Keystone Neuroinflammation 2017

Last month, I attended my first scientific research conference as a PhD student at the beautiful town of Keystone in Colorado. The Keystone Symposia on “Neuroinflammation: Concepts, Characteristics, Consequences” was a week-long meeting with around 300 participants focussed on the interaction between two complex systems – the Central Nervous System (CNS) and the Immune System. Being a relatively new area of investigation, the conference brought together neuroscientists and immunologists who’re working on unraveling the mysteries of the brain and the human body. A traditional immunologist entering this field will have to grasp the intricacies of the brain with respect to microglia, astrocytes, neural circuits, neurodevelopment, the blood brain barrier (BBB), electrophysiology, behavior, degeneration, and more. Similarly, a traditional neuroscientist new to the field is expected to be familiar with all the details of the immune system from the heterogeneous immune cell types to different markers, the complement system, cytokines and chemokines, and be able to define aging in the context of immune regulation. As for me, being new to both the disciplines, this was an exciting opportunity to discover bold ideas as well as revisit my research objectives from a fresh perspective. Not to forget how wonderful Keystone itself was and made me realize how much I love the mountains!

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Without going into much of the details, some highlights of the conference are summarized below.

Microglia – the good, the bad, and the ugly

As expected, microglia were a hot topic through the meeting. Being the only resident immune cells in the CNS, microglia make up around 10% of the total cells in the brain. Activated microglia exhibit self-renewal and proliferative capacity along with phagocytic capability by engulfing toxic misfoled proteins (such as the aggregated amyloid beta peptides in Alzheimer’s disease and the alpha syneucleins in Parkinson’s disease). These “good” microglia are therefore essential for the maintenance of brain homeostasis. But what happens when these macrophages go haywire and start chewing up healthy neural synapses and start making more pro-inflammatory cytokines than necessary? How do these “bad” microglia contribute to neuroinflammation and accelerate the neurodegenerative phenomena? More importantly, the M1/M2 phenotypic characterization of microglia that I have previously written about is now considered to be an obsolete concept. Currently, we know that these cells are much more complex than to exhibit two polarized states. Microglia are heterogeneous in that their microenvironment dictates their characterization and therefore can exhibit multiple phenotypes.

Secretome – Looking beyond the transcriptome

A common method to predict the cellular phenotype or function is to study the transcriptome profile of a group of cells (or a single cell) under different activation states. In his talk, Christopher Glass highlighted the differences between the transcriptomes of human microglia and mouse microglia, and stressed on the importance of working with microenvironment-dependent microglial gene expression data. His work also showed the major differences in the up-regulated vs down-regulated genes in human vs mouse genomes highlighting the drawbacks of utilizing mouse models for characterizing human microglial cell behavior. Hugh Perry, in his talk laid out the “secretome” profile (the cytokines and chemokines) of microglial cells in the brain. Dr. Perry described the journey of his lab’s work towards targeted immunomodulation in chronic neurodegeneration by using this data.

The BBB – Pushing the boundaries of neuroinflammation

Among many topics of discussion, an interesting area was the role of the BBB in influencing immune cell proliferation into the CNS under stress and injury. (Side note: Other interesting areas of discussion were – cool new tools being developed for drug discovery, the use of various animal models in studying neuroinflammation such as transgenic and wild type zebra fish, drosophila, induced pluripotent stem cells, humanized mouse models, and rats). Studying the BBB in the context of inflammation can lead to some interesting questions such as – “What are the differences in the markers expressed and cytokines produced by the infiltrating macrophages versus the resident microglial cells?“, “How can the resident microglia be characterized in the context of microglial functions such as phagocytosis?“, “How do the pericytes and the oligodendrocytes contribute to inflammation in different disease models?

It’s all about the microglial cross-talk with glia (astrocytes) and neurons

Recently, Ben Barres’ group showed that activated microglial cells induce the formation of toxic A1 astrocytes by releasing TNF-a, IL-1a, and C1q that causing neurotoxicity and the eventual degradation of neurons. This study was significant because it showed that the neural death occurs through astrocytes and not directly from microglia. The importance of the role of astrocytes in mediating neuroinflammation has increased since then. Of course, the entire process in itself is a cross-talk between various cells within a particular microenvironment. Hence, when we discovery and design drugs to target microglia, we should also consider the effects of the compounds on glia and neurons in vitro, before moving to the in vivo studies.

Is anti-inflammation and not pro-inflammation the culprit in Alzheimer’s disease?

Perhaps one of the most provocative talks of the conference was by David Hansen from Genentech who illuminated the role of Triggering Receptor Expressed On Myeloid Cells 2 (Trem2) and debunked the myth of pro-inflammatory cytokines in Alzheimer’s pathology (i.e., lot of inflammation causes Alzheimer’s). Dr. Hansen highlighted key genes that are evident in anti-inflammation and immune suppression to be up regulated in his studies pointing towards an alternative activation pathway for neuroinflammation. This is an example where looking into the secretome along with the transcriptome becomes crucial in characterizing the cells and their subtypes in the progression of the disease.

