Elsevier

Neurobiology of Disease

Volume 27, Issue 2, August 2007, Pages 238-247
Neurobiology of Disease

Parkin Co-regulated Gene (PACRG) is regulated by the ubiquitin–proteasomal system and is present in the pathological features of parkinsonian diseases

https://doi.org/10.1016/j.nbd.2007.04.014Get rights and content

Abstract

Mutations in parkin are a common cause of early-onset autosomal recessive Parkinson's disease. Parkin Co-Regulated Gene (PACRG) is a novel gene that was discovered because of its close genetic proximity to parkin and the two genes were subsequently demonstrated to be regulated by a bi-directional promoter. However the role of PACRG has not been well characterized and the distribution of the protein in both normal and diseased brain is not known. Here, we report that like parkin, PACRG is regulated by the ubiquitin–proteasomal system. Immunohistochemistry identified PACRG in astrocytes throughout the brain and in pigmented noradrenergic neurons of the locus coeruleus. PACRG was also detected in Lewy bodies and glial cytoplasmic inclusions in patients with Parkinson's disease and Multiple System Atrophy, respectively. Together, these results demonstrate that PACRG is regulated by the ubiquitin–proteasomal system and may play a role in the pathogenesis of Parkinson's disease.

Introduction

Parkinson's disease (PD) is a chronic neurodegenerative disorder that has been estimated to affect approximately 1% of the population at 65 years of age (Farrer et al., 2001). PD is a movement disorder characterized by rigidity, resting tremor, bradykinesia and postural instability. Pathologically, there is a loss of dopaminergic neurons, primarily in the substantia nigra pars compacta (SNpc) and the presence of eosinophilic intracytoplasmic protein inclusions, known as Lewy bodies (LB). The majority of cases of PD appear to be sporadic (Petit et al., 2005) however rare monogenic forms of the disease are useful in gaining a greater understanding of the underlying mechanisms involved in the pathogenesis of PD.

Mutations in the parkin gene, located on chromosome 6q25–27, are the most common genetic cause of early-onset Parkinson's disease (EO-PD). Parkin functions as an E2-dependent E3 ubiquitin ligase in the ubiquitin–proteasomal system (UPS). It plays a role in conjugating ubiquitin to damaged or misfolded proteins for subsequent degradation by the proteasome. It has been suggested that loss-of-function mutations may result in reduced enzymatic activity of parkin, thereby causing the accumulation of toxic parkin substrates and thus dopaminergic neuronal loss (Kubo et al., 2006). However, the mechanisms by which mutations in parkin and other PD linked genes contribute to the pathogenesis of PD are not well understood.

A study of the parkin promoter region identified the novel gene Parkin Co-Regulated Gene (PACRG). The two genes were shown to be linked in a head-to-head arrangement on opposite DNA strands and co-regulated by a bi-directional promoter (West et al., 2003). Bi-directional promoters have been previously described and the genes typically demonstrate similar tissue expression patterns or the gene products often interact or function in a common pathway (Li et al., 2006). We hypothesize that mutation of PACRG, which encodes a protein of unknown function that is abundant in Lewy bodies, also may contribute to the pathogenesis of PD. The genetic architecture of the parkin/PACRG locus, with the two genes under the control of a bi-directional promoter, is suggestive that PACRG, like parkin, may play a role in PD. Indeed, a previous study suggested that PACRG might function as a mediator of neuronal cell death (Imai et al., 2003). To date, only one study has directly investigated the potential role of PACRG in PD. However, sequence analysis of PACRG in a cohort of 112 Caucasian EO-PD patients failed to detect any pathogenic mutations (Deng et al., 2005). To date, the function of PACRG in the brain has not been well characterized and its expression and distribution in both normal and PD brain have not been reported in the literature.

Here we report that overexpressed PACRG could only be detected in HEK293 and BE(2)-M17 neuroblastoma cells at the protein level following proteasomal inhibition. Furthermore, the co-immunoprecipitation of PACRG with ubiquitin suggests that steady-state levels of PACRG are mediated by the UPS. Immunohistochemistry detected PACRG in Lewy bodies, glial cell inclusions and neurofibrillary tangles, the pathological hallmark features of PD, multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) respectively. The identification of PACRG's involvement in the UPS and its localization in Lewy bodies is consistent with reports linking this pathway to PD and suggests that this protein may also be involved in the pathogenesis of the disease.

Section snippets

PACRG constructs, cell culture and transfections

The entire hPACRG ORF was amplified by RT-PCR using SuperScript III reverse transcriptase (Invitrogen) and a 60–50 °C touchdown protocol over 35 cycles. The forward primer corresponded to base pairs 267 to 284 and the reverse primer to base pairs 1022 to 1040 of the Genbank accession no. AF546872. The 774 bp product was subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen). A similar strategy was employed to subclone the hPACRG ORF into the mammalian expression vectors

Results

PACRG has recently been shown to share a common promoter region and be co-regulated with the PD-linked gene, parkin. To determine if PACRG, like parkin, is regulated by the UPS, HEK293 cells and BE(2)-M17 neuroblastoma cells were transiently transfected with the following plasmid constructs: (1) PACRG untagged, (2) C-terminal V5His tagged PACRG or (3) N-terminal Xpress tagged PACRG. Following parallel transfections, cells were harvested for either cDNA expression or western blot analysis. Under

Discussion

Mutations in parkin are the most common cause of both familial and sporadic EO-PD. Parkin is an E3 ubiquitin ligase that is regulated by, and functions in, the UPS (Imai et al., 2000). Previously, we demonstrated that parkin was linked to and co-regulated with the novel gene PACRG (West et al., 2003). The functional significance of this genetic architecture and the role of PACRG in the brain are currently unknown. In this study we demonstrate that PACRG also is regulated by the UPS. We describe

Acknowledgments

Funding—National Health and Medical Research Council project grant #222727 to GMH and #334349 to PJL. PJL is an NHMRC RD Wright Fellow (#334346) and MBD is an NHMRC practitioner fellow (#284520).

Tissue—human brain tissue samples were received from the Australian Brain Donor Programs Prince of Wales Medical Research Institute Tissue Resource Centre, which is supported by the National Health and Medical Research Council of Australia, Australian Brain Donor Programs approved request #GH060505 to

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