are reading because they do not have all the information, so the need for the average person to be more sceptical is growing.

I personally have noticed the words 'allegedly' and 'reportedly' or 'speculating' appearing more in papers, on the news and on internet sources, these words suggest that the report has not been verified as accurate. Combing this type of publicising and interest in public health with a lack of scepticism from the lay person leads to consequences such as pressure on medical personnel because people request 'the new drug they have read about that cures this' it then has to be explained that this drug is in development or does not have enough research to even trust as a viable option. Having spoken to doctors and medical students, patients and family members often ask for 'the new treatment' that turns out to be nowhere near ready to come to market and many of them report being accused of withholding treatment because they will not administer this new drug that does not exist. This could be prevented if articles clarified the statement 'potential/new therapy'.

Potential therapies

When it comes to healthcare, there are things to look out for in the articles and news you see. The biggest one being 'potential therapies', this phrase as it is really is not the problem, many papers with implications in healthcare will contain this phrase or one similar and it is generally a true statement if the research is credible (not going into this yet). For example, I have used the phrase in my own work and will continue to use the phrase when it is required. This is not the issue, the issue is when the phrase is used in an article or news story and especially in a tag line or title. This is because when someone writing a paper suggests their research could lead to 'a potential new therapy' this is many years and advances in research after that paper is written, whereas when a person hears it as news, it is perceived as cutting edge and something that is available in the near future. Thus, it is the distinction in time and amount of research left to be done that is not stated in the article, or it is put at the end of the article once a person has clicked on it/brought it (click bait).

To put this into examples, a study by Coste et al in 2010 identified the first good evidence of a mechanically activation cation channel they named Piezo1 and Piezo2. It was described that potential treatments for touch disorders and mechanical nociception were implicated. This is indeed true as the same group later identified the channel in nociceptors and its role in mechanical pain, again suggesting targeting this channel could be a potential therapy.

This is all true but 7 years after the original study was published we are still a long way from a drug/therapy coming to market, it is not because research has stalled or it is incorrect but because research takes a long time and once a potential drug is developed, that is just the beginning.

To put it into perspective, just taking into account drug development (not the research before such as the discovery of Piezo2 and subsequent research) a target is identified for the drug, involving intricate chemistry to target specific amino acids at specific positions in the protein, this then needs to be tested with an assay to ensure binding affinity (does not prove efficacy). Based on this assay and functional assays for efficacy testing, the chemistry is adjusted and tests repeated until it is optimal.

Then animal testing for efficacy is done in at least two species of animal, usually rats and dogs (see later for issues with this). After this the drug will be adjusted chemically again. Side effects will be tested the same as efficacy then phase 1 clinical trials (side effects in humans) begin, then phase 2 trials and then phase 3 trials. Phase 1-3 alone 'clinical development' takes around 6 years, so it is often the case that the 'potential therapy' mentioned in the news or article is anywhere from a few years to over two decades from being available (figure 1). In addition a drug can fail for many reasons at any point in development and the majority do. 

Figure 1: typical drug development process.

The issue I have is not with 'potential therapy' or anything like that, it is the accidental or purposeful omission of the clarification of potential therapy in the article or news story. How far along is it? Is it one of the 5 or 10,000 compounds in figure 1? Is it before the drug discovery stage and just a theory? Because I do not think this information will ever be reported on, it is important for the people taking in that information to be sceptical of how much of a potential therapy it really is. It may seem trivial to some people but if you have a family member that has an illness and you read 'potential new therapy' you can think it could change that persons life and it is false hope if it is not clarified that it is 20 years away and it is unfair to use this false hope just to get people to read your article. Some scepticism will help combat all of the consequences, such as just finding the original academic paper and reading comments from other scientists and finding out if it is just one stand alone study as this will show the reality of the situation. That is what scepticism is, asking questions, doing your own research to find the truth. 

Scepticism; the responsibility of everyone involved and the consequences of not being sceptical

Now to the science, just because that article is based on a real study that suggests a potential therapy, does not mean that the paper is flawless or even credible. Lets start with the extreme and shocking example... 'a link between autism and vaccination' this example is important because it has continuing consequences on public health still, despite that it came from one study that was almost immediately discredited and led to the author losing their licence to research because of the falsification of data, bribery and unethical behaviour charged against him. The study that sparked the whole anti-vaccination movement was a single study by Wakefield et al from the Walker-Smith Lab that was picked up by The Lancet journal in 1998. The study stated a link between MMR vaccine and autism.

