General Flashcards
Is it original?
Yes, this is a novel approach that has never been used to monitor plaques before. Usuaully fibre photometry is used to monitor calcium signals or other genetic sensors, but has never been adapted to monitor a molecular pathology before.
Therefore, for the first time, we show an approach to monitor plaques with fibre photometry.
Can you justify the claims you have made?
Claim 1 is that fibre photometry is feasible for monitoring plaques. I believe this is justified as we show good correlation with histological quantification.
Claim 2 is that the in vitro and Norm data provides the best TF signals. I believe this is justified because we see the greatest increase from baseline in these examples, with stronger significance.
Claim 3 is that we can see depth-resolved signals. This is justified because we got a greater increase at deeper regions which are in regions of plaque dense population.
Claim 4 is that we cannot make a solid conclusion on the state of GENUS. This is because we cannot confidently confirm if this si due to the system or the treatment.
Did you do the work?
Yes, myself and my PI established the main goals of the project. From there, I developed the hypotheses and aims.
Then, I designed experiments and set-up all equipment used, with some help from Niall.
All experiments were completed by myself.
Do you know where your work is situated within the broader field?
As this is a novel approach this would sit by showing further confirmation of this optical technique - not only for use for monitoring plaques but for other neuronal signals.
In terms of AD, this provides a preclinical drug screening option.
Summarise your thesis in a sentence.
We aimed to design of a novel approach to monitor plaque pathology in AD to overcome limitations with previous approaches such as depth access and real-time feedback, by using M04 and fibre photometry.
Does the title represent the content?
Yes, we illustrate the feasibility of FP for monitoring plaques in vivo, with use of TFs showing the depth-resolved capabilities.
Describe your thesis in brief.
We aimed to design of a novel approach to monitor plaque pathology in AD to overcome limitations with previous approaches such as depth access and real-time feedback, by using M04 and fibre photometry. M04 is a BBB-permeable plaque marker than can be injected ip and fibre photometry is an optical system that can monitor fluorescence in the brain using an implantable optic fibre.
Using 5xfad mice, we proved the feasibility of this approach by comparing in vivo photometry signals to histological quantification.
Then, to exemplify the depth-resolved capabilities, we implemented TFs which allow light propagation and collection over 1.8 mm of brain tissue. By using appropriate light protocols and analytical approaches we could extract depth resolved m04 signals.
Lastly, in attempt to fully illustrate the intended use of this approach we monitored plaque signals while exposing mice to a sensory treatment approach.
How did you decide to order your thesis?
We decided to order it this way as it illustrates the story of establishment from proof of concept to implementation for monitoring a specific treatment.
What is your overall argument?
That combination of fibre photometry and m04 provides an optimal way to monitor plaque signals in vivo as it allows measurement in real-time, at depth and in freely behaving mice.
This overcomes several lmitations with existing techniques and therefore will provide a better overlook of the applkied treatments for preclinical testing.
Summarise the context.
AD has had a severe lack of treatments available and we suggest this may be due to the way we are testing pre-clinical treatments. Currently methods are limited and measn we are overlooking alot of important data when it comes to a treatment.
By implementing the real-time, depth-resolved approach you can result ina closed loop system whcih allows alot more inroamtion to be collected regarding the disease state.
What made you choose a PhD in Alzheimers/neuroscience?
Why did you choose this topic?
I chose this topic as since undergrad I have been greatly intrerested in neurodegenerative disease and this was enhanced by familial circumstances.
Then, I was able to do a really interesting AD and hearing loss project in my masters which introduced me to some really cool techniques which made me want to continue in the neuroscience and laboratory field.
This particular topic was interesting to me as I am really interested in being involved in the development and testing of potential treatments.
Why is this topic important, and to whom is it relevant?
This topic is important because AD has a massive and continuously increasing prevelance which can reduce the lemngth and livlihood of many. As there has been limited treatments approved for this, this technique is relavent for boosting and accelerating potential treatments to a clinical setting.
In other cases, this approach is releavent for any AD researcheres as this ould be used to understand pathological processes and in combination with other disease markers.
Did you enjoy your PhD?
Overall, yes. It did not come with out its challeneges and certainly there were times where I was under alot of stress and probably overworked. But now I have came out the other end I am greatly appreciative of the skills I have learnt aling the way.
What are you most proud of during your PhD?
I am probably most proud of actually getting positive results for this system.
Improving my coding skills to the point I was able to model data and write my own LABVIEW codes.
Presenting my work at conferences and getting great feedback.
What was the biggest struggle during your PhD?
