Upregulation of alphaB-crystallin expression in the substantia nigra of patients with Parkinson's disease

Abstract

Parkinson's disease (PD) is one of the most devastating neurodegenerative disorders. The underlying mechanisms of the characteristic neurodegeneration in the substantia nigra (SN) are still not fully understood. To better understand the molecular events occurring in the SN of PD brain, we used the culture-derived isotope tag–based quantitative proteomics to compare the protein expression profiles in the nigral tissue of PD patients and control subjects. We identified a total of 11 differentially expressed proteins, including alphaB-crystallin (Cryab). Both the levels and pattern of Cryab expression in the SN were validated. It was revealed that Cryab was markedly upregulated in the SN of PD brain. Cryab expression was also upregulated in reactive astrocytes and microglia in a neurotoxin-induced mouse PD model. Moreover, we showed increased expression of Cryab in cytoplasmic inclusions in a subset of glial cells in Parkinsonian brain. Thus, we identified Cryab that is highly expressed in the SN of PD brain and may be involved in the glial pathology during dopaminergic neuron degeneration in PD.

Introduction

Parkinson's disease (PD) is a common neurodegenerative disorders affecting millions of people around the world. It is characterized by selective degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Patients with PD are afflicted by severe motor symptoms including resting tremor, bradykinesia, rigidity, and postural instability. After decades of intensive investigation, several hypotheses regarding the causes and pathogenesis of PD have been proposed, such as oxidative stress, mitochondrial dysfunction, protein aggregation, and inflammation (Foltynie and Kahan, 2013, Jenner, 1991, McGeer et al., 2001, McNaught and Olanow, 2006, Mizuno et al., 1998). However, no curative therapeutics for PD has been successfully developed until now based on the current understanding of the disease, highlighting the importance of identifying new therapeutic targets.

The application of high-throughput approaches, such as DNA microarray and proteomics, has dramatically enhanced our understanding of the pathophysiology of several neurologic diseases including PD. DNA microarray analysis evaluates gene expression on a genome scale by using brain tissues from animal models or human patients. By using this powerful method, a wide variety of genes, including PGC-1α, that is linked to pathogenic changes in PD has been identified (Mandel et al., 2005, Simunovic et al., 2009, Zheng et al., 2010). However, given the discrepancy between the messenger RNA and protein abundance in the biological samples, proteomics has the advantage of allowing direct comparison of the changes in protein levels among various conditions.

One of the drawbacks of traditional proteomic approaches is the inability to make quantitative comparison on the absolute or relative protein levels across biological samples. Recent advances in the development of quantitative methods in proteomics provide unprecedented opportunity to quantify protein levels in complex biological samples. One of such methods is based on the use of culture-derived isotope tags (CDITs), in which the isotope-labeled proteins from cell culture were introduced as internal standards to quantify the protein levels in complex tissue samples (Ishihama et al., 2005). Here, we quantitatively compared the proteomes of the SN from PD patients and healthy controls by using CDIT method. We found 11 proteins that displayed >1.5-fold change between control and disease groups. Among the 11 proteins, the upregulation of alphaB-crystallin (Cryab) was validated by using western blot in human brain samples. Immunohistochemistry revealed aberrant expression patterns of Cryab in the SN of PD patients and neurotoxin-induced rodent PD model. Our study provides compelling evidence that Cryab is involved in PD pathogenesis.