I will not go into lots of depth on this as the information is readily available but I will summarise the key evidence refuting the claims. Firstly, the data set was very small with no control groups and relied almost entirely on parents beliefs that the child may have autism symptoms, rather than medical examination of the limited number of children. Secondly, in over a decade, none of hundreds of studies have reproduced results that are even close. Thirdly, Wakefield changed the family histories and accounts given to better fit his hypothesis because crediting the link between autism and MMR vaccines increased his personal financial standing and the financial standing of the institution he worked for (Royal Free Hospital) which is FRAUD. He was convicted of this fraud in a long trial and stripped of his clinical and academic credentials. Part of his conviction was simply because he was part of a group of people in an ongoing lawsuit of the makers of the MMR vaccine and that he accepted money to publish research that would help the group with their lawsuit, none of this was disclosed when the research was published. Finally (because I think I have made my point, but there are many more reasons), there were 12 authors on that paper and 10 of them retracted their involvement in the paper with most stating that they were mistaken or misinterpreted the results, whereas Wakefield would not say he was mistaken but also refused to commit to reproducing his data in controlled, independent conditions. He refused despite being offered funding and the opportunity to do the research himself to support his autism-MMR vaccine claim, you do not have to understand science or law to know that this is the mark of a guilty man who knows he would not be able to reproduce the data because he falsified it. The consequence of this one incorrect study has been a resurgence in three dangerous diseases and other diseases such as TB, polio and other fatal diseases.

So how would this be different with scepticism employed? Well firstly, the final version of the paper was written by Wakefield alone, thus most of the other authors were unaware of this false data and false conclusions. As an author on a paper, you are staking your reputation on being partly responsible for that research and the conclusions the research draws. The authors should have read and critiqued the final copy and never let it be sent to journals. Secondly, when journals receive papers they are critiqued and sent to independent researchers to critically analyse and give their opinion on the limitations and if the data is credible. The reviewers of this paper for whatever reason failed to notice the glaring errors, tenuous links and gaps in the science that if flagged up would have prevented this paper ever being published and prevented the public health epidemic it has lead to, because when the publishers sent the paper back to Wakefield to amend it, he would have failed to do so and been exposed. This to me is the first failure for scepticism as it would have been nipped in the bud either by the other authors or the journal but it was not. So the next place scepticism failed (or was totally ignored) was by the media who wrote articles on the 'potential links' between Autism and MMR vaccination. The articles were shockingly unbalanced and ignored hundreds of testimonies from other scientists that there were lots of anomalies, inaccuracies and inconsistencies in the research. Unfortunately celebrity endorsement gave further credit to the lies and swayed public opinion, if you wanted to you could argue that the celebrities in question saw endorsing anti-vaccination as a way to ensure they got extra publicity in a hot topic in the media to further their celebrity status, for now I will say it was just lack of scepticism on their part. Finally, if the lay person had been sceptical of the media reports and took the time to research it, they would have seen that it was merely one paper which should ring alarm bells and then reading testament from other scientists would have shown that it was in fact not concrete evidence and in this case just untrue. Though as I have stated, the lay person is often let down by the media who should be employing scepticism for them and in this case the paper should never have seen the printing press. Nonetheless, it is an example that proves scepticism is important for the lay person but also for researchers and publicists alike.

I would like to stress that if the MMR-autism link was true (it is not) I would still be using this as an example because it was just one obscure, poorly conducted research paper at the time so scepticism should have meant Wakefields' claims were lost because of the poor methods employed. If subsequent more scientific studies had been done that showed a significant link then so be it, but I would still be using this as an example for why scepticism is important at all levels, from researchers to the lay person.

The importance of employing scepticism as a scientific researcher!

I want to give one more example, I am not going to talk too much about it because I am publishing another entry specifically related to animal models of depression, but I am going to explain a bit about why scepticism is important to those of you who are learning at undergrad at the moment or even postgrad students and beyond.

As a bit of background, there are 3 types of anti-depressants: monoamine oxidase inhibitors, Tricyclics and selective-monoamine re-uptake inhibitors, this is in order of oldest to newest with no advance in efficacy coming since the mid 20th century (Table 1), the only improvement is fewer side effects for the selective re-uptake inhibitors. So what are the reasons? flawed hypotheses (e.g. monoamine hypothesis), lack of understanding of the disorder and poor animal models of the disorder (the last two go hand in hand).

Table 1: progression of anti-depressants from 1957-2000 (little has happened since 2000) 

‘Modern’ antidepressants are virtually no different to those first discovered by chance after the end of WW2- Leonard 2000 

I want to clarify that what I say on depression also applies to anxiety, autism, schizophrenia and any disorder that has social aspects, but the arguments are all very similar so you can use this understanding in depression and relate it to these other disorders and if it makes sense (which it should) you will see how it is so ridiculous that so many researchers are still getting funding to do this kind of research (for this you will need to read the later post 'critical analysis of animal models in depression'). Quite simply, due to a lack of scepticism by researchers and drugs companies, mice are the primary animal used for depression research, depression is now strongly believed to be at its core, a social disorder. This makes sense as the most debilitating and obvious symptoms of depression are those that affect how the person interacts with other people, they often isolate themselves and do not possess the social clues that others look for to socially interact, these include smiling, eye contact, open postures. These social behaviours are very obvious and very consistent between depression patients. Yet depression is still diagnosed by questionnaire and interview rather than observation of the behaviours that are consistent in depression patients, this is known as an ethological approach and is also ignored in animal research in favour of artificial tests that require lots of complication interpretation that is not consistent between research groups. To get all of the information read the post I will post after this about depression.

