BIOLOGICAL OVERVIEW

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of proteinaceous intraneuronal inclusions known as Lewy bodies. Little is known of the molecular mechanisms responsible for loss of dopaminergic neurons in PD; however, evidence suggests that environmental and genetic factors both play contributing roles. Mutation of a mammalian protein, Parkin, is associated with autosomal recessive juvenile parkinsonism (AR-JP). Parkin appears to be part of the cell's defense against damage caused by environmental insults (Steece-Collier, 2002). Parkin is an E3 ubiquitin ligase that degrades proteins with aberrant conformations (Imai, 2000).

To gain insight into the molecular mechanism responsible for selective cell death in AR-JP, a Drosophila model of this disorder has been created. Drosophila parkin null mutants exhibit reduced lifespan, locomotor defects, and male sterility. The locomotor defects derive from apoptotic cell death of muscle subsets, whereas the male sterile phenotype derives from a spermatid individualization defect at a late stage of spermatogenesis. Mitochondrial pathology is the earliest manifestation of muscle degeneration and a prominent characteristic of individualizing spermatids in parkin mutants. Drosophila parkin mutants also appear to have defects in a population of brain dopaminergic cells. Cells of the dorsomedial dopaminergic cell cluster reliably show shrinkage of the cell body and decreased tyrosine hydroxylase immunostaining in proximal dendrites in aged parkin mutants relative to controls. These results indicate that the tissue-specific phenotypes observed in Drosophila parkin mutants result from mitochondrial dysfunction, and as it does in mammals, mitochondrial dysfunction affects the dopaminergic neurons. These observations raise the possibility that similar mitochondrial impairment triggers the selective cell loss observed in AR-JP (Greene, 2003).

A second study in Drosophila analyses the effects of expression of a known mammalian substrates in Drosophila embryos. Panneuronal expression in Drosophila of Pael-R, a putative G protein-coupled transmembrane polypeptide, causes age-dependent selective degeneration of Drosophila dopaminergic (DA) neurons. Coexpression of Parkin degrades Pael-R and suppresses its toxicity, whereas interfering with endogenous Drosophila Parkin function promotes Pael-R accumulation and augments its toxicity. Furthermore, overexpression of Parkin can mitigate neuritic pathology induced by a second target of Parkin, alpha-Synuclein, and suppresses alpha-Synuclein toxicity. This study implicates Parkin as a central player in the molecular pathway of Parkinson's disease (PD) and suggests that manipulating Parkin expression may provide a novel avenue of PD therapy (Yang, 2003).

PD is the most common movement disorder and the second most common neurodegenerative diseases. PD patients suffer from rigidity, slowness of movement, tremor, and postural instability. The movement disorder in PD is largely due to the deficiency of brain dopamine content caused by the degeneration of DA neurons in the midbrain. Although the mechanism underlying the selective degeneration of DA neurons is still poorly understood, both exogenous environmental toxins and endogenous proteotoxins have been implicated in PD pathogenesis (Yang, 2003).

The molecular cloning of genes linked to familial forms of the disease has provided tremendous insights into the pathogenesis of PD. Missense mutations in the alpha-Synuclein (alpha-Syn) gene have been shown to be the cause of rare forms of autosomal dominant familial PD. alpha-Syn is an abundant brain protein enriched at presynaptic terminals. Mice with alpha-Syn gene deleted show increased striatal dopamine release but do not develop neurological phenotypes observed in PD. This suggests that familial alpha-Syn mutations may cause PD through a dominant gain-of-function mechanism. Significantly, wild-type alpha-Syn protein was found to be a major component of Lewy bodies, the proteinaceous aggregates found in PD and other diseases termed 'Synucleinopathies.' This suggests that the accumulation and aggregation of alpha-Syn is intimately involved in disease pathogenesis. Further support came from transgenic animal studies in which overexpression of wild-type and mutant forms of alpha-Syn in mouse and Drosophila led to alpha-Syn aggregate formation and neuronal dysfunction (Yang, 2003 and references therein).

