In the sequenced Drosophila genome, there is no clear fly homolog of human Pael-R. To address the role of Pael-R in causing neurodegeneration, the bipartite UAS-GAL4 system was used to target the expression of human Pael-R protein to different fly tissues and cell types. This system involves two transgenic lines, a UAS-Pael-R line, in which the human Pael-R cDNA is placed under the control of upstream activating sequence (UAS) for the yeast transcription factor GAL4, and a GAL4 line, which expresses GAL4 in a tissue- and cell type-specific pattern. In the progenies resulting from a cross between these two lines, Pael-R is expressed in the same tissue and cell type where GAL4 is expressed (Yang, 2003).
Multiple independent UAS-Pael-R transgenic lines were generated through P element-mediated germline transformation. To assess the levels of Pael-R expression in these different transgenic lines, these UAS-Pael-R lines were crossed to the scabrous-GAL4 line, a strong GAL4 driver line that directs transgene expression in the precursor cells and their differentiated progenies in the central and peripheral nervous system. Resulting transgenic embryos were examined for transgene expression by immunostaining with antibodies against Pael-R. Of ten independent transgenic lines analyzed, four give detectable expression of the Pael-R protein as determined by the immunostaining method. Two lines, a strong expression line and a moderate expression line, were chosen for subsequent studies (Yang, 2003).
To target the expression of Pael-R to DA neurons, the Ddc-GAL4 driver line, in which the expression of GAL4 is under the control of the DOPA decarboxylase gene promoter was used. Sections of aged control and transgenic fly brains were immunostained for tyrosine hydroxylase (TH), which specifically identifies DA neurons. Analysis was focused on the dorsomedial (DM) clusters of DA neurons, which have been well characterized and shown to preferentially degenerate in alpha-Syn transgenic flies (Feany, 2000; Auluck, 2002). The DM clusters are composed of approximately 18 DA neurons in wild-type adult fly brain. The two clusters are distributed in a bilaterally symmetric fashion with respect to the midline. The number of DA neurons in the DM clusters does not change significantly, even in 60-day-old senescent wild-type flies. In 40-day-old transgenic flies expressing Pael-R, however, it was observed that the number of DA neurons in the DM clusters was reduced compared to the controls. This reduction of DA neurons was correlated with the expression level of the Pael-R transgene, since the strong expression lines consistently showed a smaller number of DA neurons than the moderate expression line (Yang, 2003).
The loss of DA neurons could be caused by late onset neurodegeneration or early developmental abnormalities. To distinguish these possibilities, younger flies were examined. In newly eclosed transgenic flies (1-day-old) expressing Pael-R, the number of DA neurons in the DM clusters was similar to that observed in control flies. This suggests that the DA neurons are initially formed normally in the transgenic flies, but over time some of these neurons degenerate as a consequence of the overexpression of Pael-R protein (Yang, 2003).
The effects of expressing Pael-R ubiquitously in all the differentiated neurons of the fly nervous system were tested using the strong UAS-Pael-R line and the panneuronal driver elav-GAL4. Western blot analysis on adult fly heads showed that the amount of transgene-produced Pael-R protein per milligram of brain tissue in elav-GAL4 /UAS-Pael-R flies is 45%- 55% that of endogenous Pael-R protein in mouse brain. TH immunostaining revealed that, similar to that observed in Ddc-DAL4 driven transgenic flies, the number of DA neurons in the DM clusters is also reduced in elav-GAL4 driven transgenic flies (Yang, 2003).
