Assessing Proteomic Differences Between the Two Major Cell Types of the Striatum
The vast intercellular and intracellular heterogeneity of the brain presents major challenges for proteomic analysis. Regulatory events are localized to specific neuronal cell types or subcellular compartments, resulting in discrete patterns of protein expression and activity. Quantifying the proteome in brain regions with respect to this localization is therefore extremely difficult. The striatum, a region critically important in the development of addictive drug-related behavior, is a perfect example of this complexity; as it contains two major cell types (D1 and D2 type dopaminoceptive neurons) whose cell bodies and processes are intermixed throughout the region. While the use of D1 and D2 mice for ribosome immunoprecipitation (TRAP) experiments has shown significant differences in cocaine-induced mRNA expression changes between these two cell populations, little is known about changes in the levels of protein expression or modification in these neuronal populations. We are currently working to overcome these issues of cellular heterogeneity and shape by using D1 and D2 TRAP mice to assess neural protein translation dynamics through cell type specific ribosomal footprinting. However, this approach does not allow for assessment of the entire cellular proteome, only measuring rates of protein production and disallowing assessment of post-translational modifications that form the final functional state of the neural proteome. In this pilot project we propose complementary methods to address the question of the complete neural proteome.
Specific Aim 1
We will evaluate the use of fluorescence-activated cell sorting to isolate GFP-L10a positive neurons from D1 and D2 mice for proteomic assessment. FACS will first be performed on isolated nuclei from these animals. Assessing protein changes in other irregularly shaped cellular compartments poses greater challenges. We have optimized a sort method that enables assessment of 24,000 D1 neuron positive events, which we presume includes the nucleus, some cytoplasm, and rough endoplasmic reticulum from the surrounding soma. We will evaluate the proteome of these events from D1 compared to D2 mice.
Specific Aim 2
The FACS-based isolation method depends on random shearing of cellular membrane and therefore may result in irregular sampling of the cytoplasm. For comparison, we will perform Laser Capture Microdissection using tissue from D1-Flag-DARPP-32 and D2-Myc-DARPP-32 mice. The advantage of using these mice is twofold: DARPP-32 acts as a superior volume label of the cell body than GFP ribosomes, and both D1 and D2 cells types can be collected from the same animal. It is estimated that LCM dissection of 1,000 or more cells can bring neural proteomes within reach, so by collecting ~10,000 cells from each experimental condition for proteomic assessment we expect to obtain very high quality data.