Mutations in the parkin gene have been linked to AR-JP (Kitada, 1998). AR-JP patients develop the typical parkinsonian symptoms also as a result of loss of midbrain DA neurons. This usually occurs in the absence of Lewy body formation. Biochemical studies have shown that Parkin has E3 ubiquitin-protein ligase activity and that AR-JP-linked parkin mutations abolished this activity (Imai, 2000; Shimura, 2000; Zhang, 2000). Ubiquitin-protein ligases are components of the ubiquitin-proteasome pathway that degrades proteins with abnormal conformations. In this pathway, ubiquitin moiety is transferred to substrate proteins through ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and ubiquitin ligase (E3). After a polyubiquitin chain is attached to the substrate protein, it is recognized by the 26S proteasome and targeted for degradation. Substrate specificity in this reaction is largely conferred by the interaction between E3 and the substrate (Yang, 2003).

A number of proteins have been identified as Parkin substrates in vitro. CDCrel-1, a synaptic vesicle-associated protein, has been shown to interact with and can be ubiquitinated by Parkin (Zhang, 2000). Synphilin-1, which was originally identified as a alpha-Syn-interacting protein, has also been shown to be a substrate of Parkin-mediated ubiquitination and degradation (Chung, 2001). It has not been determined whether CDCrel-1 or Synphilin-1 accumulates to higher levels in AR-JP patient brain as a result of loss of Parkin activity. Parkin has also been shown to promote the ubiquitination and degradation of an O-glycosylated form of alpha-Syn (alphaSp22) (Shimura, 2001). The abundance of alphaSP22 is very low in normal brain, but it was found at modest levels in AR-JP brains lacking Parkin activity. It is not known whether O-glycosylation is a strict prerequisite for alpha-Syn interaction with Parkin and its subsequent ubiquitination and whether these modifications play any significant roles in PD pathogenesis. It is possible that through Synphilin-1, Parkin and unmodified alpha-Syn could functionally interact in the disease process (Yang, 2003).

Parkin has recently been shown to ubiquitinate Pael-R, a putative G protein-coupled transmembrane polypeptide (Imai, 2001). When overexpressed in cultured cells, Pael-R tends to become unfolded and insoluble and induces endoplasmic reticulum (ER) stress. Parkin ubiquitinates Pael-R and promotes the degradation of the insoluble form of the protein. Moreover, the insoluble form of Pael-R has been found to accumulate in AR-JP patient brains (Imai, 2001). However, the causal relationship between Pael-R accumulation and AR-JP is not clear (Yang, 2003).

To test the functional significance of Parkin's biochemical interactions with Pael-R and alpha-Syn, transgenic flies expressing human Pael-R, Parkin, or alpha-Syn were used to analyze their in vivo relationships and their roles in the pathogenesis of PD. Panneuronal expression of Pael-R results in age-dependent selective degeneration of Drosophila DA neurons, suggesting that DA neurons are especially susceptible to Pael-R toxicity. Coexpression of human Parkin causes the degradation of Pael-R and suppresses the DA neuron degeneration phenotype, whereas interference of endogenous Drosophila parkin function promotes Pael-R accumulation and enhances Pael-R-induced neurotoxicity. These data provide strong in vivo evidence that Pael-R is a genuine substrate for Parkin and that the accumulation of Pael-R is a cause of DA neuron death in AR-JP. Interestingly, overexpression of Parkin can also suppress the toxicity of alpha-Syn, at least in part by mitigating alpha-Syn-induced neuritic pathology. This study implicates Parkin as a central player in the pathogenesis of different forms of PD (Yang, 2003).

