It’s brain awareness week!

Hello all!  I wanted to take a few minutes to write something for the brain awareness week. This is important to me because my research focusses on understanding the role of the immune system in the brain. For a very long time, the brain was thought to be an “immune privileged” organ i.e., it was thought that the brain is protected from all the peripheral insults and that it is “divorced” from the rest of the body. In 2015, it was shown that there exists certain lymphatic vessels that connect the CNS to the rest of the body (1). The lymphatic system carries immune cells through a network of vessels and tissues; it connects the bloodstream and tissues in order to remove dead cells and other debris. The discovery of the new “glymphatic system” has opened new avenues to study the connection between the brain and the rest of the body. This is especially helpful in understanding the role of the peripheral immune system on the CNS during infections, injury, and other disease insults.

glymphatic system
Old lymphatic system (left) and newly discovered lymphatic system in the CNS (right). Source: University of Virginia Health System

My work focusses on a specific cell type in the brain known as microglia which are are the resident macrophages of the CNS (they eat up and clear out the bad stuff in the brain like dead cells and mis-folded proteins). Microglia are the only known immune cells of the brain. Compared to all that’s known about the cells of our body’s immune system (B cells, T cells, NK cells, neutrophils, basophils, Treg cells, MDSCs, TH1, TH2, and many many more with several subtypes of each cell), it is safe to say that cells of the CNS are poorly understood. My efforts are focussed towards understanding the role of microglial cells in neurodegenerative diseases such as Alzheimer’s Diseases (AD) , Parkinson’s Disease (PD), Multiple Sclerosis (MS), etcetera. These diseases are characterized by mis-folded proteins that aggregate in the different regions of the brain tissues causing the neurons to degenerate and eventually die. The microglial cells in these disorders play a major role in disease progression by regulating many pathways involved in cell-cell communication, cell survival, and cell death. This is a relatively new and an exciting area of study with many missing links and questions to be answered. I will try my best to keep this space alive with updates and stories! In the meantime, here’s a fun read on Leonardo da Vinci’s contributions to neuroscience: http://www.sciencedirect.com/science/article/pii/S0166223600021214

And here’s a 1504-1506 drawing of the human brain by da Vinci:

Leonardo da Vinci's contributions to neuroscience
In the upper figure, the three ventricles are labeled imprensiva (anterior ventricle, corresponding to the paired lateral ventricles), senso comune (third ventricle), and memoria (posterior or fourth ventricle). Below the ventricles, seven pairs of cranial nerves are shown. The lower figure shows a human head in an exploded view, with the skull raised over the brain and from the head. Source: 

Sources:

  1. Louveau A, et al. Structural and functional features of central nervous system lymphatic vessels. Nature. 2015;523(7560):337–341. doi: 10.1038/nature14432.
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Mountains are for climbing

Guess what? I successfully powered through my first year of grad school! My first year was all about rotating from one lab to another in hopes of finding a permanent home where I would metamorphose from being a timid first-year grad student into a fearless, hopeful, and an optimistic researcher powered by data and caffeine.

I cannot believe how much I underestimated the process moving forward. Between taking courses (and therefore preparing for exams and working on assignments), attending seminars, teaching two labs (and two recitations, one office hour, plus all that grading), writing grants and fellowship applications, AND doing my own research in any time that I find in between, it has been a CRAZY semester so far. One of the most disheartening things is how much behind I am on my reading. I am usually so tired by the end of the day that my brain freezes and will not take in any new information that’s thrown at it. My eyes burn down, my legs become numb, and my back starts yearning to crash on my cozy bed as soon as I get home. The papers keep piling up, experiments haunt me in my dreams (the night before every rat dissection, episodes of drug treatments and protein assays flash before my eyes!) and I dread the 1:1 meetings with my PI having no data to report or no hypotheses to discuss. Is this normal for a second-year grad student? I don’t know. I am trying to make up for all the research time lost due to coursework and teaching by working till late evenings and on the weekends. There is no difference between a Friday and a Saturday or a Sunday anymore. Is this grad life? Are we more than just grad students?