Other neuronal types were examined to see if they are also affected after panneuronal expression of Pael-R. The monoclonal antibody 22C10, which recognizes the Futsch antigen, is highly expressed in the visual system as well as the central brain complex in adult flies. The staining of lamina and medulla neuropils by the 22C10 antibody was similar between the control and Pael-R transgenic flies. Overall morphology of the central brain was preserved in Pael-R transgenic flies, suggesting that ubiquitous expression of Pael-R in the brain does not cause widespread degeneration. Specific neuronal subtypes were examined. Immunostaining with the antibody against choline acetyltransferase, an enzyme in the biosynthetic pathway of acetylcholine, showed that the distribution and number of cholinergic neurons in major brain cholinergic centers were similar between control and Pael-R transgenics. Immunostaining of serotonergic neurons with anti-5HT antibody also did not reveal any obvious reduction of these neurons in major brain serotonergic centers. Although the possibility cannot be excluded that a small percentage of serotonergic or cholinergic neurons degenerate in Pael-R transgenic flies, this analysis indicates that there are no clusters of these neurons that are vulnerable to Pael-R toxicity like the DM clusters of DA neurons (Yang, 2003).
Whether the expression of Pael-R is toxic to cell types outside of the central nervous system was also examined. For this purpose, the UAS-Pael-R transgenic lines were crossed to GAL4 driver lines that have known expression patterns in non-CNS tissues. Previous studies showed that expression of many human disease-causing proteins such as polyglutamine repeat-containing proteins, tau, and alpha-Syn in fly eye caused defects in eye morphogenesis and retinal degeneration. In contrast, Pael-R expression in the eye has no phenotypic consequence . Pael-R expression in the eye was targeted to different stages of eye development using the elav-, sevenless-, gmr-, and eyeless-GAL4 lines. In all four cases, Pael-R-expressing flies had normal eye morphology and retina structure. Similarly, no phenotype was observed when the dpp-GAL4 and 24B-GAL4 lines were used to express Pael-R along the anterior-posterior boundary of imaginal discs and in muscle cells, respectively (Yang, 2003).
The only phenotype outside of the nervous system was observed when the apterous-GAL4 line was used to direct Pael-R expression. apterous-GAL4 is expressed in several larval imaginal discs including the dorsal mesothoracic and metathoracic discs. In newly eclosed flies from a cross between the apterous-GAL4 and the strong Pael-R lines, body fluids leaked from two spots located in a bilaterally symmetric fashion on the dorsal thorax at the junction between the notum and scutellum. The fluids quickly solidified and formed two dark patches adhering to the cuticle. This phenotype is presumably caused by the degeneration of the epithelial tissue or the underlying musculature at those sites (Yang, 2003).
Given the propensity of overexpressed Pael-R to become unfolded and insoluble and elicit ER stress in cell culture (Imai, 2001), it is possible that the degeneration phenotype observed in Pael-R transgenic flies is caused by the accumulation of abnormal and toxic forms of Pael-R protein. To test this possibility, it was asked if coexpression of human Parkin protein could promote the degradation of these aberrant proteins and therefore suppress Pael-R toxicity. In transgenic flies that coexpressed Pael-R and human Parkin in DA neurons under the control of the Ddc-GAL4 driver, it was observed that the number of DA neurons in the DM clusters is restored to near wild-type levels. Expression of the human Parkin protein alone using the Ddc-GAL4 driver did not affect the number of DA neurons in the DM clusters (Yang, 2003).
When human Parkin and Pael-R are coexpressed using the apterous-GAL4 driver, the thoracic toxicity of Pael-R is also suppressed and no dark patches form on the dorsal thorax. Expression of human Parkin alone using the apterous-GAL4 driver had no effect on thoracic morphology. These results indicate that coexpression of human Parkin suppresses Pael-R toxicity in two different cell types (Yang, 2003).
To gain further insights into the interaction between human Parkin and Pael-R at the molecular and cellular level, immunohistochemical analysis was performed of human Parkin and Pael-R proteins expressed in neural stem cell-derived DA neurons. Isolated Drosophila embryonic neuroblasts undergo stereotyped divisions when cultured in vitro. After being in culture for 12 hr, the neuroblast progenies exit the cell cycle and form clusters of differentiated neuronal or glial cell types. The neuronal progenies, which include an appropriate proportion of TH-positive DA neurons, are stable in culture for weeks. In cultured DA neurons, Pael-R is mainly localized to the cell surface, as indicated by its colocalization with Numb, a membrane protein. There is also a lower level of expression in neuronal processes. Human Parkin protein exhibits similar localization pattern in cultured DA neurons, but its localization to neuronal processes is more pronounced and appears punctate. Double labeling with Synaptotagmin suggests that the punctate staining of Parkin in neuronal processes represents its association with synaptic vesicles. This is consistent with earlier findings (Kubo, 2001) in mammalian cell culture (Yang, 2003).
