Cerebellar Neuronal Proliferation Timeline: An Analysis Using EdU

Video Transcript:

Hello, my name is Jonah, and my project this year is focused around Alpha-synuclein producing Neurons in the Cerebellum.

Parkinsons disease is one of the most common neurodegenerative disorders in the world, and it’s symptoms significantly decrease quality of life in people living with it. It remains incurable due largely to the complex and varied causes that can bring about it’s development, like natural ageing and genetics, but remains an area of major interest for research.

One pervasive indicator of Parkinsons development is the formation of Lewy Bodies within the brain. Lewy bodies are defined as abnormal clumps of protein that hamper cell communication and ultimately lead to cell death, and the one protein always present within them is alpha-synuclein.

Despite this fact, the early development of SNCA+ neurons, that is to say alpha-synuclein producing neurons, remains largely unknown, and so for my project I set out to investigate the proliferation timeline of SNCA+ neurons in the Cerebellum.

Background

Parkinsons Disease is one of the most common neurodegenerative disorders in the world, with over 8.5 million people experiencing some form of it, causing loss of motor control, cognitive impairments, and often leading to dementia. PD remains incurable due to it's complex but mostly unavoidable causes, such as genetics and aging, but remains an area of major research in the field of neuroscience.

Knowledge Gap, Novelty of This Project

What we can say with certainty is that the presence of Lewy bodies in the brain is a sign of PD. Lewy bodies are abnormal clumps of protein that cause impaired messaging and cell death within the brain, extremely commonly found in PD patients, and alpha-synuclein is always present within these Lewy bodies. Alpha-synuclein protein is a regulator for neurotransmitter release and pre-synaptic vesicle trafficking in the adult brain, and it helps to maintain neural homeostasis, but little is known about the embryonic proliferation and differentiation of alpha-synuclein producing cells (SNCA+).

This leads into my objective, to identify the proliferation timeline of SNCA+ cells during the embryonic development of the Cerebellum using an EdU proliferative assay technique.

Value

While lots of research has been devoted to the degeneration and later stages of SNCA+ cells, there is a large gap in our knowledge of the proliferation and differentiation of these cells, which leads to a lack of broader knowledge about the issue and negatively impacts understanding. My project sets out to construct a better foundational knowledge of these cells.

Materials

For this project, we developed a novel method using an SNCA/GFP transgenic mouse as well as EdU as a marker for proliferation.

EdU, a thymidine analog detectable via Click-Chemistry-enabled fluorescent labeling, allows for a significantly more accurate quantification of cells then its predecessors. It specifically labels proliferating cells within a subject, meaning that it enables very accurate cell counting.

SNCA/GFP refers to the genetic makeup of our mice subjects. We have inserted EGFP, or Enhanced Green Fluorescent Protein, into our alpha-synuclein producing gene. This means that whenever our target protein is produced, GFP will be produced in conjunction, allowing us to fluroescently detect SNCA+ cells.

By using both GFP and EdU in our target cells, and by looking at the colabelling of both of these fluorescent tags, we can find the proliferation timeline at each day of embryonic development. GFP marks SNCA expression, EdU marks proliferation, so the colabelled cells are our newly proliferated alpha-synuclein cells.

Methods

For this project, images of the developing cerebellum were taken at different days of embryonic development, and analyzed for colabelling under a fluorescent microscope. We administered the EdU into our pregnant mouse model and then allowed a 12 hour chase time at different days of development, which enabled uptake only in proliferating cells at that stage.

Data Collection and Sample Size

For data collection, we first took sections of our mouse brain and applied them to our slides. Sectioning was performed using a cryostat machine, and sections were usually between 10-20 microns thick. To detect the prescence of EdU in our cells, we used a copper (I) catalyzed click-chemistry reaction, binding a fluorescent Azide-dye to our cells.

Primary Findings

Using a cell counting software (FIJI) for image analysis and quantification (example in figure one), through this study we were able to identify the peak proliferation timeline of SNCA+ cells in the Cerebellum to be embryonic day 9-10. After the peak proliferation time, there is a steady decline to maturation at embryonic day 12.5, as visualized in figure two. After obtaining these datasets, I performed a statistical analysis using GraphPad's PRISM software, more specifically a one-way ANOVA and Tukey's range test, to learn the statistical significance of my results.