The ability of overexpressed Parkin to suppress both Pael-R and alpha-Syn toxicity suggests that despite the difference in pathological manifestation between the recessive and dominant forms of PD, there is a common pathway of PD pathogenesis in which Parkin is a central player. The observation that Parkin is localized to Lewy bodies is consistent with this notion (Shimura, 2001; Schlossmacher, 2002). It is possible that Parkin is actively engaged in degrading abnormal alpha-Syn proteins in Lewy bodies. It is also possible that Parkin may be sequestered by alpha-Syn into inactive complexes, similar to the sequestration of transcription factor CBP by polyglutamine-repeat proteins into nuclear inclusions in Huntington's disease. This may interfere with Parkin's normal function in the ubiquitin/proteasome pathway and contribute to alpha-Syn toxicity. The latter scenario is consistent with the observation of accumulated ubiquitin immunostaining in alpha-Syn transgenic neurites and its disappearance after Parkin overexpression. A recent report has shown that overexpression of mutant alpha-Syn protein increases the sensitivity to proteasome inhibitors by decreasing proteasome function in mammalian cell culture, an effect that can be suppressed by overexpression of Parkin (Petrucelli, 2002). This is also consistent with the latter possibility. However, the observation of reduced neuritic alpha-Syn aggregates in Parkin-coexpressing flies is also consistent with the former possibility that Parkin directly acts on abnormal forms of alpha-Syn, which may constitute only a minor portion of total alpha-Syn protein in the cell. It is likely that both mechanisms may be involved in the disease process (Yang, 2003).

Previous studies have shown that in response to unfolded protein response (UPR), Parkin becomes upregulated at both the mRNA and protein level and that overexpression of Parkin can suppress UPR-induced cell death in cell culture models (Imai, 2000). Thus, Parkin may play a more general role in cellular quality control to protect neurons from unfolded protein-induced stress. It is therefore conceivable that overexpression of Parkin could also suppress neurotoxicity induced by unfolded proteins other than Pael-R and alpha-Syn. Given that overexpression of Parkin does not have obvious detrimental effects on the animal, manipulation of Parkin expression with small molecules may provide an effective strategy for PD therapy (Yang, 2003).

AR-JP has been placed into the group of neurodegenerative disorders characterized by the accumulation and aggregation of aberrant forms of proteins. This includes Amyotrophic Lateral Sclerosis, Alzheimer's disease, Huntington's disease, the late onset dominantly inherited forms of PD, and spongiform encephalopathies. In these various forms of neurodegenerative diseases, specific groups of neurons undergo degeneration as a result of the aggregation of distinct misfolded proteins. These proteins may cause cellular toxicity by affecting different aspects of neuronal physiology, from axonal transport to gene expression in the nucleus. In the case of Pael-R, abnormal forms of the protein may cause cellular toxicity through eliciting ER stress (Imai, 2001). Components of the ubiquitin-proteasome degradation pathway and molecular chaperones have been shown to associate with inclusions formed by these different proteins. This may reflect the cell's strategy to eliminate these misfolded proteins by repairing/refolding them with the molecular chaperones or degrading them through the ubiquitin-proteasome system (Yang, 2003).

The importance of the ubiquitin-proteasome system in neurodegeneration is highlighted by the association of familial forms of PD with mutations in Parkin and ubiquitin carboxy-terminal hydrolase L1 (UCHL-1), another enzyme involved in ubiquitin metabolism (Leroy, 1998), and the acceleration of spinocerebellar ataxia type 1 phenotype by loss-of-function of an E3 ubiquitin ligase in mice (Cummings, 1999). Hsp70 molecular chaperones have been implicated in disease processes by whole animal studies in Drosophila, that have shown that directed overexpression of Hsp70 attenuates whereas interference with endogenous chaperone activity exacerbates neurotoxicity associated with polyglutamine repeat-containing proteins and alpha-Syn. It remains to be determined whether Hsp70 molecular chaperones play significant roles in removing misfolded Pael-R protein. The fate of abnormal Pael-R protein depends on the relative kinetics of its interactions with the molecular chaperone pathway and the proteasome pathway (Imai, 2002). Unlike alpha-Syn, a small cytosolic protein, Pael-R is a large 7 transmembrane protein, the misfolded forms of which reside mainly in the ER. The Hsp70 class of molecular chaperones may have limited access to misfolded Pael-R or have limited ability to renature misfolded Pael-R proteins, and therefore the Parkin-mediated proteosome pathway may play a more dominant role in clearing them. Alternatively, other molecular chaperones may play more prominent roles in regulating the fate of misfolded Pael-R. One good candidate is the ER resident molecular chaperone BiP (GRP78), which associates with unfolded proteins in the ER and has been shown to be upregulated (Imai, 2000, 2001) during ER stress and in AR-JP patient brain (Yang, 2003).