A faint silver lining amidst this craziness has been the fact that I have started to formulate the research direction I want to pursue my main Ph.D. thesis on. Of course, I have been working on other projects on the side, but I have now started to connect the dots and evaluate my main project in terms of its novelty, idea, and the required experimental framework. I realized that the more I write about my work in grant/applications or the more I attempt to justify it, I start to identify the gaps in knowledge that needs to be filled. This is truly exciting. The funny thing is, I sometimes wish there was a guidebook that could tell me exactly what I need to think or how I should approach a problem. Unfortunately, there isn’t one. There is so much knowledge out there, but no guidelines for using it. Maybe this is what its all about?

Picking my permanent lab

Almost a year has passed since I started my Ph.D. journey in the land of snow and maize. After four long lab rotations across three departments and hopping from one project to another, it is time to pick a permanent lab and a research direction.

IMG_6374.JPGI am happy to announce that I have officially joined the distinguished Department of Chemistry at my university and have begun my research at the Institute for Drug Discovery where I will work for the remaining period of my doctoral degree. I couldn’t be happier with my decision which was mainly determined by three aspects – my advisor/mentor, the research area and the lab (environment and members). It feels good to finally know where I am headed towards and not feel lost or uncertain. Every one of my rotations was unique and helped me learn the nitty-gritty of grad school. Moving forward, I will focus on brain-related disorders like Alzheimer’s Disease and work towards understanding a tiny piece of a large puzzle which may aid in finding a cure/prevention/slow down the progression of the disease. Specifically, the overarching theme of my work will be to identify and test compounds predicted for the disease by taking into account all the possible interactions between biomolecules in the protein universe (aka the proteome). Traditional drug discovery methods involve targeting a specific protein or a specific pathway and thereby limiting the possibility of finding successful leads. In reality, we know that one biomolecule interacts with several other biomolecules in several different pathways. Interactome-based drug discovery is promising because of its broader and quicker approach compared to the other mainstream pipelines that exist today.

One other major factor that helped me decide my major lab was the computation aspect involved in drug discovery research. Taking the challenging Computational Chemistry course this semester helped me take the first step towards learning about some of the components of computer-aided drug discovery. It is amazing how the two channels of research (wet lab and dry lab) finally come together in solving some of the major challenges in health and medicine. Anyway, I will continue to update here more on my day-to-day lab rat adventures. I am excited to start this new chapter of my life and see where it takes me! :)

Thoughts on lab rotations

The thing with first-year rotations in a Ph.D. program is that anxiety starts kicking in somewhere along the way when you consciously identify the lab that you want to join and want to get started right away. Having realized that this is going to be a long journey and rushing into things may not help, I am now gaining patience and perspective, and hope to make the most of the remaining time of my first year.

Rotations are a great way to learn about a lab and get involved in the nitty-gritty of research. I was warned at the beginning by a few seniors that I would either love a lab or reject it within the first few weeks of the rotation. Mind you – this has nothing to do with the science pursued in the lab (one wouldn’t decide to rotate in a lab if they didn’t find the research interesting in the first place). This is more about getting comfortable with the way a lab functions and deciding if the environment is a good fit for you. An eight-week lab rotation is really like an eight-week long interview with a potential PI and the lab! It is essential to identify the kind of relationship you foresee having with your advisor for the next couple of years (and beyond). This is perhaps one of the most important aspects of a rotation for me, next to the research work. A good mentor-mentee relationship can go a long way and can be extremely beneficial to one’s academic/professional career. I prefer having an open channel of communication with my mentor and learn as much as possible from him/her.

Not all graduate programs require laboratory rotations. Many departments or programs accept or reject students simply based on their application and/or an interview. In the UK for example, students are recruited to work on specific projects and grants as a part of their Ph.D. for the time period of around 3 years. This may not benefit the candidates who wish to propose their own ideas and develop their own thesis based on their individual research interests. In the US, for most graduate programs in the life sciences (mainly biology and chemistry), the average time for graduation is around 5-6 years. I believe that the freedom and independence of this system trump the short graduation time of the other systems. Although I am certain that both sides have their set of merits and demerits, at the end of the day, the journey is unique to each one of us and what we make of the experience matters the most.