For now I will just say, employ scepticism when learning about topics and question why a lab has used the technique they have because the paper you read will give their interpretation, for that interpretation to be correct there must be no other reasonable explanation possible at that time. For example, removing a rats olfactory bulb results in aggression, a symptom of depression, thus removing a rats olfactory bulbs causes depression. This is an interpretation, can you think of any other possible interpretations that are just as possible? (you can comment them if you like) I will start you off with one possible other interpretation, the rat has lost its principle sense that it uses for everything including whether another animal is friend of foe, thus to increase its chance of survival the rat, being unable to distinguish friend or foe, attacks to give it a chance to win and survive. Therefore an equally possible (if not way more plausible) interpretation is that the rat has an extensive stress response (activating fight of flight) because it cannot distinguish friend from foe without smell. So the 'removing rats olfactory bulbs causes depression' interpretation is not conclusive and would never be the conclusion of a group of researchers who are experts in the field, right? wrong. There are hundreds of studies using exactly this method and claiming they have made the rat depressed and anti-depressants helped.

The problem I have with explaining this is that there are so many examples similar to the one above and those I talk about in my other post that I just cannot give you all the examples to watch out for, you will really need to use your critical analysis to decide if the experiment or indeed animal they are using, is appropriate or the researcher is just doing it because that is what the lab and other researchers have done before them. I will give an example specifically to do with using mice and rats in depression and any other social disorder. 

There are some studies and questionnaires you can see where researchers in depression, autism etc... have been asked questions about their animals, with the majority using mice, they are asked questions like 'are mice social creatures' and almost all the researchers (cumulative total of over 200 from all the independent questionnaires I could find) the answer was YES! If you ask that same question to a zoologist or someone who studies mice, the answer will be NO! Mice in the wild are very solitary creatures once in adulthood that generally only come together to mate. So why are the overwhelming majority of researchers using mice to test depression and other social disorders? There are a few answers to this, the labs already used mice so they just carried on without questioning it (no scepticism) and that because mice are housed together in labs, researchers think they are social. They are not, they are housed together because you can fit 5 mice in a shoebox sized enclosure. If you actually study the housing, the mice will split into dominant and subordinate mice, where few mice have lots of territory and the rest huddle in a corner so they will not keep being attacked (I have read papers in top journals by groups with good reputations that state that the mouse that is on its own is depressed because it has isolated itself from the group. NO! This mouse is dominant and it is alone because if the other mice go near it, it attacks them [figure 2]) none of this exists in wild mice as they are not social and essentially shows that the researchers have a fundamental lack of understanding of mice as a genus (I won't even go into the species differences issues here). I want to say that all of what I am saying is proven by questionnaires and studies already conducted and is not just guessing but show scepticism, research it and you will find what I am talking about. So you can see how scepticism from the new guard of researchers onto the old methods will greatly benefit science compared to just continuing on with the same old, poor methods and animals that have led to a half a century of stagnation in depression (and other areas) research that has led to no truly novel treatments succeeding since monoamine oxidase inhibitors and has caused some of the largest drugs companies to pull out of depression and anti-depressant research all together, at the cost of jobs and public health. Perhaps a change of animal and better, more socially relevant tests are the answer? but without scepticism from those going into study these disorders, research will continue to stagnate and it will continue to be trial and error where miracle treatments may appear (unlikely) but we will have no better understanding of it than we do now.

With all of this in mind, 'mice' and 'social' do not go together very well, there is some credit if the mice are juvenile as they do engage in play but far less credible for adult mice, so when you see mouse-social-autism, keep the fact that they are not very sociable creatures in mind as you decide whether you agree with the research. The same applies with anxiety and schizophrenia too.

Figure 2: Stippled areas represent territories defended by dominant mice – there are a large number of non-territory holding subordinate animals of both sexes (at the 12 O’clock position) that congregate together because they are ‘not allowed to remain elsewhere’ 

I am going to finish by saying that I am not that interested in getting comments from people who do think anti-vaccination is a good idea or that mice and the current tests in depression are fine, because my intention of this post is to get people to employ scepticism and question what they read, hear and see then research it to come up with an opinion based on evidence. My views on the MMR-autism and depression studies are my opinion based on a plethora of reliable evidence from research groups across the globe that have reproduced data, so I am confident in what I am saying... but, as with everything, don't just take my word for it and believe what you read, question anything you are not convinced on, research it and once you have, if you agree that what I have said is true/likely true then good, if you don't then also good, just don't feel the need to tell me.

Finally, YOU CAN BE SCEPTICAL AND STILL, AFTER RESEARCHING BELIEVE THE THING YOU WERE SCEPTICAL OF, TO BE TRUE! JUST BECAUSE YOU ARE SCEPTICAL, DOESN'T MEAN YOU DISAGREE, JUST THAT YOU WANT TO FIND MORE INFORMATION BEFORE MAKING YOUR MIND UP. 

