Region-specific protein abundance changes in the brain of MPTP-induced Parkinson's disease mouse model

Abstract

Parkinson's disease (PD) is characterized by dopaminergic neurodegeneration in the nigrostriatal region of the brain; however, the neurodegeneration extends well beyond dopaminergic neurons. To gain a better understanding of the molecular changes relevant to PD, we applied two-dimensional LC-MS/MS to comparatively analyze the proteome changes in four brain regions (striatum, cerebellum, cortex, and the rest of brain) using a MPTP-induced PD mouse model with the objective to identify potential nigrostriatal-specific and other region-specific protein abundance changes. The combined analyses resulted in the identification of 4,895 nonredundant proteins with at least two unique peptides per protein. The relative abundance changes in each analyzed brain region were estimated based on the spectral count information. A total of 518 proteins were observed with substantial MPTP-induced abundance changes across different brain regions. A total of 270 of these proteins were observed with specific changes occurring either only in the striatum and/or in the rest of the brain region that contains substantia nigra, suggesting that these proteins are associated with the underlying nigrostriatal pathways. Many of the proteins that exhibit changes were associated with dopamine signaling, mitochondrial dysfunction, the ubiquitin system, calcium signaling, the oxidative stress response, and apoptosis. A set of proteins with either consistent change across all brain regions or with changes specific to the cortex and cerebellum regions were also detected. Ubiquitin specific protease (USP9X), a deubiquination enzyme involved in the protection of proteins from degradation and promotion of the TGF-beta pathway, exhibited altered abundance in all brain regions. Western blot validation showed similar spatial changes, suggesting that USP9X is potentially associated with neurodegeneration. Together, this study for the first time presents an overall picture of proteome changes underlying both nigrostriatal pathways and other brain regions potentially involved in MPTP-induced neurodegeneration. The observed molecular changes provide a valuable reference resource for future hypothesis-driven functional studies of PD.

Figure 1

Flowchart showing the experimental workflow.

Figure 2

(A) Heatmap of ~500 region-specific proteins from control brain samples. Protein abundances were displayed with a normalized abundance ratio scale where the spectral count for each protein in a given region was divided by the average spectral count across the four regions. All proteins had at least five total spectral counts and the normalized abundance ratio was >3 in at least one region. (Str: striatum; ROB: rest of brain; CB: cerebellum; Cor: Cortex) (B) Selected localized proteins showing good agreement with mRNA abundance distributions available in the Allen Brain Atlas (http://www.brain-map.org/). The ROB is not included because the average data for the rest of brain region is less quantitative. The highest region abundance of each protein is set as 100%. Myosin-5A (MYO5A) is a general protein expressed the same across all brain regions.

Figure 3

MPTP induced protein abundance changes for selected proteins across the four regions. Abundance changes were displayed as fold changes where the fold change is [MPTP]/[Ctrl]-1 if abundance ratio of spectral count ≥1, or the fold change is 1-[Ctrl]/[MPTP] if abundance ratio <1. Region annotations: Str: striatum; ROB: rest of brain; CB: cerebellum; Cor: Cortex.

Figure 4

Comparison of the cellular component distributions between the total identified proteins (black) and all regulated proteins (white) that show significant MPTP-induced abundance changes. The number of proteins in each category is expressed as a percentage of the total.

Figure 5

(A) Fold changes observed for selected mitochondrial proteins in the striatum. Fold-changes from ¹⁸O labeling is detailed in a previous paper^[22]. (B) Western blot analysis of ubiquitin carboxyl-terminal hydrolase (USP9X) and vacuolar ATP synthase subunit F (ATP6v1f) in three different mouse brain regions before and after MPTP treatment. The spectral count for a protein in each brain region is labeled under each gel band. USP9X is observed to be up-regulated in all three regions. ATP6v1f is observed to be down-regulated in STR and ROB regions with MPTP treatment. The results agree with the proteome data. (STR: striatum; CB: cerebellum)

Figure 6

Comparison of the number of nigrostriatal-specific and total significant proteins in each functional category. Nigrostriatal-specific proteins contribute to the main portion (>60%) of all functional categories.

Figure 7

Heat maps of abundance changes for specific protein functional categories. (A) Dopamine signaling pathway; (B) Mitochondrial dysfunction; (C) Apoptosis and cell death; (D) the ubiquitin system; (E) Calcium signaling. Relative protein abundances are displayed as fold changes (MPTP/control).

Figure 8

A general mechanism of MPTP induced neurodegeneration.