One-way ANOVA are a method of data analysis that identifies if the differences between two or more groups means are significant or not. I chose it as a method for this project specifically because I was analyzing datasets from different days of embryonic development, and wanted to understand the peak of proliferation and how quickly it dropped off after.

Tukey's range test is a post-hoc test I used after the one-way ANOVA, used to determine where the significant drops in averages were. I chose this as a secondary analysis to better determine when in the embryonic development of our subjects the largest dropoff in proliferation occured.

From my initial One-way ANOVA, I obtained a p-value of 0.0012 (**), validating that my results are statistically significant.

Discussion

These results indiciate to us a couple key points.

Firstly, we have validated and developed a technique for an EdU based neural proliferation assay in the cerebellum. While EdU has been used previously in different regions of the brain, very few cases have been reported of cerebellar assays.

Secondly, we can now say with confidence that the peak proliferation of SNCA+ cells within the cerebellum is at Embryonic Day 9 to Embryonic Day 10. The significance of these results lies mostly in the fact that the degeneration of these cells is one of the most common causes of Parkinson's Disease. While lots of focus has been given to those later phases of SNCA+ cells, both in research and in the literature more broadly, there exists a large gap in our knowledge about the early proliferation of these cells, which is what these results aim to tackle.

These results give us a new insight into the embryonic devlopment of the cerebellum, and give us a much more solid foundational knowledge of SNCA+ cells. Now that we have developed a proliferation timeline for our target cells, we can expand on those results by investigating the migration and their functions during neurogenesis.

Discussion

Our results give us a new insight into the embryonic development of the cerebellum, and a new benchmark to measure other aspects of SNCA+ neurons development.

By investigating the early differentiation, proliferation, and migration of these cells, we can better understand the mechanisms behind why they start to misfunction, and develop more effective treatments for when they do start to fail. Proliferation is the first step in that process, as we need a timeline to sequence the changes in morphology and maturation.

Now that we understand the peak proliferation time to be at embryonic day 9-10, we can say with confidence that SNCA+ cells are playing a role in the development of the brain. Because it is such an early day of embryonic development, and by that time they are already nearing the end of their cell cycle and reaching maturation, it logically follows that they are assisting with the formation of new neurons rather than just sitting stagnant.

It has been well documented that alpha-synuclein protein is present within all Lewy Bodies, but we still don't understand the mechanisms behind why these protein clumps form and why alpha-synuclein is always present in them. However, if we can better trace their migration patterns to find where they are maturing within the brain, that will enable us to better understand their deterioration and misfunction.

Conclusion

SNCA+ are early proliferating neurons within the cerebellum, with a peak proliferation time of embryonic day 9-10 that mature by embryonic day 12.5.

Future Directions

Increase replicates for a wider dataset, allowing more accurate results: current three replicate models only allowed a more restricted look at the timeline.

Perform additional in vivo stainings, such as H&E and Dil, to better understand the proliferating SNCA+ cells morphology and migration patterns respectively.

Time-lapse imaging of mouse embryo culture to trace origin and migration of SNCA+ neurons.

While a one-way ANOVA and Tukey's range test allow for a good analysis of my data, I would like to run additional tests with the wider datasets, such as a Two-Way ANOVA and Dunnet Test.

I'd like to thank the U of M's Human Anatomy and Cell Sciences department for their continued allowance to me of their facilities and funding.

I'd also like to thank the Central Animal Care Services department for their help in maintaining the health of our mice subjects and sourcing them to the lab.

Thank you to Farshid Ghiyamihoor, who took me under his wing initially and taught me proper lab procedure, as well as being able to answer any questions that came up during my research.

Thank you to Azam Asemi Rad and Dr. Hasan Marzban for their continued support and allowing me to work in their facilities.

Thank you to my school advisor Mahalia, who has supported me throughout the projects duration.

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