One of the most intriguing features of neurodegenerative diseases is the cell type specificity of neuronal death. Previous studies suggested that the restricted tissue distribution of Pael-R might contribute to the selective degeneration of DA neurons in AR-JP (Imai, 2001). It was shown that Pael-R protein is widely expressed in the mouse brain, predominantly in oligodendrocytes. Its neuronal expression is restricted to a subset of neurons such as DA neurons in the substantia nigra and hippocampal neurons in the CA3 region. While the restricted tissue distribution of Pael-R may partially explain the selectivity of neuronal degeneration in AR-JP, this study shows that there are some features specific to the DA neurons that contribute to their particular vulnerability to Pael-R toxicity. It is possible that this selective vulnerability is due to a smaller capacity of DA neurons to handle Pael-R-induced ER stress. It is also possible that certain DA neuron-specific cofactors may contribute to Pael-R toxicity. Two risk factors, oxidative stress and dopamine, have been suggested to contribute to the selective toxicity of alpha-Syn toward DA neurons. DA neurons in the substantia nigra are known to produce excess reactive oxygen species such as superoxide anion, which in reaction with nitric oxide (NO) can generate the more potent oxidant peroxynitrite and cause damages to proteins and other macromolecules. It was also shown that dopamine can be ligated to alpha-Syn to form dopamine-alpha-Syn adducts. This adduct selectively inhibits the protofibril-to-fibril conversion and causes the accumulation of alpha-Syn protofibrils, which are the presumed toxic species. Future studies will test whether Pael-R is also modified by oxidative stress and dopamine and whether these modifications contribute to its toxicity (Yang, 2003 and references therein).

Many studies have shown that there is unanticipated conservation of signaling pathways, regulatory mechanisms, and cellular and physiological processes between flies and humans. The identification of Parkin as an important modulator of Pael-R and alpha-Syn toxicity suggests that fly models could be used to further understand the role of Parkin and its substrates in the pathogenesis of PD. The ability to perform facile genetic loss-of-function and overexpression screens in this system will allow the identification of new genes that can either attenuate or exacerbate disease phenotypes. Such studies will allow the delineation of the molecular pathways involved in the pathogenesis of PD and possibly other related neurodegenerative diseases and identify potential therapeutic drug targets (Yang, 2003).

GENE STRUCTURE

cDNA clone length - 1478

Exons - 2

Bases in 3' UTR - 299

PROTEIN STRUCTURE

Amino Acids - 482

Structural Domains

Parkin is a RING-type E3 ubiquitin-protein ligase which binds to E2 ubiquitin-conjugating enzymes, including UbcH7 and UbcH8, through its RING-IBR-RING motif (Imai, 2000).

To identify a Drosophila homolog of the parkin gene, the human Parkin protein sequence was used to query a six-way translation of the Drosophila genome sequence. From this search, a single gene was identified that encodes a polypeptide of 468 aa exhibiting 42% amino acid identity and 59% similarity overall with human Parkin. This gene is the only one in the Drosophila genome that encodes a polypeptide bearing a ubiquitin-like domain, ring-finger domains, and an IBR domain, indicating that it represents the Drosophila parkin ortholog (Greene, 2003).

date revised: 25 May 2003

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