New Posts Alert

First off, I have not been posting the last few months as I had exams and my graduation and I was interviewing for PhD positions. I am happy to confirm that I have graduated from the university of Leeds with a First Class Honours degree in Neuroscience and that I will be returning to the university as a Postgraduate researcher in the Gamper Lab where I will be studying the functional expression of M channels in nociceptors and in conjunction with Eli Lilly establish the treatment possibilities based on this research. This will gain me my PhD in Neuroscience.

With that out the way and hopefully some proof that I am at least somewhat knowledgeable I am going to be talking about employing scepticism when you are learning, researching and just in life in general. The reason this has been prompted is from recent reports I have seen that even so called 'science news pages' doing the same.

I will organise it that I will write a post explaining what I mean, what scepticism is, why it is good and the various reasons why what the various types of media are doing is not entirely beneficial. This of course will relate to science and neuroscience but it is important in life in general. After this, I will publish 2 examples in research that highlight why we need to ask questions and not take things as read because it hinders research.

These examples will be from 1: mental health focusing on depression, but also relevant for schizophrenia, autism, anxiety and other mental health issues. 2: Spinal cord injury and motor movement disorders, focusing on SCI and ALS.

I promise it will be worth a read

Discussion of the Current Theories on the Specificity of Pruriceptive Neurons and the Chemical Mediators of Pruritus

Pruritus (itch) is a sharp tingling sensation resulting from a potentially harmful stimulus, chronic and neurogenic itch are persistent bouts of itching with (chronic) and without stimulus (neurogenic). Although scratching is a reflex response to itching, if the irritation is extensive there is more likely to be a pain reflex before scratching which potentially means that scratch is to alleviate irritation rather than to protect against what has caused it. Due to the overlap with pain, historically it was thought that itch was the same mechanism and pathway as nociception but interpreted differently as it is a lower intensity (Frey, 1922), in effect a warning that pain may occur if the body stays exposed to the stimulus but is not immediately dangerous. This has largely been rejected in favour of the suggestion that there are specific neurons controlling itch (Schmelz et al., 1997). However, the overlap between itch and pain mean a specific itch system may be too simple and the neurons may conduct nociceptive signals as well (selective theory). The following review will tackle the evidence for the selectivity theory with regards to peripheral neuron types and discuss the potential mediators and corresponding receptor subtypes responsible for itch signalling at the peripheral terminals of dorsal root neurons.

Identification of itch signalling neurons

It has been hypothesised that pruritus is transmitted along primary afferents specific to itch (Schmelz et al., 1997). Yosipovitch contested specificity theory with claims that noxious heat and scratching inhibit itch as it changes ones perception from itch to pain. This claim was supported by their results identifying the intensity of itch decreasing by 0.9cm (P<0.05) (Yosipovitch et al., 2005). However, the scale used was a visual analogue scale which although they showed to be reliable in repetition, is not the most accurate way to measure itch intensity because it is designed to measure subjective characteristics. Yet itch can be quantifiably measured in the frequency and duration of scratch methods which have high construct validity, as many species including humans scratch to alleviate itch. Their claim is supported more conclusively in 2015` with the co-expression of itch neuronal markers and TRPV1 (table1) suggesting noxious heat would have an effect on itch signalling (Usoskin et al., 2015). Usoskin’s team identified neurons responding to itch by using RNA-sequencing to group dorsal root neurons into sub-populations based on RNA expression. They identified 13 sub-groups of sensory neurons, with non-peptidergic unmyelinated C-fibres having 3 sub-groups identified (NP1-3)(Usoskin et al., 2015). These groups contained populations of neurons expressing itch receptors. Different populations expressed different itch receptors at varying expression levels (figure1A-D) suggesting that the different populations are involved in different types of itch. Their results identify selectively high expression of somatostatin in NP3 neurons (0.83)(Table1), this was therefore used as a marker for NP3 neurons.

When co-precipitating somatostatin tags with the tag for isolectin-B4, a known nociceptive marker, Usoskin’s team found that IB4 did not co-localise with somatostatin (figure1E/F). The absence of IB4 in NP3 subpopulations has been documented since this discovery (Stantcheva et al., 2016). This seems to suggest that NP3 is not nociceptive and therefore itch specific. However, TRPV1 is also a nociceptive marker and is significantly expressed in NP3 neurons (0.58)(Table1). Therefore, these neurons could be prompted to elicit nociceptive signalling due to TRPV1 presence.

RNA-sequencing can produce false positives due to artefacts (Ozsolak and Milos, 2011). This can lead to mean data that is not representative of the expression of that RNA. Usoskin’s team made efforts to minimise this limitation by setting a threshold for expression for each gene and only considering the genes that exceeded that expression when grouping populations ((Usoskin et al., 2015) supplementary methods).

