Northern blot analysis using poly(A)+ RNA from embryos, larvae, and adults detected parkin transcripts at all developmental stages with particularly high abundance in adults (Greene, 2003).
To generate a disruption of the parkin gene, a transposon mutagenesis screen was conducted by using a P element mapping close to parkin. This strategy yielded a single line, designated parkEP(3)LA1, bearing an insertion 71 bp upstream from the parkin start codon. To generate more severe alleles of parkin, the parkEP(3)LA1 insertion was mobilized with transposase under conditions favoring the creation of coincident deletions extending from the insertion locus. A large collection of deletion alleles were recovered from this screen, including several that remove all of the parkin coding sequence and thus represent null alleles of parkin. This work also yielded a chromosome bearing a precise excision of parkEP(3)LA1, which was maintained for use as a control chromosome (designated parkrvA) in these studies (Greene, 2003).
Flies bearing any of the parkin null alleles in trans to the Df(3L)Pc-MK deletion chromosome, which removes the parkin gene, are viable through the adult stage of development but exhibit a slight developmental delay, typically eclosing a day later than controls, and show significantly reduced longevity (P < 0.0001). parkin null flies have an average lifespan of 27 days, with none able to exceed 50 days of age, whereas flies bearing the parkrvA precise excision chromosome in trans to Df(3L)Pc-MK have a mean lifespan of 39 days and can survive up to 75 days (Greene, 2003).
Female parkin mutants are fertile and produce normal offspring, however, males are completely sterile. This finding permitted a screen of a collection of ~1,100 ethyl methanesulfonate-mutagenized homozygous viable male sterile lines for additional parkin mutations generated independently from deletion alleles. Sequencing the parkin gene from two of the mutants recovered from this screen revealed missense and premature stop codon mutations, verifying that the male sterile phenotype results from loss of parkin function (Greene, 2003).
Analysis of testes from homozygous or transheterozygous parkin mutants indicates that the male sterile phenotype derives from a late defect in spermatogenesis. Spermatogenesis appears to proceed normally in parkin mutants until the individualization stage, at which point a 64-cell germ-line cyst that normally separates into mature sperm cells fails to do so, resulting in an absence of mature sperm cells in the seminal vesicle. Ultrastructural analysis of developing spermatids in parkin mutants revealed structural irregularities in the sperm tails. Mature sperm tails usually consist of a flagellar axoneme, with a 9 + 2 arrangement of microtubules, and a specialized mitochondrial derivative known as the Nebenkern. Although the axoneme in parkin mutants appears normal, Nebenkern integrity is severely disrupted; some spermatids have multiple Nebenkern, whereas others have only an extremely diminished component. Additionally, the electron density of the Nebenkern outer matrix is significantly diminished with respect to wild type. These results suggest that defective Nebenkern formation and/or function may underlie the spermatid individualization failure of parkin mutants (Greene, 2003).
In addition to reduced longevity and male sterility, all of the parkin null alleles, as well as those recovered on the basis of the male sterile phenotype, confer a partially penetrant downturned wing phenotype as homozygotes or as transheterozygotes with the Df(3L)Pc-MK chromosome. The penetrance of this phenotype increases with age; ~40% of newly eclosed flies exhibit abnormal wing posture, whereas by 10 days of age more than 70% of the parkin mutants display this phenotype. This finding prompted the use of an assay of the locomotor ability of parkin mutants. These analyses revealed severe defects in both flight and climbing ability in parkin mutants. Both phenotypes were also manifest in parkin mutants with normal wing posture. The climbing decay rate was similar in parkin mutants and wild-type flies, indicating that parkin mutants begin adult life with a reduced climbing ability (Greene, 2003).
To address the origin of the locomotion defects in parkin mutants, the UAS/GAL4 system was used to express parkin in defined tissues. Two GAL4 lines that drive parkin expression in mesoderm were found to rescue the wing posture, flight, and climbing phenotypes of parkin mutants to wild-type or near wild-type levels, demonstrating that Parkin function is required in the musculature (Greene, 2003).
