2.1 Introduction

Parkinson’s disease (PD) is a neurodegenerative motor disorder that is caused by progressive loss of dopaminergic neurons in the substantia nigra. Upon its discovery, PD was thought
to be only of sporadic origins with no known genetic etiology at the time.However, this has been found to be untrue; there are well-supported cases that have demonstrated the genetic contributions to this disease. Different PD phenotypes have been observed as a result of mutations in genes such as ATP13A2, DJ-1, LRRK2, Parkin (PARK2), PINK1, and SNCA. These underlying genetic factors are believed to be of great importance when trying to understand the pathogenesis of this disease, eventually resulting in a more targeted and personalized approach of treatment (ie. different treatments based on different gene expression levels). Mutations in the parkin result in a subset of parkinsonism which will be discussed in greater detail. Parkin will be the main focus of this paper (Kumar, Djarmati-Westenberger, & Grunewald, 2011). It is noteworthy that different neuropathologies arise from mutations in these genes (See figure 1).

Figure 1: Overview of genetic contributions to different neuropathologies in Parkinson's Disease. (Kumar et al., 2011)

2.2 Parkin gene overview

The parkin gene is located on the long arm of chromosome 6 (ie. 6q25.2-q27) (Matsumine et al., 1997). Mutations in the parkin gene result in a non-sporadic, familial, autosomal recessive form of PD.. They are the major cause of Autosomal Recessive Juvenile Parkinsonism (AR-JP) linked with homozygous parkin mutation , which is represented by the age-of-onset of below 45 years. The mutations in parkin account for up to 77% of PD incidents with age-of-onset of below 20 years of age (Lucking et al., 2000). This monogenic form of PD was first documented in Japan from analysis of 17 families (Ishikawa and Tsuji, 1996). The phenotypic insults of AR-JP is similar to other forms of parkinsonism, characterized by rigidity, tremor, and akinesia (the inability to initiate movement). In addition, AR- JP patients tend to respond quite well to levodopa treatments and in some cases levodopa-induced dyskinesia is observed as the treatment side effect. Also, lower limb dystonia (continuous and sustained muscle contraction) are observed in greater frequencies in these patients (Lucking et al., 1998). One prominent difference between AR-JP and other types of parkinsonism phenotypes is in the pathology of the disease where dopaminergic cell loss at substantia nigra compacta as well as locus coeruleus without much Lewy Body formation (Kitada et al., 1998). The infrequent presence of Lewy Bodies was confirmed by postmortem examination of AR-JP patients (Mori et al., 1998). It is noteworthy that the nature of mutations and the extent of the damage on the parkin gene does not lead to different clinical results in patients; missense and truncating mutations seem to be leading into the same level of severity of PD (Lucking et al., 2000).

2.3 Parkin Protein

Parkin transcription and translation will ultimately lead to a protein consisting of 465 amino acids, called Parkin. This protein can be best analyzed through its different domains. The domain at the N-terminus called the ubiquitin-like domain (UBL) has a 62% homology with ubiquitin. This domain is crucial in stability of the Parkin transcript as well as its expression levels. This domain is functional as a E3 ubiquitin ligase, involved in ubiquitination. As for the C-terminus, the presence of three RING domains has been confirmed, RING0, RING1, and RING3 as well as an IBR (inbetween-ring) domain. These domains interact with the ubiquitination machinery. Zinc ions play an important role in this proteasome-mediated ubiquitination to an extent where its removal causes Parkin unfolding (Hristova, Beasley, Rylett, & Shaw, 2009).

2.4 Proposed Molecular Mechanism of Parkin-Dependent PD

Studies have revealed the presence of another protein, Parkin interacting substrate (PARIS). In the study it was shown that Parkin interacted with PARIS and regulated its levels by the Ubiquitin-Proteasome System (known as UPS). The study further showed that PARIS is a transcriptional repressor of PGC-1α (peroxisome proliferator-activated receptor gammacoactivator-1α). One known function of PGC-1α is to act as a liaison between the external stimuli and the levels of mitochondriogenesis. In conditional mice knockout models of parkin, over-expression of PARIS (and subsequent decline in PGC-1α transcription) resulted in loss of dopamine neurons (Shin et al., 2011). (See Figure 2)
Figure 2. The PD model of the Parkin-PARIS- PGC-1α Pathway. ROS (Reactive Oxygen Species), NO (nitrosative), and DA (Dopamine) stress result in accumulation of PARIS which subsequently reduces the transcription of PGC-1α which leads to the ultimate dopaminergic loss in PD (Shin et al., 2011)