Itch receptors and itch mediators

Histamine

Histamine was the first identified itch mediator which might be expected due to the alleviation of acute itching by antihistamines, importantly histamine helped explain the antagonistic relationship between itch and pain (scratch alleviates itch). Nilsson’s team used cutaneous field stimulation (nociceptive electrical impulses) to abolish histamine induced itch in an area of skin, 4 hours after, itch intensity was still 32% lower than control (Nilsson and Schouenborg, 1999). Although histamine is used experimentally to induce itch, pathological itch is unaffected by histamine (Klein and Clark, 1999) and expression maps identify only low levels of the histamine receptor Hrh1 in NP2 and NP3 (table1). The low receptor expression and lack of effect in pathological pruritus may explain why acute itch is only a low level irritation. High receptor occupancy would be required to evoke action potentials which may also explain why high local histamine concentrations are required for itch such as in inflammation.

Interleukin-31

Previous studies identified that interleukin-31 (IL-31) was implicated in chronic disease (Dillon et al., 2004; Takaoka et al., 2006). Since then research identified IL-31 as a key mediator of atopic-dermatitis in mice (Grimstad et al., 2009). It took until 2013 to demonstrate that IL-31 induced scratching with a single acute dose (Arai et al., 2013) see in figure2A. Arai observed that lengthy scratching to IL-31 was higher if applied to lesioned skin, supporting IL-31s role in chronic itch from previous studies (figure2B) but no receptor had been conclusively identified. Usoskin et al then uncovered that NP3 neurons expressed the IL31ra receptor (figure1G) in equal abundance to TRPV1 (0.58)(table1). They investigated this further by treating mice with IL-31 and observing the frequency of scratching increase from 10 to 50 (figure2D). Usoskin also states that NP3 neurons are involved in chronic itch, therefore so is IL-31, which supports the work done by Dillon and Takaoka’s teams (Dillon et al., 2004; Takaoka et al., 2006). However, Dillon et al had to overexpress IL-31 and used transgenic mice to introduce IL-31ra into the epithelial cells. These are not usual causes of atopic-dermatitis so construct validity of the model is limited. In addition, although the model showed good face validity as the mice had similar symptoms to the human disease, their results could not be translated into wider context because no empiric evidence was provided for increased scratching, despite it being stated in the text. Takaoka later produced a similar study but including results showing increased frequency of scratch (figure2C), implicating IL-31 in physiological as well as pathological itch.

Serotonin

Serotonin was found to be involved in itch when Weisshaar applied it to human skin (Weisshaar et al., 2004). Serotonin was also shown to increase scratching in vivo (figure1H) and 5HT1f was highly expressed (0.83) in NP3 neurons and scratching increased from 10 to 80 in response to 5HT (figure2D) which supports the claim that serotonin receptors have a functional role in itch (Usoskin et al., 2015).

Morita’s team showed that 5HT7 is coupled to TRPA1 cation channels by expressing 5HT7 or TRPA1 alone in human embryonic kidney cells and showing no inward calcium current, then co-expressing them and observing an inward current (Morita et al., 2015) also see figure3A. This suggests they are coupled but does not confirm that they cause itch or that other ion channels are not involved. So the team produced knockout mice, to show that there was a significant reduction in scratching if either 5HT7 (65s reduced to 25s) or TRPA1 (35s reduced to 5s) was ablated, suggesting both are required for itch (figureB/C). In addition, the TRPA1 knockout suggests that without TRPA1, 5HT7 does not lead to itch therefore is not coupled to other ion channels involved in itch.

The role of another TRP channel, TRPV4, has been implicated in 5-HT mediated itch (Akiyama et al., 2016), yet in vivo, Morita showed reduced scratching time from 35 to 5 seconds in response to 5-HT without TRPA1 suggesting only TRPA1 is sufficient for itch. However, Akiyama’s results show significantly reduced scratching in TRPV4 knockouts (100 bouts reduced to 20) but no difference between wildtype and TRPA1 knockouts (figure3D). This discrepancy (compare figure 3C and D) is not explained in the literature so a follow-up study should be undertaken to identify 5-HT response in vivo and in voltage-clamp experiments of isolated neurons with TRPV4, TRPA1 or both ablated to identify the contribution of each.

Histamine and IL-31 induce itch along with inflammation and serotonin induces itch at micromolar concentration but pain at millimolar concentration (Morita et al., 2015). This could be evidence that itch neurons also respond to pain, however the response could be from separate classes of neurons with the same receptors.

Conclusion

In summary, it is clear that a sub-population of non-peptidergic neurons (NP3) transmit pruriceptive signals. It is still not completely clear whether these neurons are specific to itch signalling or selective for itch. The lack of co-localisation between itch markers and IB4 suggest the neurons are itch specific but involvement of TRPA1, TRPV1 and TRPV4 in pruritus and nociception suggest that the neurons could be prompted to respond to nociceptive stimuli. To conclude, the evidence from these mediators and receptors strongly suggests that itch neurons can conduct nociceptive signals but it is still possible that the neurons are itch specific. A future approach may be to use optogenetics to induce action potentials specifically in itch neurons by introducing photoactivated ion channels under the somatostatin promoter. Then record the in vivo response to identify whether when scratching is induced these neurons become silent or nociceptive.