The interaction between Pael-R and human Parkin was examined when they were coexpressed in postmitotic neurons under the control of the elav-GAL4 driver. In 1- or 2-day-old neuronal culture, Pael-R and Parkin showed colocalization, and in most neurons (135/154) the level of Pael-R protein was similar to that observed in neurons expressing Pael-R alone. In 6-day-old culture, however, the majority of these neurons (172/235) showed a significant reduction of Pael-R protein as judged by immunostaining. Interestingly, there was a significant portion (63/235) of neurons that still expressed normal levels of Pael-R protein in the presence of Parkin. Because the action of Parkin requires E1, E2, and possibly other cofactors, these neurons may miss certain cofactors required for Parkin-mediated degradation of Pael-R (Yang, 2003).
Whether DA neurons have the capacity to degrade Pael-R in the presence of human Parkin was tested. These two proteins were co-expressed in cultured DA neurons using the Ddc-GAL4 driver. Similar to that observed in elav-GAL4 neuronal culture described above, in younger cultures (1-2 days old) Pael-R and Parkin showed colocalization, and in most Ddc-GAL4-expressing neurons (27/31), the level of Pael-R was similar to that observed in neurons expressing Pael-R alone. But in older neuronal culture (4-6 days), Pael-R protein level was markedly reduced in most Ddc-GAL4-expressing neurons (21/29). In contrast, control neurons expressing Pael-R in the absence of Parkin showed robust Pael-R expression in 6-day-old culture. Thus, it is concluded that human Parkin promotes the degradation of Pael-R in selective groups of Drosophila neurons including DA neurons (Yang, 2003).
To demonstrate the in vivo relevance of these results, showing Parkin-mediated degradation of Pael-R in cell culture experiments, Western blot analysis was used to quantify Pael-R protein levels in transgenic flies with or without Parkin coexpression. Due to the small number of neurons expressing Ddc-GAL4 and the sensitivity of available Pael-R antibodies, Pael-R protein could not be detected in Ddc-Gal4 driven transgenic flies, even in the absence of Parkin. Therefore, elav-GAL4 driven transgenic flies, which express Pael-R protein in more cells, was used. In head extracts obtained from 30-day-old Pael-R transgenic flies driven by elav-GAL4, a significant reduction of Pael-R protein level was observed in the presence of coexpressed Parkin. In contrast, coexpression of Parkin has little effect on the level of a control GFP protein. Together, this immunohistochemical analysis on cultured neurons and Western blot analysis on adult fly brain demonstrate that Parkin promotes the degradation of Pael-R under physiological conditions (Yang, 2003).
