Parkinson’s disease is the second most common neurodegenerative disorder. It affects approximately 1 million people in the United States and more than 4 million people worldwide (Genetics home reference). Two markers characterize Parkinson’s disease. The first marker is the accumulation of Lewy bodies in cholinergic and dopaminergic neurons. Lewy bodies are made up of α–Synuclein protein aggregates. Cholinergic neurons produce the neurotransmitter acetylcholine, which is important for cognitive function. The second marker is the degeneration of dopaminergic neurons in the substantia nigra portion of the brain. When functioning normally, dopaminergic neurons produce dopamine, which serves as a chemical messenger that allows communication between the substantia nigra and other regions of the brain. This communication coordinates balanced and smooth muscle movement. A lack of dopamine results in a loss of the ability to control body movements and in abnormal nerve functioning (NIH senior health).
Autosomal Recessive juvenile Parkinsonism (AR-JP) is characterized as early onset, with symptoms beginning before age 40 and has been directly linked to mutations in the PARK2 gene (Kitada et al, 1998). Parkin, the protein encoded by PARK2, is expressed primarily in the nervous system and is part of the multi-protein E3 ubiquitin ligase complex, which attaches short ubiquitin peptide chains to proteins to tag them for degradation (Shadrina et al).When the parkin protein is aberrant, there is a loss of the ubiquitin-protein ligase activity and the buildup of toxic proteins can lead to neural death (Von Coelln et al, 2004).
The PARK2 gene has 12 coding exons. Alternative splicing of this gene produces multiple transcript variants encoding distinct isoforms. In the alternative splicing process, particular exons of a gene may be included or excluded from the final mRNA. The proteins that are then translated from alternatively spliced RNA will contain differences in their amino acid sequences, and many will be missing substantial portions of their amino acid sequence. This will cause variants to differ in their biological functions. Multiple PARK2 splice variants have been researched to date, but this study focuses on the splice variant that is missing exon 5.
Epigallocatechin gallate (EGCG), an antioxidant found in green tea; genistein, a compound found in soy; and digoxin, a cardiac glycoside, have all been shown to alter the splicing process (Anderson et al, 2003; Anderson et al, 2012; Liu 2012). The purpose of this study was to analyze the effects of these compounds on the splicing of exon 5 in the transcripts of the PARK2 gene in a neuronal cell line. Because these compounds have been shown to affect splicing, it was expected that they would have an effect on the PARK2 transcript. It was found in this study that EGCG has an effect on the splicing process whereby it induces the PARK2 splice variant that is missing exon 5. This finding is important since exon 5 codes for a region that is crucial for the binding of zinc ions, which are needed for the correct folding of the parkin protein. Therefore, the results of this study suggest that EGCG affects splicing of the PARK2 transcript.
Materials & Methods
Neuroblastoma cell line
JMN cells are a type of neuroblastoma cells. The JMN cell line was kindly provided by Dr. Robert Ross, Neurobiology Laboratory, Department of Biological Sciences, Fordham University. The JMN cell line is a non-N-myc amplified I- type cell line that was isolated from a patient with a stage 3 tumor. The JMN cells were cultured in a DMEM/F12 + GlutaMAX™ medium and were treated for 48 hours with either epigallocatechin gallate (EGCG), EGCG and genistein, genistein, or digoxin.
The RNA was extracted using RNeasy® Plus Mini Kit (QIAGEN) according to the manufacturer’s instructions.
RT-PCR was performed using QIAGEN® One-Step RT-PCR Kit following the kit instructions. Temperature cycles were: one hold at 50°C for 30 minutes and one hold at 95°C for 15 minutes. 40 cycles of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds; followed by a final extension at 72°C for 2 minutes. GAPDH was used as the positive control.
RT-PCR products (5 μl) were loaded on 1.0 % agarose gels. Gels were visualized in BioRad UV trans-illuminator.
PCR product purification and sequencing
PCR products were purified using QIAquick® PCR Purification Kit. PCR purified products were quantified on a Beckman Coulter DU®530 Spectrophotometer.
Purified PCR products were sent out for sequencing by GENEWIZ® and were confirmed by BLAST.
RT-PCR was performed using a specially designed primer pair. The forward primer was a exon 4,6 primer and the reverse primer was an exon 9 primer (Fig.1). The forward primer was designed so that only the splice variant missing exon 5 would be able to have the forward primer bind to it, and therefore get an amplification of that splice variant. The 5' end and the first half of the primer was complementary to the last 13 nucleotides of exon 4, and the second half of the primer and 3' end was complementary to the first 6 nucleotides of exon 6. Only the variant that had exon 4 next to exon 6 was able to have this primer bind to it.
The gel results showed that the PARK2 variant missing exon 5 was produced only by the cells that were treated with EGCG alone. (Fig.2). The cells that were treated with digoxin have a very faded band at approximately 1400bp which may be due to intronic contamination at the time of RNA extraction from the cells. It may also be due to the primer binding to a small region of either exon 4 or 6 and having minimal amplification. The cells that were treated with both genistein and EGCG did not have any bands suggesting that genistein is preventing the effect that EGCG has on splicing when cells are only treated with the EGCG compound. Sequencing confirmed that the RT-PCR products were the parkin protein (Fig.3). GAPDH was used as a positive control to ensure that the RT-PCR process worked properly (Fig.4).
When cells were treated with EGCG, it was found that the splice variant missing exon 5 was produced. Guerrero Camacho et al (2012) showed that the 28 acids that comprise exon 5 span amino acids 178-206 of the PARK2 transcript. They designated this region as RING0, which is a cysteine rich unique parkin domain.
The RING0 domain comprises two distinct, conserved cysteine-rich clusters. The positions of the cysteine residues in this region are similar to the RING1 and RING2 domains of the PARK2 gene, as well as other E3 ubiquitin-ligase domains.
Zinc binding has been shown to play an important role in facilitating the proper parkin protein folding. Eight zinc ions bind to the parkin protein in total. Two bind to each of the four domains: the RING0 domain, the RING1 domain, the IBR domain and the RING2 domain (Hristova et al, 2009). They revealed that removing zinc causes a mis-folding of the parkin protein. Therefore, it is essential to have the zinc ions bound. Missing 28 amino acids, especially the last 11 that are part of the cysteine rich region which is crucial for zinc binding, could have a negative effect on the functioning of the protein. The results of this study merit further research to confirm that EGCG has the ability to affect splicing of the PARK2 transcript. This research would specifically be important for individuals who are already carrying a mutation of the PARK2 gene. The reason for this is that the existing mutation in one allele increases the susceptibility of these individuals in the event that exon 5 is spliced from the healthy allele.
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