Hi everyone

Firstly, I know it has been 2 months since I uploaded anything for you all. My dissertation has taken over everything as well as difficult final year exams so I have not had much time to put anything together as it takes a long time to research the posts and then write them up.

I finish in 3 weeks so the plan for after that is to give a bit of an idea about what my dissertation was like and some tips for how to do well if you have one to do soon.

I will also return to writing overviews of areas of neuroscience.

In the meantime, I have a very interesting post on pruritus (itch) signalling which I will be posting later today. So look out for that, it is worth the read.

Highlighting the importance of pharmacogenetics. Follow up from TPMT deficiency

Drug development is plagued by ineffectiveness or toxicity in significant proportions of the population. This is because blockbuster drug dose is based on the results from cumulative response curves. So the dose is decided by (for example) above 80% of the population respond. However, at this point there will be people that are under-responsive and over-responsive at this dose (figure 1). So they will get no effect or get toxicity at this dose. With certain drugs this is fatal, with 100,000 deaths a year from drug toxicity and 4 million serious adverse reaction events. Variations exist through polymorphisms and drug interactions mostly, the following will discuss some contributions to variability in drug response with some experimental examples.

Absorption, distribution and excretion
There is less variation seen in these processes than metabolism but there are some important variations that can play a big role in drug effectiveness. Absorption is generally similar in most people but disease state can cause variability, for example, 32% of patients requiring irinotecan have diarrhoea, therefore oral administration would create considerable variation with those with diarrhoea absorbing a lower dose than those without. This has to be a consideration in drug development because it suggests the drug should not be developed to be delivered via this route.

Drug distribution and excretion also become variable with disease state, distribution rate is increased if as the disease progresses hypertension and tachycardia increase. In contrast, kidney damage and failure in late stages of disease reduce the body’s ability to excrete the drug, allowing it to be active longer thus increase response.

Metabolism

Metabolism activates and inactivates drugs, so changes to metabolism will cause variation in response because reducing either of these will either lose the effectiveness of the drug or increase the action well over the usual response and be toxic. Many drugs are heavily metabolised by CYP450 enzymes and when an enzyme is metabolising one it cannot metabolise the other. This can cause variability in drug response because one person may be on the drug alone whereas anoth may be taking other drugs. This is exemplified by mibefradil which is safe alone but when administered with propranolol, inhibited CYP450 enzymes, preventing the inactivation of propranolol. A study conducted looked at patients taking both and found systolic blood pressure decreasing from over 140mmHg systolic to 60-70mmHg a fatal level. This was found to be because propranolol was not being inactivated by CYP450s and thus acting excessively on the heart. This drug was soon discontinued.

Other than drug interactions inhibiting or increasing cYP450 metabolism of another drug, CYP enzymes also get polymorphisms, relatively commonly. CYP2C9*2 is the mutant allele in 20% of Caucasians, this is a significant number of people with a loss of function of an important enzyme in drug metabolism. Testing of how much variation the presence of this allele causes showed as much as 30% differences in dose of warfarin required for efficacy. This is a large variation because if a full dose is 30% more effective it is also likely to be toxic at this level. This becomes a problem in drug development because it makes it impossible to give an accurate recommended dose and makes it likely the drug will fail in clinical trials due to toxicity. Evidence that these polymorphisms contribute heavily to variation in drug response between humans, comes from studies adding CYP2C9*2 allele to transgenic mice but using a pharmacogenetics approach of genotyping and phenotyping activity and adjusting the warfarin dose given based on the result. They found that doing this required fewer adjustments to the dose and fewer incidences of toxicity due to too high doses. This suggests that CYP polymorphisms cause lots of variations to metabolism, but pharmacogenetics can be used in drug development to get the correct dose much faster and safer and almost eliminate the variation that is seen.

The future for genetic CYP testing could be in producing transgenic mice with CYP mutations as part of animal models to identify what changes in dose are required for each polymorphism. Evidence it is accurate from measuring CYP2D6 polymorphisms to quinidine and measuring the changes in response to doses and changing dose until variability is within acceptable levels.

CYP polymorphisms are not the only causes of variation in response, other important enzymes also cause variation in response between people including thiopurine methyltransferase (TPMT). TPMT*3A-C polymorphisms cause a total loss of function of this enzyme which allows a build-up of active thiopurines this in turn leads to an increase in efficacy and toxicity which eventually leads to myelosuppression. In this case variation has been fatal, because the toxicity is quickly fatal and a full dose to some heterozygote mutants is 50% higher than the dose should be and 15% of people are at least heterozygous for a TPMT mutation. Again the relationship with drug development is that drug cannot be developed to suit most but not all because it can be fatal. This could lead to a loss of millions at clinical trials or even more if it has to be discontinued. This is more evidence that variation in patients has to be accounted for in drug development, therefore pharmacogenetics may be the approach in rug development to create more individualised medicines from drugs that are technically block busters, it should increase success in clinical trials along with reducing variation.

