This is the first post in the somewhat belated start to my final project outline. For my project question, (which has yet to be formally written out) I was thinking about doing something related to neuronal growth and differentiation. During one of my co-op terms I worked with voltage-gated calcium channels (VGCCs) which have been found to play a role in synaptic transmission. Additionally, the directed studies project that I am currently working on deals in part with pH changes that take place at the synaptic cleft. It has been shown that these pH changes play a role in the long-term-potentiation of neurons as well as the formation of dendritic spines and neuronal differentiation. So it would be interesting to take this idea and expand the scope of it to consider which specific genes/proteins play a role in inducing the pH changes necessary for neuronal differentiation. For the purpose of this project I will probably focus on one specific gene.
Project Outline (Oct 23rd)
Topic chosen: Astrocytes and the synaptic cleft.
SPECIFIC QUESTION: What role does synaptic cleft pH play in influencing astrocyte development and function?
HYPOTHESIS: Increase in pH at the synaptic cleft will act to dampen astrocyte potassium channel function, thus having a major impact on neuronal and astrocytic function.
EVIDENCE ON WHICH THE HYPOTHESIS IS BASED (INCLUDE REFERENCES):
The synaptic cleft provides an enclosed environment which allows for localized changes in the pH within the cleft. Furthermore, transient changes in synaptic pH have been shown to influence neuronal differentiation. In particular, sodium/hydrogen exchanger 5 (NHE5) has been shown to influence cleft pH in a significant manner (Diering and Numata, 2014). Astrocytes are glial cells that were long thought to play little more than a structural role in the brain. However, more recently astrocytes have been found to play a central role in regulating transmission at neuronal synapses. (Haydon and Nedergaard, 2015). Astrocytes are known to highly express the inwardly rectifying potassium channel (Kir4.1), which is expressed in the surface membranes surrounding the neuronal synapse (Moroni et. al.). The function of Kir4.1 have been shown to be influenced by small ions like H+. (Hibino et al. 2010). Moreover, Kir4.1 expression may have an important role in the development of the brain (Olsen et. al. 2015). Considering this, in addition to the close proximity of astrocytes to the synaptic cleft, then localized pH changes at the cleft could induce changes in astrocyte function via Kir4.1 that may potentially have ramifications with regards to the development of the central nervous system.
PREDICTION(S): Alkalinisation of the cleft should show an increase in the output of Kir4.1, which will in turn should act to hyperpolarize the axon and as a result this should lead to increased synaptic transmission. Thus knockout of NHE5 should influence astrocyte function in a significant way.
EXPERIMENTAL APPROACH TO TEST PREDICTION (INCLUDE ANY DETAILS THAT YOU HAVE WORKED OUT SO FAR):
LIST OF RELEVANT PRIMARY AND REVIEW ARTICLES READ, AND SUMMARY OF RELEVANT INFORMATION FROM EACH (this is the start of an annotated bibliography):
Haydon, P.G. and Nedergaard, M. How do astrocytes participate in Neural Plasticity? CSH perspectives in biology. 2015. 7(3): 1-15.
http://cshperspectives.cshlp.org/content/7/3/a020438.full.pdf+html
This article provides insight into the manner by which astrocytes contribute to synaptic transmission; it also outlines the history of the field as well as recent developments. For the most part astrocytes seem to do contribute to transmission by releasing calcium ions. They also raise an interesting point regarding the sensitivity of neurons to changes in potassium ion concentration. In addition to this they bring up ways in which astrocytes are generally studied, which could be useful to the project.
Hibino, H., Inanobe, A., Furutani, K., Murakami, S., Findlay, I. and Kurachi, Y. Inwardly Rectifying Potassium Channels: Their Structure, Function, and Physiological Roles. 2010. Phys. Rev. 90(1): 291-366.
http://physrev.physiology.org/content/90/1/291.long
This paper was primarily useful for explaining to me what exactly Kirs were and how they are regulated. According to the paper there are a number of Kir genes, whose respective proteins can be combined to form heteromeric and homomeric tetramers. They can be regulated by a number of different molecules, including polyamine compounds and potassium ions. Most interestingly, the Kir4.1/Kir5.1 heteromer which is expressed in astrocytes found at the synaptic processes has a pka of 7.5. The paper also indicates that acidification of the area surrounding the channel results in a significant decrease in Kir4.1/Kir5.1 activity.
Olsen, M.L., Khakh, B.S., Skatchkov, S.N., Zhou, M. Lee, C.J. and Rouach, N. New Insights on Astrocyte ion channels: Critical for Homeostasis and Neuron-Glia signalling. 2015. JNeurosci. 35(41): 13827-13835.
http://www.jneurosci.org/content/35/41/13827.long
This paper provided evidence that Kir4.1 is actively involved in the development of the nervous system. It also serves to elucidate the manner in which it modulates synaptic transmission. Knockout of the Kir4.1 gene in mice resulted in postnatal death and mutations of this gene in humans results in seizures and mental retardation.
Diering, G.H. and Numata, M. Endosomal pH in neuronal signaling and synaptic transmission: role of Na+/H+ exchanger NHE5. 2014. Front Physiol. 4(412).
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3888932/
This paper provided information regarding the importance of synaptic pH balance in neuronal function. It also provides information regarding a mechanism by which this pH is regulated (ei. NHE5). NHE5 is an ion transporter that is primarily found in the brain. It most commonly involved in the regulation of endosomal pH and interestingly contributes to the localization of TrkA; a well-characterized receptor tyrosine kinase involved in neuronal growth to the synaptic cleft. This transporter is also found, in a lesser extent at the synaptic cleft where it has been shown to adjust the pH there.
