High-throughput Screen to Characterize and Understand the Role of Synaptic Cell Adhesion Proteins in Neurons
Many proteins control and affect the billions of connections among neurons in the brain. At the simplest level, the mapping of such neuronal interactions dictate how we learn, think, and remember. Moreover, when certain proteins are altered, neurological diseases can result ranging from learning disorders to memory impairment. This work will integrate high-throughput imaging technologies to characterize and systematically understand the role of certain synaptic cell adhesion proteins in neurons. For each protein, high-throughput microscopy methods will be used to image and quantify the cell morphology, visualize calcium signalling, and correlate the protein location with other neuronal markers having known functions. The results and broader impacts of this work may shed fundamental insight on how the brain is wired and how different neurons connect to and recognize each other.
The postdoctoral fellow, from a materials science and engineering background on the development and use of massively parallel scanning probe tools, will gain extensive experience in neurobiology, molecular biology, large-scale data analysis, and neurobiology in the context of imaging calcium signalling within cells. In addition to scientific training, the fellow will mentor undergraduate students in how to conduct research. Educational outreach activities such as teaching classes about recent scientific advances, writing web articles about biology intended for a non-science audience, and designing museum exhibits highlighting the intersection of biology and engineering will help engage public awareness.
The extraordinary abilities of the brain arise from the complex system of connections between neurons. What determines how neurons connect and the strength of such interactions is incompletely understood, but importantly, forms the foundation of learning, memory, consciousness, but also neurological diseases and disorders. To address such neurobiology questions, researchers conduct experiments at various levels: from molecules, cells, circuits, to entire organisms. Indeed, many transmembrane cell adhesion proteins have been separately identified as critical for brain function, but little is known about entire families of proteins. In this project, we have chosen calcium activity as a read-out for protein function. Importantly, it also serves as a single assay from which one can uniformly and quantitatively compare effects across many proteins. We have chosen to study transmembrane proteins that possess certain structural motifs (e.g. leucine rich repeat and immunoglobulin), believing that these proteins are likely to mediate neuronal connectivity. In about two years, we were able to generate and confirm the sequences of viral constructs for 200 genes. We have expressed these proteins in mouse hippocampal neurons and recorded spontaneous calcium activity from around half a million cells. The entire body and breadth of calcium imaging data is several terabytes in size; given this massive data set, custom computer programs process, analyze, and quantify all of this information in an unbiased manner. Some interesting results include the observation that certain Alzheimer’s disease (AD)-related proteins consistently increase overall calcium activity, while mutated AD proteins dramatically decrease calcium activity. We also saw that a family of immune system-related proteins, which are not extensively studied in the brain, affect neuron activity. We anticipate that this calcium activity platform could not only be useful for related neurobiological studies, but in other biological systems as well. Furthermore, this work will likely lead to downstream in-depth investigations of certain protein mechanisms. While it is necessary to continue exploring this massive data set, we are also cognizant of publishing this work and making the viral constructs and computer programs available so that both people within and outside of the field can benefit. In that vein, the complexity and challenge of understanding the brain truly requires interdisciplinary approaches. The execution of this project merged individuals from diverse backgrounds at the postdoctoral fellow and undergraduate levels, including biology and engineering. Even though the formal presentation of this project has occurred within academic networks, the larger discussion of science issues such as publishing and gender has taken place in the greater community as well.