Histological analysis of the major flight muscles [the indirect flight muscles (IFMs)] from parkin mutants revealed severe disruption of muscle integrity, consistent with the role of muscle dysfunction in the parkin flight defect. Muscle integrity was almost completely restored in parkin mutants ectopically expressing parkin in muscles. Analysis of proboscis muscle from parkin mutants revealed pathology similar to that of the IFM, indicating that this phenotype is not specific to the flight muscles. However, the tergal depressor of trochanter muscle, involved in the jump response, and the larval body-wall muscles involved in larval locomotion are morphologically and functionally normal in parkin mutants, indicating that only a subset of muscles are affected by loss of parkin function (Greene, 2003).
Ultrastructural analysis of the IFM in 1- to 2-day-old parkin mutants revealed an overall decrease in the density of myofibrils, a broadening of the myofibril Z-line, and a shortening of the sarcomere length. However, the myofibril structural alterations were variable with some indistinguishable from those of control flies. By contrast, swollen mitochondria manifesting severe disruption and disintegration of the cristae were a uniform feature of the IFM in parkin mutants. Transgenic expression of parkin in the musculature restores the myofibril integrity and mitochondrial morphology. The temporal relationship between mitochondrial and myofibril pathology was investigated by analyzing IFM ultrastructure in parkin mutants at the pupal stage of development shortly after IFM formation. At 96 or 120 h after puparium formation the integrity of the myofibrils in parkin mutants was similar to controls, showing no signs of degeneration. The only detectable difference in IFM ultrastructure between parkin mutants and control animals at the pupal stage of development was a disintegration of the mitochondrial matrix in parkin mutants. These results demonstrate that mitochondrial pathology is an early indicator of muscle dysfunction and that the muscle pathology is degenerative in nature (Greene, 2003).
To determine whether muscle degeneration in parkin mutants proceeds through an apoptotic mechanism, the IFM in parkin mutants and age-matched control flies were subjected to terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) staining. At 96 and 120 h after puparium formation, no TUNEL staining was detected in parkin mutants and control flies. However, a dramatic increase in TUNEL-positive nuclei was observed in the IFM of 1-day-old adult parkin mutants relative to age-matched control flies, suggesting that the muscle mitochondrial defects ultimately result in cell death through an apoptotic mechanism (Greene, 2003).
To examine the role of parkin in the brain, sections were prepared from flies at 1, 10, and 30 days of age. Standard histologic analysis revealed appropriate development of the major brain centers. No obvious loss of neuropil integrity or cell cortical number was seen in aged parkin mutant flies compared with controls. Because dopaminergic neurons are a preferential target in AR-JP, brain sections were also immunostained for tyrosine hydroxylase. Dopaminergic neurons of the dorsomedial, dorsolateral, and anteromedial clusters and the medulla were assessed. No clear neuronal loss was observed in any of these cell groups. Tyrosine hydroxylase immunoreactive terminal density was also generally preserved. However, cells of the dorsomedial dopaminergic cell cluster reliably showed shrinkage of the cell body and decreased tyrosine hydroxylase immunostaining in proximal dendrites in aged parkin mutants relative to controls. No such changes were observed in other dopaminergic cell groups. The preferential effect on the dorsomedial cluster is intriguing given the enhanced toxicity of alpha-synuclein (Feany, 2000; Auluck, 2002), another protein implicated in familial PD, in this cluster of dopaminergic neurons (Greene, 2003).
An obvious difference between Drosophila parkin mutants and AR-JP concerns the tissues affected by loss of parkin function. Dopaminergic neurons in the substantia nigra appear to be the primary tissues affected in AR-JP individuals, whereas the most striking phenotypes in Drosophila parkin mutants derive from muscle and germ-line pathology. Nevertheless, the underlying molecular mechanisms responsible for pathology in these different tissues may be highly conserved. Indeed, ultrastructural examination of the male germ line and IFM in parkin mutants reveals mitochondrial defects as a common characteristic of pathology in these distinct tissue types. Although further work will be required to establish the relevance of mitochondrial pathology to the spermatid individualization defect, studies of IFM pathogenesis strongly indicate that mitochondrial pathology is a primary defect. Thus, these results suggest that the Drosophila parkin phenotypes derive from a common origin of mitochondrial dysfunction. There are a variety of cellular insults capable of producing the specific mitochondrial structural alterations observed in parkin mutants, and further work will be required to elucidate the mechanism by which loss of parkin function triggers mitochondrial pathology and ultimately cell death (Greene, 2003).
