Author Archives: Justin Savage

Investigating the Role of PTPRZ1 in Astrocytes

Astrocytes are a non-neuronal cell population in the brain which perform many important functions, including regulating synapse formation and function and helping to form the blood brain barrier. Despite the importance of these cells in vivo, little is known about how they carry out these critical functions. Protein Tyrosine Phosphatase Receptor Z1 (PTPRZ1) is a transmembrane protein which is heavily expressed in astrocytes and oligodendrocytes, another non-neuronal cell in the brain. While some function has been found for PTPRZ1 in controlling oligodendrocyte maturation, little is known about the function of the protein in astrocytes. The present study investigates the role of this protein by determining its location of action, its expression across development, and finally what it is doing in astrocytes. To determine the location in which PTPRZ1 is acting, immunohistochemistry (IHC) was used on mouse brain sections. These tests showed expression of PTPRZ1 along the branches of astrocytes, with a co-localization between the PTPRZ1 and our astrocyte marker GFP. We also found the protein to be present on western blot in the brains of mature animals. We plan to continue this work by investigating PTPRZ1 levels over different developmental time-points using western blot and quantitative PCR analysis.

An Engineering Perspective

I thought that Iris’ research into Traumatic Brain Injury (TBI) was quite interesting. I was initially interested in the project because I am also doing research in neuroscience. What I quickly discovered through her talk, however, was that her research was much less molecular and much more mechanical. Thinking about the different kinds of acceleration that could damage the brain was a very new experience for me. The idea of thinking of the brain in as a rigid structure that could be thrown and broken was a very new idea.

Rather than repeatedly breaking our prized model subject, Iris first models these assaults in a computer program. I thought it was really cool that they had this predictive power to know what particular injury would occur given a certain type of trauma. I thought this could be of great clinical relevance as a reconstruction of the trauma, quickly ran on a computer, could be a great aid to a surgeon to know where bleeding would occur.

I was also surprised when Iris said that she would use a dead pig brain to test these forces and strains. As her questions are purely on the forces the brain was enduring, physiological responses are not necessary.

The final thing that was different about Iris’ talk was her ending it with ideas about the potential impacts of her research. While I’m sure my PI must be thinking of these things while writing grants, I had never thought of a reason for my research other than to learn more about the brain. Iris’ specific mention of applications to headgear and neurodegenerative disease, which are implicit in any neurobiological research, were an interesting consideration to make.

Where is PTPRZ1?

My project comes out of the work of my mentor Katie Baldwin. Katie used data collected from the Barres lab at Stanford to compile a list of genes that were highly expressed in astrocytes relative to other cells in the brain. She then took this list and looked into the identity of each gene to see what was known about it and if it could be interacting with synapses (the focus of the lab being astrocytes’ influence upon synapses). She then looked at the effect of knocking out some of these genes on astrocytes to see if there were any clear differences in the shape of the cells. Once she had made this list of research subjects, Katie, as well as some other members of the lab, began to investigate what these genes are doing in astrocytes.

When I came to the lab, Katie told me about a few genes from this list that hadn’t been investigated yet and would be good projects for me to work on. One such gene she was particularly interested in was PTPRZ1. PTPRZ1 is a membrane-bound receptor that removes phosphate modifications from proteins. This sort of process can be important for controlling existing proteins, so I was very intrigued by this research target. Katie had also already seen that losing this gene caused astrocytes to look strange.

This summer I have been working on trying to figure out where this protein is expressed and developing techniques to be able to see it both in cells and in dead tissues. I hope to have good protocols for microscopy and western blotting to learn more about this protein’s function. I also plan to return to the lab when the semester starts to further our understanding of PTPRZ1 role in astrocytes.

Exciting Questions

As we sat in the dark room behind the great and powerful Olympus microscope, I decided it would be a good time to ask my mentor, Dr. Katie Baldwin how she had gotten to this point. Starting college at the University of Michigan, Katie had first wanted to study biochemistry. When confronted with the obscene mathematics of this field, however, Katie made the sensible choice of switching to Cell and Molecular Biology. After completing her undergraduate degree, Katie went on to study stem cells as a graduate student at U Michigan. She was most interested in how the body could repair itself and started work in a lab to study stem cells in the spinal cord, an organ notorious for its inability to repair. Late in her grad school career, Katie went to a Cold Spring Harbor Laboratories conference to learn about the cool and exciting things going on in the world of stem cells. The problem was that there “wasn’t much cool and exciting”. Katie found many of the questions being asked in the field to be subtle variations of those asked in the early days of stem cells a decade ago. She then decided for her postdoctoral work to head in a different direction.

