Super-Resolved Imaging of the Functional Microarchitecture within Single, Living Dendrite Spines

Thomas A. Blanpied, Ph.D.

University of Maryland School of Medicine

Department of Physiology
Funded in June, 2009: $200000 for 3 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY .

LAY SUMMARY

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Imaging How Alzheimer’s-related Beta-Amyloid May Disrupt Synaptic Communication

This study will explore how beta amyloid, a protein heavily implicated in Alzheimer’s disease, may disrupt synaptic communication in the brain.

Neurons communicate with one another at the synapse, the junction between them. Brain synapses have two properties that enable cells to store and exchange information. First, they maintain stable transmission strength between two connected neurons over very long periods of time. Second, they can undergo rapid changes (in seconds) in the strength of that connection. Some researchers, therefore, suspect that the inability to form new memories in Alzheimer’s disease is initially due not to brain cell death but rather to altered molecular events at synapses that disrupt communication of information to be stored as memory.  Specifically, the neurotransmitter glutamate is ordinarily released from one cell in the hippocampus and activates its connected neuron at the cells’ “dendritic spines,” miniscule finger-like protrusions.

Dendritic spines vary widely in size and shape, correlated with the strength of their synaptic connection. According to the investigators, because many forms of synaptic plasticity involve changes to only one synapse, it is likely that single spines contain internal molecular processes that enable their modification.  This study will use laboratory cultures of rat hippocampus to test how beta amyloid may disrupt these processes.

The Maryland investigators hypothesize that a protein called “actin” plays a key role in coordinating the shape and size of dendritic spines and the number of glutamate receptors on them. They will use a newly developed molecular imaging technique, called “photoactivated localization microscopy” (PALM), in laboratory rat hippocampal tissue. PALM, which has a resolution that is 10 to 100 times greater than conventional confocal microscopes, shows where each of many fluorescently-labeled molecules is located. It therefore can track individual actin molecules within a single dendritic spine.

First, they will determine whether actin is separately regulated at the synapse or at other points in the dendritic spine. Next they will see whether synaptic plasticity directly involves changes in actin in contact with the synapse. Thereafter, they will test the effects of amyloid protein on the internal organization of the dendritic spine to see whether it hijacks a process in healthy plasticity or induces a harmful form of plasticity.

ABSTRACT

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Super-Resolved Imaging of the Functional Microarchitecture within Single, Living Dendrite Spines

Neuronal dendritic spines comprise a set of spatially segregated functional subdomains, notably the synapse and the endocytic zone, each of which is strongly influenced by the spine actin cytoskeleton. During learning and behavior, rapid regulation of ongoing actin polymerization underlies diverse forms of synaptic plasticity, notably the control of receptor numbers in the synapse and the morphology of the spine itself. 

We hypothesize that spine function is maintained by an internal, dynamic microarchitecture such that: (1) the spine cytoskeleton is organized to allow independent regulation of actin polymerization at functional subdomains; (2) plasticity of healthy synapses involves modification of the dynamic relationship between actin and the postsynaptic density; and (3) disease processes such as Aβ-induced synaptic hypofunction involve disruption of this micro-organization.

However, the submicron dimensions of synapses and spines have made it difficult to measure whether dynamic actin structures exist at the synapse itself, or whether polymerization is regulated at synapses independently from other points in the spine.

We have developed live-cell, super-resolution imaging methods based on photoactivated localization microscopy (PALM) to generate the nanometer spatial resolution necessary to test these hypotheses about the internal organization of living spines. By tracking single molecules of actin as they move within spines, we can now map the distribution of actin polymerization in the spine with respect to the functional landmarks that define spine organizational substructure. This approach will allow us to determine whether plasticity of healthy synapses involves modification of their internal microarchitecture, particularly the dynamic relationship between actin and the postsynaptic density.

Based on this highly resolved view of normal spine function, we will proceed to examine whether toxic species of misprocessed amyloid precursor protein, thought to underlie Alzheimer’s disease pathology, induce changes of spine internal organization that are unique or shared with other forms of plasticity.

These results will provide fundamental insight to synapse dysfunction in Alzheimer’s disease. By forging methods to track cytosolic protein position and movement in living neurons with unprecedented precision, they offer the hope of new assays of pathology in numerous psychiatric and neurological diseases.

INVESTIGATOR BIOGRAPHIES

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Thomas A. Blanpied, Ph.D.

Dr. Blanpied graduated from Yale University with a bachelor’s degree in psychology and concentration in psychobiology. From Yale, he moved to the University of Pittsburgh Department of Neuroscience, where he obtained a Master’s Degree under the mentorship of Theodore W. Berger, Ph.D. and subsequently a doctoral degree with Jon W. Johnson, Ph.D. 

He then undertook postdoctoral training with George Augustine, Ph.D. and Michael Ehlers, M.D. Ph.D. at Duke University in the Department of Neurobiology. Dr. Blanpied joined the Department of Physiology at the University of Maryland School of Medicine in Baltimore as an assistant professor in 2005.

His work is supported by the National Institute of Mental Health and the Katherine D. and Theodore J. Carski Memorial Foundation. He was the recipient of NARSAD Young Investigator Awards in 2003 and 2005, and was granted their Freedman Award for Outstanding Basic Research in 2007. He was honored in 2008 to be awarded the Presidential Early Career Award for Scientists and Engineers.

His research focuses on elucidating molecular mechanisms which underlie synaptic communication in the brain, particularly by applying advanced, live-cell imaging techniques to visualize events of synapse plasticity that mediate memory formation and go awry during psychiatric and neurological disorder.

When not working in the lab, Dr. Blanpied often fantasizes about a trip to Tibet.