Although only a few of the factors contributing to the PD disorder have currently been identified in mammals, significant insight into the mechanism of neuronal death in PD has come from studies of the PD-inducing compound 1-methyl-4-phenylpyridinium (MPP+). MPP+ is a specific toxin of dopaminergic neurons that induces cell death by inhibiting mitochondrial complex I. This finding led to the identification of other mitochondrial complex I inhibitors that trigger death of dopaminergic neurons, and prompted studies of mitochondrial integrity in individuals with idiopathic PD. These studies revealed a correlation between PD and mitochondrial dysfunction, and together with the studies of mitochondrial toxins, provide strong support for mitochondrial dysfunction as a major component of PD (Greene, 2003).
The finding that mitochondrial pathology and apoptosis are prominent features of indirect flight muscle degeneration in Drosophila raises the possibility that similar mechanisms underlie dopaminergic neuron loss in AR-JP. Although previous studies have not addressed a role for parkin in mitochondrial integrity or apoptosis, a substantial body of evidence suggests that mitochondrial dysfunction and apoptosis are important factors underlying neurodegeneration in idiopathic PD. Thus, these findings provide a potential mechanistic link between AR-JP and the broader spectrum of idiopathic PD (Greene, 2003).
Panneuronal expression of Parkin substrate Pael-R in transgenic flies causes age-dependent selective degeneration of Drosophila dopaminergic (DA) neurons. The initial observation that in more than half of the Pael-R transgenic lines the protein is present at a low level undetectable by immunostaining suggests that there are mechanisms in Drosophila that limit its accumulation. Recent sequencing of the Drosophila genome has revealed a fly homolog of human Parkin. To test whether Drosophila parkin genetically interacts with the Pael-R transgene, the RNA interference (RNAi) technique was used to inhibit Drosophila parkin expression and examined the effect on Pael-R-induced DA neuron degeneration. To inhibit the expression of parkin in DA neurons of adult fly brain, UAS-IRParkin transgenic flies were generated that express double-stranded Parkin RNA as hairpin RNA from an inverted repeat of Parkin cDNA. Ubiquitous expression of hairpin Parkin RNA using a heat shock-GAL4 driver line resulted in a significant reduction of endogenous Parkin mRNA as determined by quantitative RT-PCR analysis (Yang, 2003).
When IRParkin and Pael-R transgenes were coexpressed in transgenic flies using the Ddc-GAL4 driver, a dosage-dependent acceleration of Pael-R-induced DA neuron degeneration was observed. Transgenic flies coexpressing Pael-R and two copies of the IRParkin transgene showed the degeneration phenotype when they were analyzed at 14 days of age. At this age, transgenic flies expressing Pael-R alone or coexpressing Pael-R and one copy of the IRParkin transgene showed a relatively normal number of DA neurons in the DM clusters. Furthermore, transgenic flies coexpressing Pael-R and two copies of IRParkin showed more severe DA degeneration phenotypes when analyzed at 30 days of age, with the average number of DA neurons in the DM clusters reduced by more than 50%. Thus, inhibition of endogenous Parkin function accelerates the kinetics and enhanced the severity of Pael-R-induced degeneration of DA neurons. In transgenic flies that express IRParkin alone, the number of DA neurons in the DM clusters is not significantly affected in 30-day-old flies. This indicates that activation of the RNAi pathway by itself is not deleterious to DA neurons. The lack of DA neuronal loss by Parkin RNAi alone may be due to the absence of toxic substrates such as Pael-R in Drosophila or that endogenous dParkin substrates may never accumulate to levels sufficient to kill DA neurons. However, since RNAi in IRParkin transgenic flies results in a reduction but not complete loss of Parkin transcripts, it is possible that the residual Parkin may still provide neuroprotective function. Generation of genetic null mutations in the dParkin locus could address this issue (Yang, 2003).
The acceleration of DA neuron degeneration after Parkin RNAi could be due to defects in the turnover or subcellular distribution of Pael-R protein. To investigate the mechanisms involved, Pael-R protein was analyzed by immunostaining cultured DA neurons that coexpressed IRParkin and Pael-R. Coexpression of IRParkin and Pael-R in DA neurons results in the accumulation of Pael-R to higher levels compared to expression of Pael-R alone. Some of the accumulated Pael-R protein was unevenly distributed. Thus, interfering with endogenous Parkin function results in increased stability and accumulation of Pael-R protein. This may be responsible for the acceleration and enhancement of Pael-R-induced DA neuron degeneration observed in Parkin RNAi transgenic flies (Yang, 2003).