Biochemical interactions between Parkin and Pael-R or an O-glycosylated form of alpha-Syn suggest that Parkin may be a common player in the proteasome pathway that regulates the metabolism of these two proteins. The detection of Parkin in Lewy bodies of Parkinson's disease and dementia with Lewy bodies (Shimura, 2001; Schlossmacher, 2002) further suggests that either Parkin is actively engaged in degrading abnormal alpha-Syn proteins or that Parkin is sequestered by alpha-Syn into inactive complexes in the aggregates. In either case, overexpression of Parkin would be predicted to enhance the degradation of abnormal alpha-Syn or to compensate for the reduction of Parkin activity caused by alpha-Syn sequestration and therefore suppress alpha-Syn toxicity. To test this hypothesis, alpha-Syn was coexpressed together with human Parkin in DA neurons. While the expression of wild-type or pathological forms of alpha-Syn (A30P or A53T) consistently caused a 50% reduction of DA neurons in the DM clusters in 20-day-old flies, this loss of DA neurons was suppressed when Parkin was coexpressed with alpha-Syn. Interestingly, unlike Parkin suppression of Pael-R toxicity, which is accompanied by a dramatic reduction of overall Pael-R protein level, immunohistochemical analysis shows that alpha-Syn level is comparable in transgenic flies with or without Parkin coexpression. This was further confirmed by Western blot analysis. Detailed examination of alpha-Syn distribution in DA neurons in the DM clusters reveals that while alpha-Syn forms numerous grain-like structures in the processes, there is a marked reduction of such structures in Parkin-coexpressing flies. Although it is not known whether all of these grain-like structures represent pathological forms of alpha-Syn, the suppression of alpha-Syn toxicity and concomitant reduction of these structures by Parkin coexpression suggest that at least some of them correspond to toxic forms of alpha-Syn. To further characterize the effect of Parkin overexpression on neuritic pathology induced by alpha-Syn, alpha-Syn transgenic fly brain was stained with anti-ubiquitin antibody, which can recognize Lewy bodies and Lewy neurites in human PD patient brain. In Ddc-GAL4 driven alpha-Syn transgenic fly brain, numerous ubiquitin-positive neurites could be detected. In Parkin-coexpressing flies, however, such abnormal neurites were almost completely eliminated. Therefore, mitigation of alpha-Syn toxicity by Parkin overexpression is associated with reduced alpha-Syn-induced neuritic pathology and reduced aggregation of alpha-Syn (Yang, 2003).
Parkinson's disease, a prevalent neurodegenerative disease, is characterized by the reduction of dopaminergic neurons resulting in the loss of motor control, resting tremor, the formation of neuronal inclusions and ultimately premature death. Two inherited forms of PD have been linked to mutations in the α-synuclein and parkin genes. The parkin protein functions as an ubiquitin ligase targeting specific proteins for degradation. Expression of human α-synuclein in Drosophila neurons recapitulates the loss of motor control, the development of neuronal inclusions, degeneration of dopaminergic neurons and the ommatidial array to provide an excellent genetic model of PD. To investigate the role of parkin, transgenic Drosophila were generated that conditionally express parkin under the control of the yeast UAS enhancer. While expression of parkin has little consequence, co-expression of parkin with α-synuclein in the dopaminergic neurons suppresses the α-synuclein-induced premature loss of climbing ability. In addition directed expression of parkin in the eye counteracts the α-synuclein-induced degeneration of the ommatidial array. These results show that parkin suppresses the PD-like symptoms observed in the α-synuclein-dependent Drosophila model of PD. It is concluded that the highly conserved parkin E3 ubiquitin ligase can suppress the damaging effects of human α-synuclein. These results are consistent with a role for parkin in targeting α-synuclein to the proteasome. If this relationship is conserved in humans, this suggests that up-regulation of parkin should suppress α-synucleinopathic PD. The development of therapies that regulate parkin activity may be crucial in the treatment of PD (Haywood, 2004; full text of article).
Parkinson’s disease (PD) patients show a characteristic loss of motor control caused by the degeneration of dopaminergic neurons. Mutations in the genes that encode α-synuclein and parkin have been linked to inherited forms of this disease. The parkin protein functions as a ubiquitin ligase that targets proteins for degradation. Expression of isoforms of human α-synuclein in the Drosophila melanogaster nervous system forms the basis of an excellent genetic model that recapitulates phenotypic and behavioural features of PD. Using this model, the effect was analyzed of parkin co-expression on the climbing ability of aging flies, their life span, and their retinal degeneration. Co-expression of parkin can suppress phenotypes caused by expression of mutant α-synuclein. In the developing eye, parkin reduces retinal degeneration. When co-expressed in the dopaminergic neurons, the ability to climb is extended over time. If conserved in humans, it is suggested that upregulation of parkin may prove a method of suppression for PD induced by mutant forms of α-synuclein (Haywood, 2005).
