Imaging Protein Dynamics at Synapses

Bernardo L. Sabatini, M.D., Ph.D.

Harvard Medical School

Funded in June, 2005: $300000 for 3 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Visualizing Molecular Communication at Brain Synapses in Degenerative Brain

In the laboratory, Harvard researchers will use two-photon laser microscopy to visualize how activated proteins normally move into and out of brain cell synapses to enable one brain cell to communicate with another.  Then, the investigators will image this process in a mouse model of a degenerative disease to determine how the disease disrupts this communication.

Degenerative brain diseases, such as Huntington’s, Parkinson’s, and Alzheimer’s diseases,are characterized by abnormalities that occur at the synapses, which connect one brain cell to another to facilitate neural communication.  The researchers hypothesize that physical changes at the synapse affect the movement of proteins from one cell to another, interfering with brain cell communication.  Concurrently using two sets of two-photon laser microscopes, the researchers will determine the processes governing the rearrangement of the synapses as the proteins become incorporated into them.  To see if their hypothesis is correct, researches first will conduct laboratory studies of normal brain tissue culture, and then they will study synaptic changes in a mouse model of a human degenerative disease called Tuberous Sclerosis Complex, which causes mental retardation.

Significance:  If degenerative brain diseases involve physical changes at the brain synapse that compromise cellular communication, the findings could lead to new methods for diagnosing and treating degenerative brain diseases.

ABSTRACT

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Imaging Protein Dynamics at Synapses

We hypothesize that perturbations of protein trafficking into and out of synapses contribute to the manifestations of human neurological diseases. In order to test this hypothesis, new methodologies need to be developed that allow real-time monitoring of protein movement in small cellular compartments of living neurons. The specific aims are to:

1. Develop a method to directly visualize the movement of signaling and structural proteins into and out of synapses in living neurons.

2. Identify sets of stimuli that trigger rapid rearrangements of protein constituents of the synapse.

3. Determine if perturbation of synaptic protein movement are present in a mouse model of the human disease Tuberous Sclerosis Complex.

We propose to accomplish these aims using a novel microscope that makes use of precisely controlled laser pulses to simultaneously manipulate and monitor synaptically relevant proteins and signaling cascades deep in brain tissue. The microscope focuses and scans two lasers independently over the specimen. One laser is tuned such that its activation triggers a chemical reaction that results in the release of bioactive molecules or the activation of fluorescent proteins. The second laser is used to image the structure of the neuron, follow the movement of synaptic proteins, or monitor intracellular signaling cascades. Both the imaging and photoactivation properties of the microscope rely on 2- photon excitation and therefore have high spatial and temporal resolution. Thus they can be used to study synaptic function in small parts of the neuron that are not accessible with other techniques.

We will generate a panel of plasmids encoding photoactivatable green fluorescent protein (PAGFP) fused to proteins known to be associated with the synapse. The dynamics of each protein at the synapse will be determined by activating the linked PAGFP using 720 nm laser light and following the movement of the activated fluorophore using 910 nm excitation. The regulation of protein dynamics by action potential firing and synaptic activation will be investigated. Lastly, neurons lacking Tsc1 will be examined to understand if the defects in spine morphology seen in this mouse model of Tuberous Sclerosis Complex perturb the dynamics and trafficking of synaptic proteins.

HYPOTHESIS

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Hypothesis:
We hypothesize that perturbations of protein trafficking into and out of synapses contribute to the manifestations of human neurological diseases.  New methodologies need to be developed that allow real-time monitoring of protein movement in small cellular compartments of living neurons.

Goals:
1. Develop a method to directly visualize the movement of signaling and structural proteins into and out of synapses in living neurons

2. Identify sets of stimuli that trigger rapid rearrangements of protein constituents of the synapse

3. Determine if perturbation of synaptic protein movement are present in a mouse model of the human disease Tuberous Sclerosis Complex

Methods:
We propose to use combined simultaneous 2-photon laser-scanning microscopy (2PLSM) and 2-photon laser-photoactivation (2PLP) to directly monitor the movement of proteins within dendritic spines.  Here we propose to use this technology in conjuction with a fluorophore called photoactivatable-GFP (PAGFP).  PAGFP will be used to tag synaptic proteins and monitor the movement of protein populations during synaptic stimulation.

FINDINGS

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Lay Results:
Brain cells pass information to each other through highly specialized contact points called synapses. The experience dependent regulation of synapses is thought to underlie our ability to form memories and learn new behaviors. On the other hand, the long-term stability of some synapses is thought to underlie our ability to remember experiences for a life time. How synapses can at times be remarkably stable and at other times be easily modified is only poorly understood. In order to examine the processes of synapse stability and modification, we developed technologies to tag synaptic proteins with light-emitting molecules and to follow the movement of these tagged proteins over time. We identified regions of synaptic proteins that are necessary to form the structural lattice that keeps synapses stable. However, we also found that activity of neurons triggers a signaling system that leads to the rapid but partial disassembly of this otherwise stable lattice. When this pathway is active, proteins are quickly removed from the synapse and replaced by proteins coming from different parts of the cell. These new proteins can be in different functional states than the replaced proteins. Therefore, this system of stable but regulated structural elements allows for both long-term storage of information, as well as context-dependent plastic changes necessary to store new information.

Scientific Results:
Activity-dependent remodeling of the postsynaptic density (PSD) is critical for the formation and plasticity of glutamatergic synapses. We tagged proteins with photoactivatable green fluorescent (PAGFP) and used combined 2-photon laser scanning microscopy and 2-photon laser photoactivation to monitor the dynamics of synaptic proteins in individual spines of CA1 pyramidal neurons in organotypic rat hippocampal slices. We find that PSD-95, a major scaffolding protein of PSD, is stably tethered in the spine compared to other PSD-associated proteins such as CaMKllα, CaMKllβ, GluR2 and Stargazin.  N-terminal palmitoylation of PSD-95 is critical for its incorporation into stable structures with the spine head. Furthermore, the stability of PSD-95 in spines is enhanced by its first 2 PDZ domains but is independent of the 3rd PDZ, SH3, and GK domains. CaMKll-dependent phosphorylation at serine 73 within the first PDZ domain regulates the stability of PSD-95 as well as rapid, NMDA receptor dependent changes in the numbers of PSD-95 molecules in the spine head. Expression of mutants of PSD-95 that either mimic or block phosphorylation at this site also controls the stability of the AMPAR receptor subunit GluR2 in the spine head. We propose that the CaMKll-dependent phosphorylation of PSD-95 is one of the molecular events that destabilizes the PSD and thus permits the activity-dependent rearrangement of synaptic protein complexes.

 

SELECTED PUBLICATIONS

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Carter A.G and Sabatini B.L. State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons. Neuron. 2004 Oct 28;44(3):483-93.

Ngo-Anh T.J., Bloodgood B.L., Lin M., Sabatini B.L., Maylie J., and Adelman J.P.  SK channels and NMDA receptors form a Ca(2+)-mediated feedback loop in dendritic spines. Nat Neurosci. 2005 May;8(5):642-9.