Mutations in the gene parkin in humans (PARK2) are responsible for a large number of familial cases of autosomal-recessive Parkinson disease (PD). A Drosophila homolog of human PARK2 has been isolated and its expression and null phenotype have been characterized. parkin null flies have 30% lower mass than wild-type controls; this is in part accounted for by a reduced cell size and number. In addition, these flies are infertile, show significantly reduced longevity, and are unable to jump or fly. Rearing mutants on paraquat, which generates toxic free radicals in vivo, causes a further reduction in longevity. Furthermore, loss of parkin results in progressive degeneration of most indirect flight muscle (IFM) groups soon after eclosion, accompanied by apoptosis. However, parkin mutants have normal neuromuscular junction recordings during the third larval instar stage, suggesting that larval musculature is intact and that parkin is required only in pupal and adult muscle. parkin flies do not show an age-dependent dopaminergic neuron loss in the brain, even after aging adults for 3 weeks. Nevertheless, degeneration of IFMs demonstrates the importance of parkin in maintaining specific cell groups, perhaps those with a high-energy demand and the concomitant production of high levels of free radicals. parkin mutants will be a valuable model for future analysis of the mechanisms of cell and tissue degeneration (Pesah, 2004).
parkin mutant animals have reduced body and cell size at eclosion, suggesting possible defects in cell growth, proliferation and/or cell survival. This phenotype is particularly interesting given the interaction of human Parkin with cyclin E, an important regulator of cell cycle progression. The reduced body size of parkin mutants is similar to the phenotypes of insulin growth factor (IGF) receptor mutant flies and mice. Physiological effects of insulin in the brain are not limited to regulation of food intake and control of glucose uptake, but are also important in trophic actions on neurons and glial cells. Administration of the N-terminal tripeptide of IGF1 prevents loss of DA neurons after chemically (6-hydroxydopamine)-induced DA cell lesion in rats. In addition, IGF1 also protects against DA-induced neurotoxicity in vitro. These observations suggest parkin may play a role in the insulin signaling pathway during development or in adults (Pesah, 2004).
parkin mutant flies have increased sensitivity to paraquat toxicity. Paraquat, with its two N-methyl pyridinium moieties, is structurally similar to MPP(+), a toxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; when converted to the active toxin MPP+ by monoamine oxidase B it can induce parkinsonism in primates). In humans, toxins such as MPTP cause DA neuron-specific death in the substantia nigra because of production of oxygen radicals, resulting in Parkinsonian symptoms. Glutathione and superoxide dismutase (SOD) inactivate H2O2 and superoxide radicals, respectively, thereby reducing MPTP neurotoxicity in mice. Paraquat also causes high oxygen radical production and interferes with mitochondrial respiration, resulting in cell death. The effects of paraquat are not specific to DA cells and sensitivity to its toxicity reflects a general impairment of oxygen radical defense. Loss of Drosophila SOD also results in sensitivity to low doses of paraquat, reflecting a similar defect in oxygen radical defense. The results suggest that antioxidant defenses in parkin mutant flies are also impaired and that parkin may play a role in the oxidative stress response. One likely mechanism of Parkin function is to help rid the cell of specific proteins that are misfolded because of oxygen radical damage. This is consistent with the identification of human Parkin as an E3 ligase (Pesah, 2004 and references therein).
parkin mutant flies show a progressive apoptotic degeneration of indirect flight muscles (IFMs). Furthermore, this degeneration is accompanied by mitochondrial disintegration and loss of christae. Drosophila IFMs are groups of specialized muscle that are in a constant state of vibration. They require a high oxygen supply to sustain their respiratory activity, making this tissue especially susceptible to mitochondrial dysfunction. It is possible that the mitochondrial degeneration observed in parkin mutants results in increased susceptibility to oxygen radical damage because of the impairment of antioxidant defenses by mitochondria, culminating in cell death. The data from animal and tissue culture models of Parkinson disease suggest that mitochondrial dysfunction and oxygen radical damage are two crucial factors in the development of PD pathology. The data underscore the importance of the apoptotic pathway in DA cell loss. Oxidative stress such as that produced by MPTP can trigger apoptosis. Transgenic mice overexpressing the anti-apoptotic gene Bcl2 and mice null for the proapoptotic gene Bax are resistant to MPTP. Loss of parkin results in similar phenotypes in flies: increased sensitivity to oxygen radical stress and IFM apoptosis, suggesting that underlying mechanisms of cellular dysfunction may be similar between flies and humans (Pesah, 2004).