These experiments demonstrate that the directed expression of parkin in the developing eye negates the retinal defects resulting from mutant α-synuclein expression. In addition, increased parkin expression in the dopaminergic neurons extends the climbing ability of aged flies that express mutant α-synuclein. This suggests that parkin can suppress the degeneration resulting from mutant α-synuclein expression in spite of the amino acid substitution present in the mutant form of α-synuclein that is thought to lead to a conformational change in the protein. Although the exact mechanism of phenotype suppression is not clear, it does indicate that mutant α-synuclein is likely a target of parkin’s ubiquitin-ligase activity. Co-immunoprecipitation studies have suggested that the parkin protein does not interact with or ubiquitinate unmodified α-synuclein but will ubiquitinate O-glycosylated α-synuclein (Haywood, 2005).
This study established the suppression of mutant α-synucleininduced retinal degeneration by the ectopic expression of parkin. Therefore, it is believed that mutant α-synuclein protein is modified in Drosophila in a manner that will enable it to be ubiquitinated by the parkin ubiquitin protein ligase then targeted to the proteasome for degradation. Transgenic Drosophila that express either the wild type or mutant form of α-synuclein in their central nervous systems, via the pan-neural elav-Gal4 transgene, have shown an age-dependent reduction in climbing ability when compared with control flies. Notably, flies that express mutant α-synuclein under the control of elav-Gal4 show a greater reduction in climbing ability. Although flies that express wild-type α-synuclein in their dopaminergic neurons show a marked premature loss of climbing ability, expression of mutant α-synuclein results in only a slight premature loss of the ability to climb. Regardless, over-expression by both of the parkin transgenes had the effect of extending the climbing ability of flies expressing A30Pα-synuclein when compared with the controls. Thus the premature loss of climbing ability arising from a mutant form of α-synuclein that is known to cause PD in humans was prevented by the directed expression of parkin (Haywood, 2005).
Over-expression of parkin suppresses the PD-like symptoms induced in Drosophila by wild-type and mutant α-synuclein with no apparent adverse consequences. It is suggested that the manipulation of the ubiquitin-proteasome degradation pathway in such a specific manner acts to remedy the toxicity of the accumulation of α-synuclein. Activation of parkin may be a viable treatment for PD caused by increased levels or mutant forms of α-synuclein and it is suggested that the selection of therapeutic strategies should be directed towards this end (Haywood, 2005).
Mutations in the parkin gene are a predominant cause of familial parkinsonism. Although initially described as a recessive disorder, emerging evidence suggest that single parkin mutations alone may confer increased susceptibility to Parkinson's disease. To better understand the effects of parkin mutations in vivo, transgenic Drosophila were generated overexpressing two human parkin missense mutants, R275W and G328E. Transgenic flies that overexpress R275W, but not wild-type or G328E, human parkin display an age-dependent degeneration of specific dopaminergic neuronal clusters and concomitant locomotor deficits that accelerate with age or in response to rotenone treatment. Furthermore, R275W mutant flies also exhibit prominent mitochondrial abnormalities in their flight muscles. Interestingly, these defects caused by the expression of human R275W parkin are highly similar to those triggered by the loss of endogenous parkin in parkin null flies. Together, these results provide the first in vivo evidence demonstrating that parkin R275W mutant expression mediates pathogenic outcomes and suggest the interesting possibility that select parkin mutations may directly exert neurotoxicity in vivo (Wang, 2007).