In terms of lab work, probably working to establish a reliable way to ake the signals as consitent as problem. I found the laser to be really unstable and wworked with other groups to try improve this.
What are the key findings?
FP is feasible to monitor plaque signals.
In vitro and norm provides optimal TF data.
TF allow depth-resolved monitring of plaques.
GENUS can induce 40-Hz band in cortex and hippocampus but acute stimulus did not change fibril levels.
What justifies this thesis as a doctorate?
I believe the fact a novel approach that can be adapted for various aspects of pathology of neurodegenrative diseases being established is justification.
What three publications would you say have been most influential in your work?
What did they do?
Klunk 2002 - development of Methoxy-x04. They established the development of this drug, tested the BBB permeability and established an appropriate dosing concentration for i.p injections.
Pisano 2019 - use of TFs in depth resolved manner with confirmation of use in vivo for monitoring dopamine signals.
Iaccarino 2019 - first etablishement of snesory setimulus showing the reduction of plaque pathology.
Who are the key names in this area?
For M04 - Klunk
For TF - Pisanello, Pisanno, Di Vittorio
For GENUS - Tsai, Singer, Buscaki
Do the findings confirm, extend, or challenge any of the literature?
My FP findings are consistent with previous examples of being able to use the TF to extract depth-resolved signals. They extend to this by showing their use for monitoring molecular pathologies marked using an ip drug rather than a genetically encoded sensor.
In terms of GENUS, my findings challenge the literature as we found no changes in plaque load after 1-h treatment. At first, I thought this may be due to our approach monitoring insoluble fibrils which is consistent with no change in the original paper. But since I wrote this, another paper has been published which shows that 1-h treatment reduced the area of plaques monitored using confocal microscopy.
How does your work connect to that of your reviewers?
Trevor - alot of interest in astrocytes and how they may regulate disease - this illustrates an approach that could be transferrable for monitoring astrocytes. Also, it shows a treatment that can potentially be altering their activity.
Micheal - completed studies using a freely behaving miniscope so this ullustrates a capability of this but across depth. Also, he illustrates the importance of PV and SST neurons in the generation of theta and gamma oscillations and how they can be interuppted in AD by AB. Also, he has shown how optogenetically stimulating PV neurons can restore gamma functionality and memory functions in 5xfad mice in the RSC. This exemplified a new way to interogate the RSC, while providing insight into the GENSU treatment.
Summarise your research design.
As I was establishing a novel protocol I needed to complete a proof of concept, then show I could montiro across depth and then illustrate the ability to use this for monitoring treatment capabilities.
Did you think about applying a different design?
When choosing this particular approach we did consider other dyes and we done a literature review on other optical techniques, but we found that this was the optimal approach for the three pillars we desired: real-time, depth resolved and freely behaving.
Is there anything novel in your method?
Yes, this is using a previously calcium imaging approach and tranferring its use for molecular pathologies.
This also shows for the first time monitoring of plaque pathology in real-time across depth and in freely behaving animals.
What problems did you have?
Some issues throyghout the project surrounding around the laser stability.
We first had an issue with the first laser where it deteriorated over time due to an electrical fault which held us back a few months until we could retrive a new laser. Then, the new laser had spme high and low frequrency noise which interefered with the stability across days.
What are the weaknesses of your sample?
When thinking about human AD pathology, the 5xFAD model is not representative as it is not common for a human to have alll 5 mutations. Also, this mouse model does not have tau pathology and therefore is not a fully rounded perception of AD pathology.
What are the strengths and weaknesses of your data?
Strengths: show really nice correlation with FF data, show increase in fluorescence upon M04 injection, show some depth resolution, achieved signals in freely behaving animals
Weaknesses: unable to correlate TF data, lack of strong significance in PK data, unable to detect changes inGENUS
What other data would you like (or have liked) to collect?
I would have liked to process the data further into different groups such as age, sex to uklllustrate durther the ability to detect changes in plaque pathology.
I would like to further confirm the feasibility of this approach for monitoring the response to treatments by monitoring the fluorescence when administering a pharmacological drug.
Or I would like to make the system more stabel to allow monitoring of GENUS with a more chronic treatment paradigm - 7 days.
How confident are you in your findings and conclusions?
I am confident that we are monitoring plaque signals and are achieving depth-resolved signals.
I do not feel confident enough on the GENUS findings to make a solid comment on whether or not 1. genus works or 2. this system is sensitve enough to detect changes in this way.
What the implications of your findings?
This provides an alternative method to monitor the pathology of AD.
This will help uncover alternative targets for AD as well as helping gather more understanding of the effects of potential therapeutics.