Individuals with Autosomal Recessive-Juvenile Parkinson's (AR-JP) exhibit a loss of DA neurons in the substantia nigra similar to that of idiopathic PD. Therefore, a similar phenotype in parkin mutant animals would be expected. Focus was placed on the dorsomedial cluster (DMC) neurons because they were shown to be affected in other Drosophila models of PD. However, no significant loss of DA neurons was observed at three weeks of age in parkin mutant animals compared with controls. In addition, no changes were seen in DA cell morphology in parkin mutants. Loss of mouse PARK2 also does not result in DA cell loss. However, increased extracellular DA concentration, abnormal neurophysiology and motor and cognitive behavioral deficits are observed in PARK2 mutant mice. By contrast, only motor deficits have been observed thus far in Drosophila. In another model of PD, overexpression of alpha-synuclein in mice does not result in DA cell loss but does cause DA neuron dysfunction. Similarly, in Alzheimer's disease mouse models employing overexpression of amyloid precursor protein, cognitive defects are present in the absence of cortical neuronal loss. The absence of cell loss in multiple models of neurodegenerative disease may reflect the shorter life span of flies and mice compared with humans. Although the mean onset of PD symptoms in AR-JP is the earliest of all genetically defined forms of Parkinson's disease, it is not clear how to compare fly, mouse and human life spans in this regard (Pesah, 2004).
Even though no DA loss is seen in parkin mutant animals, the mechanism of cell loss may be similar between humans and Drosophila. Neurons and muscle are two of the most energy-dependent tissues, and therefore have high numbers of mitochondria and are highly sensitive to mitochondrial insults. Although the mechanisms of cell death in IFMs and in DA neurons might be similar, it is possible that Drosophila IFMs are more sensitive than DA neurons to mitochondrial defects. The nature of this defect is still unclear; however, three mechanisms are likely. (1) It is possible that parkin functions in a trophic factor pathway that promotes cell survival, and in its absence cells become more susceptible to insults such as oxygen radicals. (2) parkin might be important in the stress response pathway, and in its absence the cell becomes more susceptible to various stimuli such as oxygen radical damage that triggers apoptosis. (3) It is possible that parkin is part of the cell death pathway, and its absence results in susceptibility to proapoptotic insults. Drosophila parkin mutants will serve as an invaluable model for understanding the biological role of parkin and may provide important clues concerning the molecular mechanisms of PD (Pesah, 2004).
Loss-of-function mutations of the parkin gene are a major cause of early-onset parkinsonism. To explore the mechanism by which loss of parkin function results in neurodegeneration, a genetic approach was used in Drosophila. Drosophila parkin mutants display degeneration of a subset of dopaminergic (DA) neurons in the brain. The neurodegenerative phenotype of parkin mutants is enhanced by loss-of-function mutations of the glutathione S-transferase S1 (GstS1) gene, which were identified in an unbiased genetic screen for genes that modify parkin phenotypes. Furthermore, overexpression of GstS1 in DA neurons suppresses neurodegeneration in parkin mutants. Given the previous evidence for altered glutathione metabolism and oxidative stress in sporadic Parkinson's disease (PD), these data suggest that the mechanism of DA neuron loss in Drosophila parkin mutants is similar to the mechanisms underlying sporadic PD. Moreover, these findings identify a potential therapeutic approach in treating PD (Whitworth, 2005; full text of article)
Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1 ; PARK6 ) and parkin (PARK2 ), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. This study shows that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1–parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease pathogenesis (Clark, 2006).