Non-metabolic genetics

Aside from metabolism genetics comes into other variations between patients. The same cancer can have different responses to the same drug in different people. This is also a type of variation in response. Also taken into account is the stage of cancer because as the cancer progresses mutations are random so the same cancer can look very different by stage 4. A good example of variation to the drug is the drug Herceptin for breast cancer. It acts on the HCN2 receptor and is very effective but only in 25% of cases because men do not have the receptor and the receptor can be down regulated early in the cancer. This shows the variation in drug response because the drug is very effective at treating breast cancer, but the receptor is not present in all people or all breast cancers. This causes a problem in drug development because it is obvious the drug is very effective but getting significant results is difficult because of how variable cancers are.


In summary, variation in drug response is seen more commonly in metabolism but absorption, distribution and excretion also lead to variation between people, mainly due to what state the disease is in. metabolism variation exists mainly through one drug changing the enzymes action on another drug or mutations to important enzymes. However, it is important to note that sometimes the genetics of the disorder are variable as in cancer.

Interesting slightly different post

I hope you enjoy the TPMT deficiency post, it is a typical pharmacogenetic interaction and I will post a follow on writing that will take this into a wider context and into the difficulties pharmaceutical companies have when developing new drugs. 

Please give me some feedback on the two posts as if the consensus is good I will do a few more similar things that are not strictly Neuroscience. 

Enjoy! 

Thiopurine S-methyltransferase (TPMT) Deficiency

Introduction:

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyses the methylation of aromatic or heterocyclic thiol (sulphydryl) compounds (Peng et al., 2008).This function is ubiquitous, therefore the enzyme could be active in methylating toxic compounds as part of immunity such as against bacterial toxins. Its ubiquitous function is mimicked by its tissue expression and expression levels (Figure 1A). RNA-sequencing reveals that not only is it present in all tissues but it is present in specialised areas within those tissues (Figure 1B) and there is little variability between the expression levels in all tissues (Figure 1A and B vertical black lines show range), with the average expression around 0.55 FPKM (Figure 1A and B vertical red lines). The narrow range and ubiquitous nature of this enzyme show that TPMT has been highly conserved throughout all cell types and plays an important role in cell protection. The TPMT protein is coded for by the TPMT gene and has numerous polymorphisms which cause varying levels of loss of function of the expressed protein (Otterness et al., 1997). Interestingly, even those that are homozygous seem to live normal lives regardless of complete TPMT deficiency, unless they are treated during their life with the immunosuppressive thiopurine drugs, which in the absence of TPMT lead to extensive cell apoptosis. This report will discuss the TPMT gene and its mutations and assess the pharmacogenetic interaction between thiopurine drugs and the mutated alleles of this gene. 

TPMT Genotype:

TPMT is located at position 22.3 on the P-arm of chromosome-6 (figure 2a), it is therefore autosomal so a person can be homozygous for one allele or heterozygous. In heterozygotes the two alleles will be expressed equally so the alleles are co-dominant but the majority of the global population are homozygotes (Weinshilboum and Sladek, 1980). Complete TPMT deficiency is autosomal recessive as it requires two copies of the mutant to produce low to absent function, however heterozygotes with one mutant allele will have a varying response to thiopurines. In the general population 89% are homozygous, 11% heterozygous and 0.3% homozygous mutant (Weinshilboum and Sladek, 1980). In 2011, Booth included evidence of 30 alleles (Booth et al., 2011) with relatively few mutations in total suggesting the allele differences are due to the combination of mutations rather than separate mutations. This observation is supported by genetic information from the Uniprot database which also shows a limited number of mutations (Uniprot, 2015). Despite a limited sample, Stanulla provides evidence that TPMT2 and TPMT3A-D account for 95% of TPMT deficiency (Stanulla et al., 2005), which is supported by equivalent studies (Collie-Duguid et al., 1999; McLeod et al., 1999). TPMT*3A is most common in Caucasians with Tai identifying 75% of the sample with TPMT*3A allele (Tai et al., 1996).In contrast, TPMT*3C is the most common allele in Asia (Collie-Duguid et al., 1999; Hiratsuka et al., 2000; Lu et al., 2006) and Africa (McLeod et al., 1999; Ameyaw et al., 1999). TPMT*3C is characterised by a single missense point mutation of tyrosine to cysteine at position 240 due to an adenine to guanine substitution in exon 10 (Figure 2B). TPMT*3A has this same mutation, in addition to an alanine to threonine mutation at position 154 (Figure 2B).