HOW DOES THE QUESTION FIT INTO THE BROADER PICTURE, AND WHAT IS ITS IMPACT?
Understanding something about the way in which these two cell types communicate could potentially elucidate something about how they may communicate during normal development. Additionally, pH changes at the synaptic cleft have already been shown to inhibit the function of NMDA receptors, which are important ion channels that are involved in long-term potentiation and memory. As such this could provide another way in which synaptic pH could influence synaptic plasticity. Research into LTP and memory development could also have an impact on our understanding of neurodegenerative diseases such as Alzheimer’s.
POTENTIAL WAYS TO MAKE YOUR QUESTION KNOWN TO THE PUBLIC AT LARGE (OR TO YOUR NON-BIOLOGIST FAMILY AND FRIENDS):
If I were to explain this to family or friends I would probably start by explaining how two neurons communicate and how that is related to synaptic transmission. If I can explain it in a way that relates cellular communication at the synapse as a way in which the brain can transmit information to the rest of the body then it will be more interesting. Neurological research is interesting to me as part of it is linked to why we behave the way we do and I feel that that is one of the principle reasons as to why the public is also interested in the subject. If the question can be taken away from being associated with something as esoteric as the pH at the synaptic cleft between two neurons and explained in a way that relates to relevant disease symptom then it could be relatable and interesting to the public. Additionally, if the question can be related to some aspect of human psychology which granted would probably be very difficult, and then it could also generate interest from the public.
ANY OTHER PARTS OF THE PROJECT COMPLETED SO FAR:
ANYTHING YOU WOULD LIKE SPECIFIC FEEDBACK ON:
I feel that this is far from ideal as far a project outline goes as I feel like I have kind of put it together at the last minute and I don’t have much in the way of an experimental plan in mind yet, but I feel that getting some kind of feedback is better than having none at all and going into the project completely blind. That way if my project as described here is bad, I can at least find a new topic and start over, rather than continuing down this path. In particular, I was hoping to receive at least some feedback with regards to whether my question is relevant in terms of what is expected for the class. If it isn’t relevant enough do you have any advice as to how I could modify it to make it more relevant? If it is relevant then I was hoping to determine whether it was specific enough or if it needs to be focussed a little more. As for the experimental part I have yet to truly work that part out. Part of me feels that the basis for the project is somewhat tenuous and I was wondering if you had any feedback regarding that aspect of it (ei. if it is doable or if I am barking up the wrong tree, so to speak.)
Biology 463 final project-draft 1.
Biology 463 final project-Mitchell Beattie
Comment/Reflection:
The final project… If I could start over I would have started work on that right away, before things started to get busy. It would have saved me a lot of anxiety and stress and in addition to that, it would have inevitably turned out much better than it did.
By developing the outline I learned just how hard it is to develop a novel research question. I spent a lot of time reading papers just to come up with material so that I could come up with a novel question, unfortunately, I was not able to put all of that research to good use. As the project outline indicates, I had initially decided to work in a neurobiology direction, I feel like the experiment could have been feasible, but I had a lot of trouble finding research that was done in mice, which are the animals that are most commonly used for producing knockouts in mammalian systems (most studies performed on Kir4.1/5.1 channels are performed in rats). I have never heard of a rat knockout system and there is probably a reason for that. That wasn’t the biggest hurdle though, If I were to analyse the flow of ions through Kir4.1/5.1 channels under NHE5 knockout I would have needed to learn how to conduct an electrophysiology experiment. I have essentially no knowledge of how electrophysiology works and in order come up with a proper experimental procedure I would have needed much more time than I had to work with, considering the circumstances. If I had been much more prepared this wouldn’t have been a problem, but unfortunately this was not the case. I should have asked for more help when I had the chance. So as a result I fell back on a technique that I had learned more about: chIP-seq. The resulting project ended up being much more biochemistry related than I had hoped, and although histone variants are important to epigenetic gene regulation, the project has more implications regarding cancer then it does developmental biology. It is pretty safe to say I have a lot of regrets surrounding this project, it could have been handled a lot more smoothly, but that doesn’t mean I can’t take something away from it.
In terms of the learning goals provided during the course, I feel like the knowledge that I gained during my research for the project wouldn’t have been out of place in Phoebe Lu’s guest lecture on epigenetic mechanisms. As I learned during my research histone variants play a major role in regulating gene expression, so much so that when they are de-localized they can cause major problems. As I pointed out in my first draft, H2A.Z is a lethal mutant in vertabrates. Although I didn’t look specifically at the roles played by post-translational modifications in modulating the function of the variants covered in my study (in hindsight that would have been a good topic for the discussion in the paper) It was pretty clear from the research that I did that they play a significant role in regulating gene expression. So in terms of course material, the research that I did for my project increased by understanding of epigenetic modifications considerably. I feel that histone variants were not discussed that often during the class but they definitely could have been discussed. Just as an example H2A.Z knockout mice die quite early in development, thus implicating the importance of this particular variant in development.
In the first draft of the paper I originally had a number of experiments lined up, until I realized that it could be much more easily tested with one much more easily performed experiment. Here I learned that a proper, well-thought out experiment takes time and research to develop. My initial idea had far too many experiments and more research was needed to sort out the best way of testing the hypothesis.