Mutations in Pink1, a gene encoding a Ser/Thr kinase with a mitochondrial-targeting signal, are associated with Parkinson’s disease (PD), the most common movement disorder characterized by selective loss of dopaminergic neurons. The mechanism by which loss of Pink1 leads to neurodegeneration is not understood. This study shows that inhibition of Drosophila Pink1 (dPink1) function results in energy depletion, shortened lifespan, and degeneration of select indirect flight muscles and dopaminergic neurons. The muscle pathology was preceded by mitochondrial enlargement and disintegration. These phenotypes could be rescued by the wild type but not the pathogenic C-terminal deleted form of human Pink1 (hPink1). The muscle and dopaminergic phenotypes associated with dPink1 inactivation show similarity to that seen in parkin mutant flies and could be suppressed by the overexpression of Parkin but not DJ-1. Consistent with the genetic rescue results, , in dPink1 RNA interference (RNAi) animals, the level of Parkin protein is significantly reduced. Together, these results implicate Pink1 and Parkin in a common pathway that regulates mitochondrial physiology and cell survival in Drosophila (Yang, 2006).
What could be the molecular mechanism underlying the degeneration phenotypes induced by Pink1 dysfunction? At lease three possibilities could be envisioned. First, Pink1 may regulate energy metabolism. When Pink1 function is compromised, tissues that have the greatest demand for energy, presumably the IFM and dopaminergic neurons, may become particularly vulnerable. The observation that inhibition of Pink1 leads to ATP depletion is consistent with this possibility. The second possibility is that Pink1 may be normally required to guard against the mitochondrial pathway of apoptotic cell death, as suggested earlier. In this regard, it is interesting to note that Parkin has also been shown to prevent mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death. Thus, Parkin and Pink1 may participate in a common pathway that protects cells against mitochondria-dependent cell death induced by toxic insults. The abnormal mitochondrial morphology associated with Parkin and Pink1 inactivation also suggests the third possibility that they may play fundamental roles in regulating mitochondrial biogenesis or mitochondrial dynamics, such as mitochondrial fusion or fission events. A connection between aberrant mitochondrial fission/fusion and neurodegeneration has been appreciated before. Further studies are needed to distinguish among these possibilities (Yang, 2006).
in vivo rescue studies clearly showed that the C terminus of hPink1 is required for hPink1 to rescue dPink1 RNAi phenotypes. Thus, although the C-terminal deleted form of Pink1 may have higher in vitro kinase activity in terms of autophosphorylation, it is incapable of providing the full spectrum of Pink1’s biological activity. It is possible that C-terminal deletion may affect the binding of Pink1 to its substrates or other cofactors. Alternatively, deletion of Pink1 C terminus may contribute to disease pathogenesis by causing deregulation of Pink1 kinase activity (Yang, 2006).
The in vivo biochemical study showed that in dPink1 RNAi animals the level of dParkin is significantly reduced. This result provides one explanation of why Pink1 and Parkin mutants give very similar mutant phenotypes and why Parkin overexpression can rescue dPink1 RNAi phenotypes. It further supports the notion that Parkin acts downstream of Pink1 in a common pathway. The biochemical mechanism by which Pink1 regulates Parkin protein level requires further investigation. In summary, the mitochondrial pathology and IFM and dopaminergic neuron degeneration phenotypes observed in dPink1 RNAi animals and the clear genetic interaction between Pink1 and Parkin suggest that further genetic analysis of the cellular pathway involving Pink1 and Parkin will reveal fundamental mechanisms governing mitochondrial and cellular maintenance. Such mechanisms will likely be applicable to mammalian systems (Yang, 2006).
Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder. Mutations in the PINK1 gene are linked to the autosomal recessive early onset familial form of PD. The physiological function of PINK1 and pathological abnormality of PD-associated PINK1 mutants are largely unknown. Inactivation of Drosophila PINK1 (dPINK1) using RNAi results in progressive loss of dopaminergic neurons and in ommatidial degeneration of the compound eye, which is rescued by expression of human PINK1 (hPINK1). Expression of human SOD1 suppresses neurodegeneration induced by dPINK1 inactivation. Moreover, treatment of dPINK1 RNAi flies with the antioxidants SOD and vitamin E significantly inhibits ommatidial degeneration. Thus, dPINK1 plays an essential role in maintaining neuronal survival by preventing neurons from undergoing oxidative stress, thereby suggesting a potential mechanism by which a reduction in PINK1 function leads to PD-associated neurodegeneration (Wang, 2006; full text of article).
