Abstract: Great advances in our knowledge are often the result of innovative technological advances. For example, combining light microscopy with Golgi staining led to the birth of modern neuroscience. My proposal aims to provide a major leap in the methodology of neuroscience research that holds the potential of transforming future studies of brain function and dysfunction. Brain function relies on tiny specialized structures at the synapse where protein molecules are arranged with nanometer precision. Small changes in synaptic machinery are viewed as early manifestations of many neurological disorders and neurodegenerative diseases, which are increasingly prevalent as the population ages. However, it has been challenging to examine synaptic protein organization at a sufficiently high level of resolution, especially in brain tissue. Surprisingly, this limitation does not lie in the microscopic methods. Fluorescence super-resolution microscopic methods such as the photoactivated localization microscopy (PALM) that can probe with 10-nm resolution have been developed. However, obstacles associated with protein labeling, sample preparation and data interpretation have prevented their application to brain tissue. Herein, I propose to develop innovative methodologies to label endogenous synaptic proteins and prepare brain samples for PALM. We will also establish a novel approach for visualizing the super-resolution protein organization within a cellular context to aid the interpretation of the data. We will do so by combining innovative developments across multiple disciplines, including molecular biology, biochemistry, genetics, super-resolution fluorescence microscopy, electron microscopy and computer programming. If established, our techniques will revolutionize the methodologies used in neuroscience research by providing unprecedented abilities to obtain fine details of synaptic architecture. These methods will be applicable to the study of other cellular proteins. The ability to identify previously undetectable subtle changes will also enable early diagnoses and an enhanced mechanistic understanding of neurological diseases affecting synaptic and other cellular structures. Public Health Relevance: Subtle changes in protein organization in the brain are an early manifestation of many neurological disorders and neurodegenerative diseases, which are increasingly prevalent as the population continues to age.
We aim to develop methods that can visualize these previously-undetectable changes to enable early diagnoses and an enhanced mechanistic understanding of neurological diseases affecting synaptic structures.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
NIH Director’s New Innovator Awards (DP2)
Project #
1DP2OD008425-01
Application #
8145426
Study Section
Special Emphasis Panel (ZGM1-NDIA-S (01))
Program Officer
Basavappa, Ravi
Project Start
2011-09-30
Project End
2016-06-30
Budget Start
2011-09-30
Budget End
2016-06-30
Support Year
1
Fiscal Year
2011
Total Cost
$2,310,000
Indirect Cost
Name
Oregon Health and Science University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Tillo, Shane E; Xiong, Wei-Hong; Takahashi, Maho et al. (2017) Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates. Cell Rep 19:617-629
Hunnicutt, Barbara J; Jongbloets, Bart C; Birdsong, William T et al. (2016) A comprehensive excitatory input map of the striatum reveals novel functional organization. Elife 5:
Zhong, Haining (2015) Applying superresolution localization-based microscopy to neurons. Synapse 69:283-94
Long, Brian R; Robinson, Danielle C; Zhong, Haining (2014) Subdiffractive microscopy: techniques, applications, and challenges. Wiley Interdiscip Rev Syst Biol Med 6:151-68
Fortin, Dale A; Tillo, Shane E; Yang, Guang et al. (2014) Live imaging of endogenous PSD-95 using ENABLED: a conditional strategy to fluorescently label endogenous proteins. J Neurosci 34:16698-712
Hunnicutt, Barbara J; Long, Brian R; Kusefoglu, Deniz et al. (2014) A comprehensive thalamocortical projection map at the mesoscopic level. Nat Neurosci 17:1276-85