Although parkin-linked disease transmission is presumed to occur in a recessive manner, the expanding number of reports associating single parkin mutation with increased risk for PD has raised questions on the mode of disease transmission by parkin mutations. To date, several parkin mutations occurring on different regions of the protein have been found in heterozygous carriers. Interestingly, heterozygous PD cases harboring the R275W mutation are a recurrent theme in these studies. However, the identification of heterozygous parkin mutations remains controversial. Indeed, single parkin mutation cases are often overestimated in the literature. Importantly, single parkin mutations exist as bona fide heterozygous mutations (West, 2004). Another concern over heterozygous parkin mutation carriers is that they may occur incidentally. However, at least two recent case control studies of the parkin gene in early- and late-onset PD reveal the presence of several heterozygous parkin mutations, including R275W, that are not found in control individuals, thereby providing a direct association between heterozygous parkin mutations and increased susceptibility for PD. Notwithstanding the controversy surrounding the existence of single parkin mutations, whether and how heterozygous parkin condition affects dopaminergic neuronal survivability remains obscure (Wang, 2007).
The importance of functional parkin to dopaminergic neuronal survival is probably related to the multitude of neuroprotective roles it serves. Supporting this, it was found that flies overexpressing wild-type human parkin are more resistant to rotenone-induced degeneration and associated locomotor defects. Moreover, expression of wild-type human parkin in parkin null flies effectively prevents select dopaminergic neurons from degenerating. Although Ddc-GAL4-driven hparkin/- flies recorded similarly poor climbing scores to parkin null flies, this is likely attributable to the inability of Ddc-GAL4-driven hparkin expression to compensate for the widespread muscle pathology in parkin null flies. Notably, when driven by 24B-GAL4, wild-type but not R275W human parkin expression in parkin null flies significantly mitigates the mitochondrial phenotype of the latter. This is consistent with previous reports by others showing that expression of wild-type Drosophila parkin using 24B-GAL4 could rescue dparkin null phenotype. The apparent protection afforded by wild-type human parkin against dopaminergic neurodegeneration and mitochondrial pathology in parkin null flies suggests a certain degree of functional conservation between human and fly parkin (Wang, 2007).
Given its broad-spectrum neuroprotective roles, one could envisage that parkin haploinsufficiency could increase the risk of heterozygous parkin mutation carriers for PD. Consistent with this, it has bee demonstrated that age, mutations, and PD-linked stress could deplete the availability of soluble functional parkin in the brain (LaVoie, 2005; Sriram, 2005; Wang, 2005a; Wang, 2005b; Wong, 2007) and, as such, may underlie the progressive susceptibility of the brain to degeneration. Furthermore, a promoter variant of parkin associated with a lower expression of parkin occurs more commonly in PD cases than in controls. However, the possibility that dominant-negative mutations might account for a proportion of single parkin mutation-linked PD cases cannot be excluded. In particular, a recent family-based study conducted in Germany implicates the transmission of the R275W mutation as an autosomal dominant trait, thereby providing additional support for the notion that single R275W parkin mutation might be sufficient to cause disease (R. Krueger, personal communication to Wang, 2007). Supporting this, the current study demonstrates that the overexpression of parkin R275W in Drosophila is toxic to dopaminergic neuronal survival, albeit in select clusters. Furthermore, R275W expression in Drosophila flight muscles also promotes mitochondrial abnormalities. Thus, heterozygous parkin R275W expression in vivo may contribute to pathogenecity. Interestingly, these observations corroborate with a very recent study conducted by Sang (2007) who showed that expression of parkin T240R and Q311X mutants in Drosophila causes age-dependent, selective degeneration of DA neurons accompanied by progressive motor impairment. The dissimilar outcomes mediated by R275W and G328E mutants in flies may be related to their different properties, as observed in a previous in vitro study (Wang, 2005a; Wang, 2007 and references therein).
