Download Research Proposal: Nivedita Chatterjee

Research Proposal: Nivedita Chatterjee
With the completion of human genome mapping, the focus of scientists seeking to
explain the biological complexity of living systems is shifting from analyzing the
individual components (such as a particular gene or cell type) to understanding the set
of interactions amongst the large number of components that results in the different
functions of the organism. This has led to an integrated, multi-disciplinary approach
to biological questions that focuses on the network of interactions instead of
considering only the characteristics of isolated elements.
I am pursuing a research program that combines genomic, cell and molecular biology
approaches with those of simulation in order to address important questions on brain
function. My aim is to reveal basic principles of intercellular communication in the
nervous system, specifically related to the role of neuron-glia interactions in health
and disease. With this goal, I will be working on multiple model systems, including
cultured neuron and glia, organotypic explants, biological samples from patients and
the nematode Caneorhabiditis elegans. In the following I briefly outline a few
highlights of my research plan for the immediate future – separately listing the main
objectives for the next two years (2012-2014) and the next five years (2012-2017).
1. Background
Recent research has challenged the classical view of glia as being only “supporting
cells” of the brain, and has instead shown them to be active components in the
functioning of both the central nervous system (CNS) and the peripheral nervous
system (PNS). Glial cells can regulate neurons by release of soluble ligands or
retrograde signalling leading to modulation of various factors, among them internal
calcium. In view of this ability of glial cells, it has been established that they play a
major role in coordinating cell-cell communication in the brain. It is also an intriguing
feature of evolutionary neurobiology that climbing up the evolutionary ladder one
sees an increasing glia to neuron ratio, a fact which might have implications for the
robustness and efficiency of information processing in the brain. Knowledge of the
changes in dynamics of intercellular communication would therefore throw light on
questions pertaining to both spontaneous network activities of cognitive processes and
brain disorders.
2. Methods
I am using a variety of experimental biology techniques to probe into glial roles.
Microscopy (both live imaging and on fixed samples) functions as one of my basic
tools. We have standardized Calcium imaging and use of other dyes, and used them
for studying dynamic processes. I shall be using Total Internal Reflection Microscopy
(TIRF) in the near future in order to study cell surface phenomena. Basic biochemical
protocols as well as cell biology assays are routinely done by my group. I have also
established homogenous cell cultures on which much of our experimental work is
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based. Functional studies on tissues are also performed on organotypic explants. Our
current cell culture work is based on retinal tissue. I am also familiar with brain
cultures, both glial, neuronal and co-cultures. We are also establishing cell cultures
from the nematode C.elegans. Routine molecular biology is carried out by my group
for both PCR-based and micro-array work. Given the direction of our work, I shall be
increasingly using genomic tools to study regulation of changes in tissues during
pathological conditions. My work with endocannabinoids would also entail setting of
a lipidomic platform. As my research is necessarily interdisciplinary, we also use biophysical, bioinformatics and systems biology tools regularly.
Details of instruments necessary for my current and future research are given later.
3. The Retina: a model system for understanding neuroprotection by glia
Principal Research Objectives
2012-2014
 Studying effects of HIV-1 soluble proteins on retinal degeneration
 Investigating Endocannabinoid involvement during retinal inflammation
 Mueller cells and microglia in retinal neuroprotection
 Signal transduction pathways involved in neuroprotection in lipid raft
microdomains
2012-2017
 Studying blood-retinal barrier changes on exposure to HIV proteins
 Studying microRNA profile in retinitis
 Investigating Endocannabinoid levels in retinitis
One of the central themes of my present research is using Mueller cells of the retina as
a target for regulating neuroprotection. Mueller cells play an important role in
pathologic processes of retinal wound healing and neovascularization. Activation and
massive local proliferation of Mueller cells is a key feature of retinal disorders. The
immune privileged nature of the retina protects it from the infected immune system
cells to a large extent. Retinal glia undertakes many of the functions of the immune
system and is a major source of cytokines. I will continue to study the effects of
endocannabinoids in the retina. As part of my effort to understand how
endocannabinoid levels change in patients with retinitis, I will be establishing a
lipidomics programme to study lipid profile, especially endocannabinoids. We also
intend to study the microRNA profile in biological samples from patients with
retinitis.
I am planning to experimentally test the hypothesis that the extensive
neurodegeneration at the retina, seen at late stages of HIV infection is due to presence
of inflammatory cytokines.
I intend to investigate whether the effects of
inflammation caused by HIV-1 proteins can be immunomodulated by
endocannabinoids towards beneficial factors for retinal cell survival. I also plan
extending this work to study the blood-brain/retinal barrier (BRB) and how it is
affected by inflammation during HIV infection. My work will elucidate the
mechanism behind neurodegeneration in the retina and ways to alleviate it, in the
context of inflammation after Highly Active Anti-Retroviral Therapy (HAART).
Identifying the changes occurring at the BRB which allows infection to progress can
serve to suggest new ways to tackle the disease. I am also in the process of initiating
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a collaboration to study optic neuritis and changes in BRB in EAE. This will involve
microRNA profiling as well as immunocytochemistry in animal models.