Chasing cancer cells

Glioblastoma_-_MR_sagittal_with_contrast
Glioblastoma (astrocytoma) WHO grade IV – MRI sagittal view, post contrast. 15-year-old boy. Image courtesy: Wikimedia commons

Glioblastoma multiforme (GBM) is one of the most invasive forms of malignant brain tumors. By the time the tumor is removed from a region in the brain, the cancer cells rapidly metastasize and spread throughout the brain. Common treatment procedures such as chemotherapy and radiotherapy are not completely effective due to the aggressive nature of the tumor invasion. Therefore, many treatments also target the migratory properties of the tumor cells. Several proteins (focal adhesion kinase, paxillin, vinculin) are over-expressed in the extracellular matrix of the tumor microenvironment and help the GBM cells proliferate through the brain tissues. A treatment approach is to target these proteins and hopefully prevent -or at least reduce- the tumor cell invasion.

Studying this type of a cancer model is tricky. Most of the work that has been done in the traditional 2-dimensional cell culture environment cannot be translated into the 3-dimensional environment of the brain. We need a system that mimics the brain to realistically model the tumor cell growth and migration. As a part of my third rotation, I have been investigating the migration characteristics of GBM cells in tissue-engineered, 3-dimensional cell culture matrices that mimic the brain environment. The cells are grown in a collagen matrix containing components of the brain extracellular matrix (hyaluronan, astrocytes, etcetera) and the tumor cell migration is studied by tracking the focal adhesion proteins. However, this is not easy. Cancer cells have shown to modify their migratory patterns based on the physical conditions of the tumor microenvironment (Herrera-Perrez et al. 2015 Tissue Engineering Part A). This makes it more difficult to target the adhesion receptors to ultimately inhibit tumor invasion. It is also challenging to prevent the disruption of the cross-talk between the targeted receptor protein and other important signaling molecules during evaluation of the treatment procedures. Overall, new innovative strategies are required that focus on the diversity and adaptability of tumor cell invasion and migration.

Blots, cultures and assays concludes rotation two

This week officially concludes my second laboratory rotation in the neuropharmacology lab with research focussed on  G protein-coupled receptors and their application in several neurological disorders such as depression and anxiety. In the eight week duration of my rotation, a few things were achieved with respect to validating the activity of the newly developed M4R-DREADD (a designer M4 muscarinic receptor exclusively activated by a designer drug). Designer receptors are engineered such that they are solely activated by a synthetic ligand. This opens new avenues in the activation and control of G protein-coupled receptors’ function in vivo.

After a long break from my Master’s research, I got back to maintaining two cell lines – CHO (Chinese Hamster Ovary) and HEK293 (Human Embryonic Kidney) cells, in which the opioid receptors were expressed for all my experiments. These cells were used to characterize the receptor signaling by western blot analysis of the downstream MAPK/ERK signaling  upon stimulation by a few agonists/drugs of interest. Luckily, the lab acquired a new fluorescence microscope during this period which helped us observe the recruitment of the β-arretin2 protein by δ-opioid receptors in HEK293 cells stimulated with clozapine-n-oxide, a synthetic ligand.

mrrd-gfp barrest-cherr cno 0 min 20x_Overlay copy
HEK293 with M4R-dreadd 20x
mrrd-gfp barrest-cherr cno 10 min 20x_Overlay copy
HEK293 with M4R-dreadd 20x

This week, I had a lot of difficulty in handling the mice. Being my first experience with animal work, watching the mice anxious and struggle while we held them down was hard. I am still pretty unsure about how I feel about animal work (if I HAVE to do it to save my research in the future, I will) but I definitely need more exposure and practice with them.

Overall, this lab taught me a lot, even if some days were stressful and  tiring. I feel like I learned and enhanced many skills in the process (primer design, restriction analysis, cell culture, cloning, western blot, cAMP assay), and got a feel for the lab at the same time. Through the course of these past two rotations, I have met some really smart and dedicated people. In the end, I am grateful to have had this opportunity.

The Human Kinome

The entire set of 518 protein kinases in the human genome makes up one of the largest of all human gene families. These enzymes catalyze the phosphorylation of proteins, specifically serine/threonine and tyrosine residues – an important reaction that regulates key cellular functions like cell division, metabolism, and apoptosis in normal and disease states. This makes kinases key therapeutic targets in several diseases such as cancer, neurodegeneration, behavioral disorders, diabetes, and cardiovascular diseases. Interestingly, both the labs that I’ve been in so far are focussed on kinases involved in pancreatic/prostrate cancer and GPCR signaling in the light of alcohol/drug addiction. Leaving this nice phylogenetic tree here as a reminder and reflection of kinome research!Map of the Human Kinome