This study applied an unbiased confocal microscopy-based quantitative method to detect subtle neuronal loss in the fly brains. Similar to the observation made by Whitworth (2005) with parkin null mutants, comparable loss in the number of dopaminergic neurons within the PPL1 cluster was detected in parkin null flies at 20 d after eclosion. Furthermore, an obvious loss of dopaminergic neurons was observed in the PAM cluster of these flies. Importantly, R275W parkin-overexpressing flies show the same degeneration pattern as parkin null flies, and both these mutant fly species exhibit marked mitochondrial pathology, suggesting that heterozygous parkin mutations could result in similar outcomes to that arising from the overt loss of parkin function. It is noteworthy that flies expressing the R275W mutant also appear to be more susceptible to rotenone-induced neurotoxicity. Although accelerated neuronal loss is observed in all the dopaminergic clusters in the various fly strains after rotenone treatment, the PPL1 neurons in R275W are significantly more affected. Interestingly, the selective impact on neuronal integrity exerted by the expression of R275W in flies correlates well with their impaired climbing ability compared with age-matched control flies. Although previous studies have attributed compromised locomotory activity observed in parkin null flies primarily to defects in their musculature, the current results with Ddc-driven parkin R275W flies show that dopaminergic neurodegeneration in select clusters alone is sufficient to trigger climbing defects. Furthermore, the protection of similar clusters of neurons by the Ddc-driven expression of wild-type human parkin translates to better climbing scores. It is thus tempting to suggest a direct association between dopaminergic neurodegeneration and locomotory dysfunction in flies, a phenomenon that is seen in PD patients (Wang, 2007).
How the expression of R275W mutant influences the function of endogenous parkin remains to be elucidated, but its expression does not appear to aggravate the dopaminergic neurodegeneration process when expressed in parkin null flies. Although the latter is consistent with the R275W mutant parkin acting as a dominant-negative protein, the poorer climbing scores of R275W/- flies compared with R275W and parkin null flies is intriguing. Furthermore, the abnormal mitochondrial phenotype observed in the flight muscles of R275W mutant flies is qualitatively different from that seen in parkin null flies, suggesting that R275W parkin mutant may exert toxic effects in vivo via a different mechanism from that produced by the overt loss of parkin expression. As with all experiments involving the expression of transgenes in a heterologous system, the possibility cannot be excluded that the phenotypic characteristics exhibited by R275W mutant flies arise from neomorphic manifestations associated with the expression of a foreign gene. However, several observations made in this study would argue against R275W mutant acting as a neomorph. (1) Ddc-driven expression of R275W mutant in flies affects the same clusters of dopaminergic neurons as those in parkin null flies; (2) although the effects mediated by R275W and parkin null mutant on Drosophila flight muscle are not exactly identical, they nonetheless promote a similar outcome, i.e., mitochondrial abnormalities; (3) compared with the R275W mutant, the better-expressed parkin G328E mutant behaves as a relatively benign foreign protein in Drosophila. Importantly, when wild-type parkin-expressing flies were crossed with R275W mutant flies, it was found that their coexpression significantly mitigates the loss of PPL1 dopaminergic neurons observed in R275W flies. Although the exact mechanism of R275W mutant-mediated toxicity remains to be clarified, it is apparent that the R275W fly model recapitulates the essential symptomatic features of PD and thus represents an ideal in vivo model of parkin dysfunction. Given the demonstrated pathogenicity of R275W mutant parkin expression in flies, these results may also help explain the increased susceptibility of heterozygous parkin carriers to develop PD (Wang, 2007).
In summary, this study has provided important in vivo evidence demonstrating that select parkin mutations could generate marked neurotoxicity in vivo. Whether the neurotoxic effects mediated by these mutants could indeed account for a proportion of single parkin mutation-linked PD cases remains to be established. Additional study should help elucidate the pathogenic mechanism caused by these mutations (Wang, 2007).
