Blog 2: Effect of Diet of Microglial Dynamics in PTEN-ASD: Wildtype Fish Preliminary Results

For my project so far I have analyzed microglia dynamics of wildtype fish that were fed 3 different diets (high fat, high glucose, and high protein (control). So far I have found differences between microglia dynamics (area and movement) of wildtype fish fed a high fat diet compared to the other two diets. I have spoken with my faculty mentor about the possible explanations behind these observed differences and we have came up with a few hypotheses that we are planning to test next semester. I have also been looking at scientific literature about microglia motility and the effects of a high fat diet on microglia as well, to help explain my preliminary results. I have also presented my preliminary findings at the monthly biology research group meeting we have here at Pace and I received some input from other professors on what we could do to get a better understanding of what our results mean. 

My results of my experiment have found that the microglia of wildtype fish fed a high fat diet had a significantly larger cell size as well as faster velocity. Additionally, I have found that microglia of fish in this condition moved greater distances and also moved for less time overall. Other research done on the effects of high fat diet have also found signs of microglial dystrophy, one of them being soma enlargement. While this helps validify my findings, I plan to analyze more fish (my current number of fish analyzed is 6) to see if this trend holds up. Interestingly, the research conducted on the effects of high fat diet on microglia did not analyze motility, which makes it difficult to verify my findings. However, I found that there was some research conducted on epilepsy’s effect on microglial motility in a mouse model, which could help give me some basis to compare my results, as epilepsy is characterized by neuroinflammation and microglia activation, which is similar to the effects of high fat diet on the brain. 

This study found that microglia in epilepsy model mice moved greater distances, while maintaining their velocity. This partially matches my findings, as I also found that microglia in the high-fat diet condition moved overall greater distances, however I also found that they moved at higher velocities. Again, I plan on analyzing more fish to see if this trend continues. Overall, after speaking my faculty mentor, we devised a few more tests to get a greater grasp on what are the effects of diet on microglia. 

Some of these include acridine orange staining (will help us analyze neuronal death to see if microglia are reacting to neuronal damage induced by certain diets), sholl analysis (will help us analyze differences microglial morphology in response to certain diets), annexin-v staining (will help us see if microglia are undergoing phagocytosis), cell counts (will help us see if microglia are proliferating in response to certain diets) and western blots (will help us see if microglia are producing pro-inflammatory cytokines in response to certain diets). Additionally, next semester we are getting a new microscope in the lab, which will help us get a total view of the zebrafish tectum, allowing us to analyze all of the microglia within it. 

In closing, my current hypothesis based on my results is that a high fat diet causes increased microglial activation, which can be evidenced by their enlarged cell bodies, and lower duration of movement, as both imply engulfment of cellular matter, which microglia are known to do in their activated state. However, further analysis using the methods I described above will give me more of a clue if this is what is indeed occurring.

 

Blog 1: Effect of Diet on Microglia Activation and Dynamics in PTEN-ASD

The title of my research project is “Effect of Diet on Microglia Activation and Dynamics in PTEN-ASD”. The purpose of this research is to analyze the impact of diet on the role of microglia in synaptic plasticity in a phosphatase and tensin homolog Autism Spectrum Disorder (PTEN-ASD). From this research, I expect to learn if microglia has a role in the synaptic abnormalities observed in individuals with PTEN-ASD and if dietary composition has an effect on both microglia function and the general brain anomalies observed in individuals with PTEN-ASD. Based on reviewing previous scientific studies, I hypothesize that microglia motility will be negatively impacted in my PTEN-ASD model which will lead to an increase in synaptic numbers. Furthermore, I hypothesize that diet will impact synaptic numbers in both WT and PTEN-ASD models via microglia motility and activation. 

Research Design (Methods):

To test this hypothesis, I will use the zebrafish as a model organism, as they are vertebrates, undergo rapid development, and are transparent early in life. Due to genome duplication they have two genes encoding PTEN: ptena and ptenb. These two genes have redundant function and removing either version yields a viable fertile zebrafish. Using fish that are lacking the ptena or ptenb will be my PTEN-ASD model, as using partial knockout of PTEN closely captures the reduced PTEN protein observed in PTEN-ASD.  I will quantify microglia dynamics in ptena-/- or ptenb-/- fish crossed with a transgenic macrophage/microglia reporter line (Tg(mpeg1:gfp)) and compare it to WT fish crossed with the reporter line Tg(mpeg1:gfp). At 5 days postfertilization (dpf) I will administer one of three diets to feed each the PTEN-ASD and WT fish: a high glucose diet, a high fat diet (egg yolk), and a high protein diet (control).  Following feeding, I will perform time-lapse imaging of the microglia and observe any changes in their dynamics and morphology. I currently have preliminary data showing that a high glucose diet in wild type zebrafish causes aggregation of microglia, as well as, a lack of interaction of microglia with retinal ganglion axons, something that was not observed in the control protein diet.  I believe this research will be beneficial in understanding and examining the role of diet on the activity of microglia in a PTEN-ASD model. I believe if a link to diet and microglia activity is found, this knowledge may be useful in making dietary recommendations for individuals with PTEN-ASD.