Proteolytic degradation and potential role of onconeural protein cdr2 in neurodegeneration.

J-Y Hwang,C-K Oh,H W Kang,W-Y Park,K C Chung,K Kim,H C Park,J Lee,Y J Oh,S Kim,H-D Um,I-Y Hwang,R B Darnell,J W Um,J-H Shin

doi:10.1038/cddis.2016.151

Abstract

Cerebellar degeneration-related protein 2 (cdr2) is expressed in the central nervous system, and its ectopic expression in tumor cells of patients with gynecological malignancies elicits immune responses by cdr2-specific autoantibodies and T lymphocytes, leading to neurological symptoms. However, little is known about the regulation and function of cdr2 in neurodegenerative diseases. Because we found that cdr2 is highly expressed in the midbrain, we investigated the role of cdr2 in experimental models of Parkinson's disease (PD). We found that cdr2 levels were significantly reduced after stereotaxic injection of 1-methyl-4-phenylpyridinium (MPP+) into the striatum. cdr2 levels were also decreased in the brains of post-mortem PD patients. Using primary cultures of mesencephalic neurons and MN9D cells, we confirmed that MPP+ reduces cdr2 in tyrosine hydroxylase-positive dopaminergic neuronal cells. The MPP+-induced decrease of cdr2 was primarily caused by calpain- and ubiquitin proteasome system-mediated degradation, and cotreatment with pharmacological inhibitors of these enzymes or overexpression of calcium-binding protein rendered cells less vulnerable to MPP+-mediated cytotoxicity. Consequently, overexpression of cdr2 rescued cells from MPP+-induced cytotoxicity, whereas knockdown of cdr2 accelerated toxicity. Collectively, our findings provide insights into the novel regulatory mechanism and potentially protective role of onconeural protein during dopaminergic neurodegeneration.

Highlights

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a selective loss of dopaminergic neurons in the substantia nigra (SN) pars compacta that is associated with both motor defects and nonmotor symptoms.[15]
We found that cdr[2] is downregulated by calpain and the ubiquitin proteasome system and that the restoration of cdr[2] levels renders dopaminergic neurons less vulnerable to 1-methyl-4phenylpyridinium (MPP+)-mediated cytotoxicity
Using both cultured MN9D dopaminergic cells and primary cultures of mesencephalic neurons challenged with MPP+, we found decreased levels of cdr[2] in tyrosine hydroxylase (TH)-positive dying dopaminergic neurons but not in GABAergic neurons

Summary

Results

Cdr[2] is highly expressed in the midbrain of normal adult rats. Previous studies show that cdr[2] is normally expressed in cerebellar Purkinje neurons but is ectopically expressed in breast and ovarian tumors of PCD patients.[4,5,32] To further characterize the normal expression pattern of cdr[2], lysates from various tissues from adult rats were immunoprobed with anti-cdr[2] antibody. Similar pattern of cdr[2] reduction in the ipsilateral side of the midbrain was observed in rats that received stereotaxic injection of MPP+ into the middle forebrain bundle, another rodent model of PD (data not shown) This MPP+-induced decrease in cdr[2] levels was associated with a reduction of TH protein, indicating that decrease in cdr[2] levels may be ascribed to a loss of TH-positive dopaminergic neurons or lower expression levels of cdr[2] in dying dopaminergic neurons or both. Exposure of primary cultures of mesencephalic neurons to 3 μM MPP+ for 36 h induced morphological changes such as neurite retraction and fragmentation (Figure 4a) In this condition, quantitative analysis indicated that cdr[2] levels were significantly reduced in TH-positive dying neurons following MPP+ treatment (Figure 4a, right panel). We observed that 6-OHDA-induced cell death was significantly blocked in cdr2-overexpressing MN9D cells

Discussion

Materials and Methods