Abstract
Many neurological disorders and diseases including drug addiction are associated with specific neuronal cell types in the brain. The striatum, a region that plays a critically important role in the development of addictive drug-related behavior, provides a good example of the cellular heterogeneity challenges associated with analyses of specific neuronal cell types. Such studies are needed to identify the adaptive changes in neuroproteomic signaling that occur in response to diseases such as addiction. The striatum contains two major cell types, D1 and D2 type dopaminoceptive medium spiny neurons (MSNs), whose cell bodies and processes are intermingled throughout this region. Since little is known about the proteomes of these two neuronal cell populations, we have begun to address this challenge by using fluorescence-activated nuclear sorting (FANS) to isolate nuclei-containing fractions from striatum from D1 and D2 “Translating Ribosome Affinity Purification” (TRAP) mice. This approach enabled us to devise and implement a robust and reproducible workflow for preparing samples from specific MSN cell types for mass spectrometry analyses. These analyses quantified at least 685 proteins in each of four biological replicates of 50 K sorted nuclei from two D1 mice/replicate and from each of four biological replicates of 50 K sorted nuclei from two D2 mice/replicate. Proteome analyses identified 87 proteins that were differentially expressed in D1 versus D2 MSN nuclei and principal component analysis (PCA) of these proteins separated the 8 biological replicates into specific cell types. Central network analysis of the 87 differentially expressed proteins identified Hnrnpd and Hnmpa2b1 in D1 and Cct2 and Cct7 in D2 as potential central interactors. This workflow can now be used to improve our understanding of many neurological diseases including characterizing the short and long-term impact of drugs of abuse on the proteomes of these two dopaminoceptive neuronal populations.
Highlights
The striatum plays an important role in a wide range of neurological disorders including obsessive-compulsive disorder, Huntington’s and Parkinson’s disease, and drug addiction [1,2,3,4,5,6].Substance abuse is one of several neurological diseases that involves specific cell types in the brain.For example, psychostimulants such as cocaine block the reuptake of dopamine leading to increased synaptic concentrations of neurotransmitter
After pooling the tissue obtained from two mice as described below, the resulting four D1 and four D2 biological replicates were frozen on dry ice before being homogenized and subjected to fluorescence-activated nuclear sorting (FANS) isolation
Accumulation of EGFPL10a during ribosome assembly in the nucleolus imparts a bright green fluorescence that enables fluorescence-activated sorting of the corresponding nuclei (FANS) from the D1R or D2R-containing striatal medium-sized spiny neurons (MSNs)
Summary
Substance abuse is one of several neurological diseases that involves specific cell types in the brain. Psychostimulants such as cocaine block the reuptake of dopamine leading to increased synaptic concentrations of neurotransmitter. This leads to aberrant signaling in specific sub-types of the γ-aminobutyric acid producing (GABAergic) medium-sized spiny neurons (MSNs) that comprise 95% of the neurons of the striatum [5,7]. D1- or D2-classes of dopamine receptors [8]. The “direct” pathway dMSNs express relatively high levels of the D1 dopamine receptor (D1R), “indirect” pathway iMSNs express high levels of the
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