My poster – Combination drug repurposing for the synergistic effect of enhancing microglial phagocytosis and reducing neurotoxicity in Alzheimer’s disease 

I presented a poster on one of my ongoing projects on combination drug repurposing for Alzheimer’s disease. I am grateful for everyone who stopped by and for all the valuable feedback and comments that I received on my work. Many questions were aimed towards the computation wing of the project (that I don’t directly work on). Briefly, these compounds are identified by utilizing the interactome-based drug discovery pipeline (called CANDO) that maps the signatures derived from the interaction between all the proteins in the proteome and all the human approved compounds. The overlapping mechanisms or pathways between Alzheimer disease with other diseases such as diabetes, heart failure, inflammation, among others can be utilized to determine drug behavior and therefore utilized for predicting novel targets.

And finally – Colorado!

I cannot leave out Colorado in this conversation about Keystone Symposia. I have been living on the plain lands among endless corn fields for almost four years now. Being around snow-capped mountains was an absolute treat to the eye (and to the soul). During the afternoon break sessions, we drove to various locations such as the Loveland pass which is around 11,990 feet above sea level in the Rocky mountains and the riven run area.  Intense winds and long stretches of snow welcomed us after a beautiful drive up the Rocky’s. I’ll let the photos do rest of the talking. Overall, attending the Keystone conference at this time in my research career was a great decision. I now have a better understanding of the field and my own project. I should really thank my advisor since it was he who pushed this idea and gave me valuable insight and advice throughout.

[Almost] one year milestone – my first advisory committee meeting

Advisory committee meetings are held once every year (or twice every year, if the student or the committee chooses to do so) to asses the progress of a grad student’s PhD thesis. The meeting involves a written report that is to be submitted to the committee a week prior to the meeting and an oral presentation on the D-Day. During the presentation, the validity of the research work is thoroughly discussed along with the future direction(s) of the project(s) being undertaken. The advisory committee meetings are extremely important for the successful advancement and completion of a thesis – it is where brutal yet honest feedback is conveyed. We as grad students are forced to think critically of our work and defend our hypotheses as well as our results.

My first advisory committee meeting was an intense two-hour long session on a rather dull Tuesday afternoon. As I explained the premise of my work and my goals for the next year, my committee members brought up important questions that I had not previously ever considered. All the members of my committee, including my advisor, were supportive and encouraging. I learned some valuable lessons from the entire experience and got some great feedback from everyone. Some interesting and important points highlighted in my feedback assessment were –

  • Think carefully about how to present data and set up an argument in my presentation.
  • Work on clearly identifying the premise that sets the stage for my hypotheses.
  • Be critical about my data.
  • Continue to read literature: more reading, and reading more critically.
  • Focus on developing more robust immunological assays to answer the questions in my aims.
  • Interact more with colleagues on campus and at other schools to learn and get insight into techniques and relevant assays (wrt understanding what works and what doesn’t).
  • Explaining the experiments in detail before delving into my results (every assay is unique and has a question to be answered).
  • Think about how I want to present the previous studies done in the field that are relevant to my questions.
  • My hypotheses should be provided with a context (what is the data in support or against my hypotheses?)

These were just some of the significant parts of the feedback that I received. Now it’s time to put these into action and definitely work on continuing to build on my project more confidently. More later.

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.

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

Orientation week: What do I want out of grad school?

The first week of grad school was intense and exhaustive with all kinds of information being tossed at us from all directions. We started off with a formal introduction to the school, the department, and all the resources available at our disposal like the libraries, mentors, health benefits, and so on. Besides all this, a main objective of the orientation week was to decide the first two labs that we are interested to rotate in. The process involved meeting with several professors, going on lab tours, meeting other grad students and evaluating if a lab was a good fit for us or not. Although I knew the direction of research I wanted to pursue, discovering so many options and learning about cool new research areas left me wondering if I really knew what I wanted to be doing for the next five years! Right now, I feel like a first grader starting school for the first time and constantly being exposed to many things I never knew existed.

Grad school 101 - What I don't know
Grad school 101 – What I don’t know

Being in a big umbrella program, there are ten different training groups to choose from. First year graduate students pick four labs within any of the groups to rotate in during their first year. This is very different from a departmental graduate program where a student can only rotate in labs within that respective department. After all the decisions and evaluations, I have chosen my first two labs for the semester and I am looking forward to be officially starting next week.

This process has made me question some decisions that I’ve taken in the last couple of years. “What do I want out of grad school?” seems to be the most significant one. Before beginning my journey, I knew that I wanted to train to be a good scientist, learn how to think, develop skills unique to my field, master techniques that will make me employable, learn how to learn, and be an overall well rounded researcher. Now I’m not sure if there is a definite answer to the question. It is something that I’d have to figure out on-the-go.