Enjoying her work with the spinal cord and being intrigued by the role of microglia in its repair, Katie searched for laboratories that were studying the poorly understood glial cells of the brain and how these cells could be playing a much greater part than previously recognized. This led Katie to ask Dr. Cagla Eroglu to meet with her at a conference they were both attending. Katie said she knew from that first meeting that Cagla was a “person she could work with” and that she was fascinated by the “simple questions [in glial biology] that had not so simple answers”. This led Katie to the Eroglu lab where she is currently working on studying proteins expressed by astrocytes to see how they are affecting neuronal synapses in the brain.

After tracking her path in science, our conversation began to shift to more general advice about the profession. Katie mentioned how she enjoyed being in a smaller lab with good mentoring and how she enjoyed being able to take risks in her work to answer the tougher questions that no one knew hardly anything about. She described the best part of her job as the moment of discovery when she’s able to find something new and interesting. She then said a lab proverb that stuck with me: “If you do everything right, every time you’re not learning”. This idea that failure was an inherent part of success made my own efforts in the lab seem more meaningful. It takes a large body of work to make a breakthrough, and much of this work goes into figuring out what doesn’t work.

With these themes in mind, we looked at the computer screen to set up for the next picture and saw an interesting morphology that seemed to only be present in one of our conditions. It seems the next discovery was before us.

Keep Calm and Kick Astrocyte

My first week of research in the Eroglu lab has been filled with a lot of information. Having done research before, I didn’t quite expect to find myself spending so much time as the student. However, I have learned so many new techniques in just 5 days. These have started to quickly reshape my plans for the summer. I’ve already started to see how scientists in the Eroglu lab go about investigating a new protein, like I will be doing for PTPRZ1. First, we thinly section brains into 20 um slices, stain these with fluorescent antibodies, and look and image them with a microscope. This process can be repeated for different kinds of mice, like individuals of different age, sex or genetically engineered strains. I expect to repeat these methods many times over the summer to try to get a better idea of what PTPRZ1 might be doing.

Another exciting part of my research has been learning how the different members of the lab go about their work and learning how to plan experiments. Katie has already done a good job of showing me how the whole process of experimentation works and I hope to build on the things we are able to learn each week in order to come closer and closer to understanding PTPRZ1.

I’ve also already started making plans with Katie and my PI Dr. Eroglu to continue my research when the semester starts so I hope to be able to learn a good amount of techniques so that I can easily flow into doing work over the semester. I hope that I am able to make good progress over the next 7 weeks so that I may have a better idea of where PTPRZ1 is acting and can focus on figuring out what it is doing by the time the semester starts.

My first week of research in the Eroglu lab has been filled with a lot of information. Having done research before, I didn’t quite expect to find myself spending so much time as the student. However, I have learned so many new techniques in just 5 days. These have started to quickly reshape my plans for the summer. I’ve already started to see how scientists in the Eroglu lab go about investigating a new protein, like I will be doing for PTPRZ1. First, we thinly section brains into 20 um slices, stain these with fluorescent antibodies, and look and image them with a microscope. This process can be repeated for different kinds of mice, like individuals of different age, sex or genetically engineered strains. I expect to repeat these methods many times over the summer to try to get a better idea of what PTPRZ1 might be doing.

Another exciting part of my research has been learning how the different members of the lab go about their work and learning how to plan experiments. Katie has already done a good job of showing me how the whole process of experimentation works and I hope to build on the things we are able to learn each week in order to come closer and closer to understanding PTPRZ1.

I’ve also already started making plans with Katie and my PI Dr. Eroglu to continue my research when the semester starts so I hope to be able to learn a good amount of techniques so that I can easily flow into doing work over the semester. I hope that I am able to make good progress over the next 7 weeks so that I may have a better idea of where PTPRZ1 is acting and can focus on figuring out what it is doing by the time the semester starts.