Protein structure and function in wildtype and mutant alleles:

TPMT is a 245 amino acid protein consisting of 10 exons that form 9 beta sheets and 8 alpha helices (figure 3A) that fold to form the tertiary structure in Figure 3B. This figure also shows the binding site for adenosyl methionine and an adjacent binding site for 6-mercaptopurine from Peng’s study, identifying that adenosyl methionine forms at least seven hydrogen bonds here but 6-mercaptopurine only forms one (Peng et al., 2008). This study of the structures and binding sites concurs with the predicted function that adenosyl methionine binds as a methyl donor to the thiopurine, producing methylated thiopurine and adenosyl homocysteine in the reaction (figure 3C) (Peng et al., 2008). The same mechanism is expected for endogenous substances, however, no endogenous substances have been identified. It is possible that TPMT is a cellular defence to exogenous substances that attack through DNA integration. Evidence for this is highlighted by the toxic effects of thiopurine drugs in those with TPMT deficiency. In wildtype, TPMT inactivates prodrug thiopurines before they can be converted to active compounds (figure 3D) (Mlakar et al., 2016). Active compounds such as methylthioinosinemonophosphate inhibit the purine biosynthesis pathway which prevents DNA synthesis and repair or other active metabolites that insert into DNA forming interstrand crosslinks and single strand breaks that lead to apoptosis due to extensive changes in gene expression and DNA damage (figure 3D).

The tyrosine to cysteine mutation in TPMT*3C changes the structure because tyrosine has a large side chain that forms hydrogen bonds and Van der Waals interactions with residues on beta7, beta9 and alpha8. Mutation to cysteine means alpha8 interactions are lost, causing it to pull away (Rutherford and Daggett, 2008). This makes the thiopurine binding site more flexible so the thiopurine is exposed to solution. Making the thiopurine less likely to accept methylation and instead become unbound. In the TPMT*3A allele this structural deformation is coupled with a loss of hydrogen bonding between alanine and tyrosine, the double mutation forms a flattened protein and destabilises the protein causing it to be degraded by proteasomes (Rutherford and Daggett, 2008). Therefore has a half-life of 20 minutes compared to 11 hours in TPMT*3C allele (Rutherford and Daggett, 2008). This seems to be the major contributing factor as to why TPMT activity is almost absent in TPMT*3A homozygotes rather than changes in enzyme action which is the explanation for TPMT*3C allele loss of function.



Clinical features, treatments and diagnosis of TPMT deficiency:

Clinical features of TPMT deficiency are the same as the toxicities of the drugs, only the incidence and severity is higher in those with the disorder so fatality is higher (Dewit et al., 2010). To control the toxicity in those with the deficiency the dose is reduced by 50% for heterozygous and 90% for homozygous mutants (Coenen et al., 2015).

The main adverse effect is myelosuppression, a reduction in erythrocytes (anaemia) and leukocytes (leukopenia) due to the destruction of newly differentiated stem cells in bone marrow (Colombel et al., 2000). The crossover between efficacy and toxicity is evident here because when thiopurines are used to treat lymphoblastic leukaemia (increased level leukocyte derived cells), they act to reduce the number of these cells but the white blood cell loss can be excessive, causing leukopenia. This means there is a narrow therapeutic window. In those with the deficiency, leukopenia is more severe due to the excess active metabolites.

Approaches to diagnosis include phenotype testing which surveys TPMT enzyme activity by adding thiopurines and measuring the metabolite level in a purified sample (Burnett et al., 2014). Or genotype tests which sequence the gene to identify mutations that could change TPMT activity. However, a novel mutation may not be picked up (Burnett et al., 2014).

Treatments tend to deal with the toxicities as there has been no co-agonist identified that can increase the endogenous activity of the non-mutant allele in heterozygotes. Future therapy may be to reduce the degradation rate of TPMT allowing it to have a longer action. However, the most effective way would be through inhibition of the protease pathway which is ubiquitous and heavily relied on in the body. This would likely lead to cell apoptosis due to a build-up of toxic substances, so is not a viable option until another way to reduce its degradation can be found. Myelosuppression can be managed through managing anaemia, by stimulating erythropoietin with Darbepoetin alpha and neutropenia can be rescued with granulocyte colony stimulating factors as well as prophylactic treatments of infection. There has been a suggestion that treating with thioguanine may provide a less toxic alternative to other thiopurines. This has shown to be the case for inflammatory bowel syndrome, unfortunately, the licencing has been rejected due to hepatotoxicity but a derivative with some structural changes could be a future therapy. 

Conclusion:

In summary, the limited treatments and genetic variation of the TPMT gene in the population highlight the need for individualised treatments especially in pharmacogenetics. The wildtype allele functions as a methylating enzyme expressed ubiquitously throughout the body and may have a role in foreign immunity as no endogenous substrates have been identified. Mutant alleles for TPMT lead to damaging effects due to an inability to reduce the cytotoxic metabolites of thiopurine prodrugs and therefore cause fatal infections through myelosuppression. TPMT*3A and C are the most common mutant alleles but TPMT*3A disrupts function primarily due to an increased degradation rate, whereas TPMT*3C has a lack of function due to a change in protein structure. The negative impacts of this drug to mutant interaction are severe but their benefit outweighs the negatives because the majority of the population do not have any mutation and they are used in the treatment of conditions that are fatal.