2.5 The functional role of Parkin

As mentioned, Parkin has a neuroprotective role, shielding cells against dopaminergic degradation and subsequently death (Henn et al., 2007). Nonfunctional Parkin protein can result in lower level of mitochondrial activity, reduced expression of oxidative stress-related proteins, and decreased respiratory capacity in mitochondria. All of these factors will subsequently result in increased oxidative damage (Palacino et al., 2004). In order to prevent mitochondrial damage and perform its neuroprotective function, Parkin needs to be transported to the damaged mitochondria. In an study by Vives-Bauza and colleagues it was shown that the damaged mitochondria experience membrane potential loss which in turn will induce the recruitment of Parkin and its relocalization. It was shown that this recruitment is PINK1-dependent, by using PINK1 siRNA (silencing RNA), both in vitro and in vivo rodent models. Last but not least, they found that the Parkin-PINK1 complex promotes mitochondrial clustering. Over-expression of PINK1, Parkin, or both in SH-SY5Y cell lines (which have lots of neuronal resemblance) resulted in clustering of mitochondrial biomarkers (Vives-Bauza et al., 2010). PINK1, as mentioned earlier, is another gene implicated in the monogenic models of PD. The mutation in either of these genes will result in accumulation of dysfunctional mitochondrial which arises from impaired mitophagy (specific mitochondrial autophagy). Some argue that the PINK1-Parkin pathway demonstrates the significance of mitochondrial dysfunction in the etiology of PD (Clark et al., 2006). In another experiment, it was shown that PINK1 and Parkin work with DJ-1 (another gene in the mongenic models of PD, see Figure1) forming a PPD complex. It was found that in addition to Parkin, DJ-1 is also being recruited to the mitochondrial membrane by PINK1 (Xiong et al., 2009).

Figure 3. The interaction between PINK1, Parkin, and DJ-1 results in the formation of the PPD complex which may be involved in enhancing the ligase activity of Parkin.

2.6 Animal Models

As you will come to understand, the experiments in search of finding the role of the parkin gene came to inconclusive results. parkin knockout in mice resulted in not so severe, yet unpleasant phenotypes while its knockout in the fruit flies resulted in a much more severe phenotype. Hence, it would make intuitive sense for a third animal model to be used in studying functions of parkin in loss-of-function experiments. The general census shared between the findings from these three animal models was the undeniable function of Parkin in neuroprotection and prevention of mitochondrial dysfunction. However, the progressive dopaminergic loss was not correlated with the gene knockout/knockdown.

mouse.jpg2.6a Parkin knockout in mice model

In one study, the proteomics of the brain in the parkin-deficient mice was observed by means of fluorescence 2D difference gel electrophoresis and mass spectrometry. 87 different proteins with expression level differences of at least 45% between the wild-type and the knockout model were observed. This study revealed the participation of septin and MAGUK protein families in parkinsonism disorders (Periquet, Corti, Jacquier, & Brice, 2005).
In another study, the behavioural insults of parkin-deficeint mice were observed. The mutation in the parkin gene in these rodents led to motor and cognitive deficits. An increase in the MAO activity in the limbic lobe was observed in these mice. Surprisingly, no statistically significant reduction in the nigrostriatal dopaminergic neurons was observed (Itier et al., 2003).
In addition, in another study using microscopic analysis, no striatal mitochondrial abnormalities correlated with the parkin null model (Palacino et al., 2004).

drosophila.jpg2.6b Parkin knockout in Drosophila model

In comparison to the mouse models, the parkin-defecient drosophila models with generally a reduced lifespan demonstrated more severe phenotypes such as, locomotor deficiency, and sterility in males. The locomotor deficits were the result of programed death of muscle cells, while the sterility of the null model males was due one faulty stage during spermatogenesis. Mitochondrial dysfunction was one of the first manifestations of muscle atrophy. The authors hypothesized the role of mitochondrial dysfunction in triggering these cell losses are conserved between species and might be partially one of the factors leading to AR-JP (Greene et al., 2003).


2.6c Parkin knockdown in Danio rerio model

In another paper, the authors claimed that study of the role of parkin in another
animal model is crucial since there are discrepancies in functional and physiological deficits between the two mostly used animal models, mouse and drosophila. They found that the role of Parkin is quite similar to that in humans, having a protective role against stress conditions. However, they did not find any correlation between the Parkin malfunction and loss of dopaminergic neurons in substantia nigra. No loss of dopaminergic neurons was observed in these animals (Fett et al., 2010).


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akinesiaIn one study, it was shown that Parkin, through interacting with PARIS and regulating its levels by the Ubiquitin-Proteasome System (known as UPS). The study further shoed that PARIS is a transcriptional repressor of PGC-1α (peroxisome proliferator-activated receptor gammacoactivator-1α). One known function of PGC-1α is to act as a liaison between the external stimuli and the levels of mitochondriogenesis. In conditional mice knockout models of pakin, overexpression of PARIS (and subsequent decline in PGC-1α transcription) resulted in loss of dopamine neurons.