Ongoing work on retinal syncytium model of Mueller glia and microglia will
investigate the effect of demyelination and intercellular communication. Apart from
immune-modulation caused by demyelination in such an in vitro model we will study
calcium dynamics in this system.
In connection with the research on calcium signaling pathway in Mueller glia that I
am carrying out at present, I will also be exploring in the future the presence in lipid
rafts of signal transduction components involved in neuroprotection. I will focus on
calcium mediated excitotoxicity induced by purinergic neurotransmitters.
Expected impact:
Agonists of cannabinoid receptors activate signalling pathways in the brain that are
linked to neuronal repair and cell maintenance, and endogenous ligands can also
activate neuroprotective responses. Our work investigating regulation of innate
immunity by endocannabinoids has the potential of being used as a therapeutic after
inflammatory damage in the retina. Future work will investigate the effect of HIV on
the retinal vasculopathy, notably at the blood retinal barrier, on exposure to HIV
proteins. Our anticipated collaboration in microRNA profiling, would be aimed at
discovery of putative regulatory molecules.
4. Caenorhabditis elegans: A model system for understanding neuron-glia
interactions at the level of an entire nervous system
Principal Research Objectives
2012-2014
 Development of C.elegans strains with specific dopaminergic neurons and
their associated glia-like cells, labelled for visualisation and measurement of
activity-induced calcium dynamics.
 Development of nematode strains labelled to identify specific glia for
development studies.
 Construct a dynamical neural network simulator for the entire C. elegans
somatic nervous system using physiologically realistic single-compartment
models of individual neurons.
2012-2017
 Investigating mutant strains specific for neuronal genes for studying neuronglia associations during development of the C.elegans nervous system.
 Theoretical investigation of how adaptive learning occurs in C.elegans.
The C.elegans nervous system provides an elegant model for studying the dynamical
activity of multiple activated neurons. The reciprocal neuronal connections and the
glial cells have been mapped fully and the data is available. In spite of being a
relatively simple organism, it is capable of a surprising variety of behaviors. It shows
adaptability to changing conditions as well as learning. I will be developing tools and
strains which would allow studying changes in neuronal/glial activity that occur in an
intact animal in response to particular stimuli. The association of the neurons and their
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sheath or socket cells are surprisingly similar to myelinated neurons in the
mammalian nervous system. I plan to use the labeled strains with techniques such as
laser ablation to study developmental changes in the nervous system on ablation of
glia-like cells. This work will also use existing mutant strains to study genes involved
in neuron-glia interactions during nervous system development.
So far, there exists little information as to how the activities of individual neurons
might correlate with particular behavioral patterns. In collaboration with theoretical
scientists, I intend to construct a dynamical neural network simulator for the entire C.
elegans somatic nervous system using physiologically realistic single-compartment
models of individual neurons. This will allow us to connect electrical activity at the
level of individual neurons to the behavior of the organism in response to specific
stimuli, something which is experimentally still challenging. This will also permit us
to predict hitherto unidentified neuronal members of specific behavioral circuits from
activity patterns of the network. C.elegans has been shown to be capable of adaptive
learning – but it is not yet clear what changes in the nematode nervous system that
allows this. We would like to formulate a generalization of the spike-timing
dependent plasticity based learning for C. elegans and test it in the somatic system
simulator.
To verify the predictions obtained from the results of model simulations, I plan to
develop C.elegans strains with cameleon-labeled dopaminergic neurons/glia for
dynamical calcium imaging. A further aim is to image and investigate activityinduced calcium changes in core cells of specific behavioral circuits. Optical methods
provide a non-invasive way to monitor the activity of neurons and muscles in C.
elegans. Although optical techniques are of use in many experimental systems, they
are of particular interest for studying C. elegans as it is optically transparent. Thus, it
can be imaged while fully intact, and a variety of genetically encoded indicators are
available which can be targeted to cells of interest with appropriate promoters. Optical
calcium indicators appear to provide a good indirect measure of the activity of
neurons and muscles. I intend to use these techniques to observe calcium-dependent
signaling in cells of the C elegans nervous system.
Expected impact:
In combination with connectivity information, activity patterns in the neuronal
network should allow informed hypotheses to be proposed about how entire
behavioral patterns arise, from sensation to motor output. Generation of the activity
pattern also has the potential of providing insight into novel behavioral circuits. The
evolutionary importance of glial cells during development of the nervous system will
be tested in the context of the dopaminergic neurons I am planning to study. Though
the nematode cells are apparently dissimilar to mammalian oligodendrocytes, my
planned study will elucidate the underlying reasons behind the development of very
similar measures for increasing the efficiency of different nervous systems.
Concluding Remarks:
A recurring theme of my research is to elucidate the role of neuron-glia interactions in
health and disease. This research agenda is driven by an urge to reveal the principles
of inter-cellular communication involved in the robust and efficient functioning of the
nervous system. My work can offer insights into potential therapies for
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neurodegenerative disorders, provide tools for understanding the design constraints
underlying the evolution of neuronal networks and the evolutionary role of increasing
glia-neuron ratio. I envisage using a variety of approaches ranging from cell and
molecular biology to systems biology (involving computational modeling) and
collaborating with clinicians, experimental and theoretical scientists in pursuing these
aims.
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