Mutations in human parkin have been identified in familial Parkinson's disease and in some sporadic cases. Expression of mutant but not wild-type human parkin in Drosophila causes age-dependent, selective degeneration of dopaminergic (DA) neurons accompanied by a progressive motor impairment. Overexpression or knockdown of the Drosophila vesicular monoamine transporter, which regulates cytosolic DA homeostasis, partially rescues or exacerbates, respectively, the degenerative phenotypes caused by mutant human parkin. These results support a model in which the vulnerability of DA neurons to parkin-induced neurotoxicity results from the interaction of mutant parkin with cytoplasmic dopamine (Sang, 2007; full text of article).
Most motor symptoms of PD are the result of progressive degeneration of DA neurons originating in the substantia nigra. The molecular pathways that lead to the death of this population of DA neurons are not known, and understanding the basis of selective mesencephalic DA neuron vulnerability may aid the rational design of therapeutics. Because all identified PD-linked genes are expressed ubiquitously in the CNS, it is unclear why such mutations give rise to selective pathology in the nigrostriatal system. It has been suggested that the pathogenesis of monogenic and perhaps idiopathic PD might involve proteins or neurochemicals that are particularly abundant in DA neurons. In vertebrates, nigral DA neurons are characterized by a distinct set of proteins that play a role in DA synthesis and metabolism, such as TH, Ddc, monoamine oxidase (MAO), and the plasma membrane dopamine transporter, as well as other proteins such as VMAT that may be differentially expressed. And of course, unlike other cells, DA neurons also store and release dopamine. The neurotoxic effects of dopamine may be mediated through general oxidative effects and perhaps more specific interactions with proteins implicated in familial PD. It has been observed that alpha-synuclein-induced neurotoxicity is observed in primary cultures of DA neurons but not in non-DA cortical neurons. Moreover, mutant forms of alpha-synuclein mutation form neurotoxic adducts with DA quinone. In addition, the oxidative effects of dopamine may increase protein nitrosylation, and parkin activity is altered by this modification both in vitro and in vivo (Sang, 2007).
Fruit flies use similar sets of genes for dopamine synthesis and transport, but they metabolize dopamine differently from mammals. Insects do not express MAO and are thought to use conjugation as the primary route for amine degradation. This difference may be related to the unique use of dopamine for the hardening and pigmentation of cuticles in insects and other arthropods. Indeed, the need to maintain adequate levels of cytosolic dopamine for these pathways may account for the somewhat surprising effects of Drosophila vesicular monoamine transporter (DVMAT) in total head concentrations of dopamine. Overexpression of DVMAT decreases total dopamine and inhibition of DVMAT using RNAi increases total tissue dopamine. Overexpression of mammalian VMAT2 in cultured cells decreases cytosolic dopamine, and decreased VMAT2 activity in mammals reduces total tissue dopamine. It is suggested that the cytoplasmic pool of dopamine may account for a larger proportion of total tissue dopamine in Drosophila, perhaps because of the requirement of dopamine for cuticle formation in other tissues. It is possible that this may render DA neurons in the fly particularly sensitive to neurotoxic mechanisms involving the conjugation of dopamine to cytoplasmic targets (Sang, 2007).
Based on previous findings in mammals, it is speculated that dopamine and/or oxidized derivatives might contribute to mutant parkin-induced degeneration. If so, mutant parkin phenotypes should be relatively specific for DA neurons and sensitive to modulation of cytoplasmic dopamine levels. Indeed, it was observed that DA but not cholinergic or histaminergic neurons degenerate in response to expression of mutant parkin. Interestingly, a more limited degenerative phenotype was observed in 5-HT neurons, consistent with the loss of other aminergic cell types in PD. DVMAT knockdown enhances mutant parkin phenotypes, suggesting that an increase in cytoplasmic dopamine increases the vulnerability of neurons to mutant parkin. Conversely, increasing DVMAT partially rescues pupal lethality and DA neuron degeneration, presumably by reducing cytoplasmic dopamine. These data therefore suggest that mutations in parkin either increase susceptibility of neurons to dopamine or its metabolites or that dopamine is permissive for the toxic effects of parkin. The Drosophila model, which provides robust behavioral and neuropathological phenotypes, suggests that some mutations may give rise to disease by dominant mechanisms. Moreover, this model may prove a useful addition to other genetically based vertebrate and invertebrate models aimed at understanding PD and identifying potential therapeutic targets (Sang, 2007).
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date revised: 30 March 2008
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