Loss-of-function mutations in the PINK1 or parkin genes result in recessive heritable forms of parkinsonism. Genetic studies of Drosophila orthologs of PINK1 and parkin indicate that PINK1, a mitochondrially targeted serine/threonine kinase, acts upstream of Parkin, a cytosolic ubiquitin-protein ligase, to promote mitochondrial fragmentation, although the molecular mechanisms by which the PINK1/Parkin pathway promotes mitochondrial fragmentation are unknown. This study tested the hypothesis that PINK1 and Parkin promote mitochondrial fragmentation by targeting core components of the mitochondrial morphogenesis machinery for ubiquitination. The steady-state abundance of the mitochondrial fusion-promoting factor Drosophila Mitofusin (Mitochondrial assembly regulatory factor, Marf or dMfn) is inversely correlated with the activity of PINK1 and Parkin in Drosophila. dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and dMfn co-immunoprecipitates with Parkin. By contrast, perturbations of PINK1 or Parkin did not influence the steady-state abundance of the mitochondrial fission-promoting factor Drp1 or the mitochondrial fusion-promoting factor Opa1, or the subcellular distribution of Drp1. These findings suggest that dMfn is a direct substrate of the PINK1/Parkin pathway and that the mitochondrial morphological alterations and tissue degeneration phenotypes that derive from mutations in PINK1 and parkin result at least in part from reduced ubiquitin-mediated turnover of dMfn (Poole, 2010).
In previous work, it has been shown that genetic manipulations that promote mitochondrial fragmentation, including increased drp1 gene dosage and decreased opa1 or dmfn gene dosage, dramatically suppress the PINK1 and parkin mutant phenotypes in Drosophila. These findings, coupled with previous work demonstrating that PINK1 acts upstream of Parkin in a common pathway, led to a hypothesis that PINK1 and Parkin influence mitochondrial integrity by regulating core components of the mitochondrial morphogenesis machinery through ubiquitination. The current results provide direct support for this hypothesis by demonstrating that dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and that the steady-state abundance of dMfn is increased in PINK1 and parkin mutants and decreased in PINK1- and Parkin-overexpressing flies. These findings suggest a model in which PINK1 phosphorylates either dMfn or Parkin and thereby increases the efficiency with which Parkin is able to ubiquitinate dMfn. The subsequent ubiquitin-mediated turnover of dMfn would then inhibit mitochondrial fusion, and thus promote mitochondrial fragmentation. While the current findings were in review, another study primarily using cultured Drosophila S2 cells (Ziviani, 2010) also reported that dMfn is a substrate of the PINK1/Parkin pathway, thus providing additional support for these conclusions (Poole, 2010).
The finding that the PINK1/Parkin pathway promotes mitochondrial fragmentation led to a proposal that this pathway may act to segregate damaged portions of the mitochondrial reticulum for turnover through an autophagic mechanism (Poole, 2008). Several recent studies provide compelling support for this hypothesis by demonstrating that treatment of cultured vertebrate cells with mitochondrial damaging agents triggers PINK1 to selectively recruit Parkin to damaged mitochondria, where Parkin acts to promote the autophagic turnover of these mitochondria, presumably by ubiquitinating particular mitochondrial targets. These studies, together with the current findings raise the possibility that the selective Parkin-mediated ubiquitination and subsequent degradation of dMfn on damaged portions of the mitochondrial reticulum, coupled with ongoing mitochondrial fission serves to sequester the mitochondrial damage to small fusion-incompetent mitochondria that are subsequently eliminated through autophagy. However, the size of ubiquitinated dMfn suggests that it is triply ubiquitinated and previous work indicates that a chain of four or more ubiquitins is required for efficient targeting to the proteasome. Thus, alternative interpretations of the findings, although not mutually exclusive, are that ubiquitination of dMfn inactivates the fusion-promoting activity of dMfn, or serves as a tag marking the damaged mitochondria for destruction by autophagy. The finding that the ubiquitination of a peroxisomal surface protein is sufficient to signal the autophagic degradation of this organelle is consistent with the latter model. Experiments are currently underway to distinguish these possibilities (Poole, 2010).
While a model in which the PINK1/Parkin pathway promotes mitochondrial fragmentation through the ubiquitination of dMfn is completely consistent with previous work on PINK1 and Parkin in Drosophila, recent findings from vertebrate cell culture studies challenge this model. In particular, several of the studies of PINK1 in vertebrate systems have found that reduced PINK1 activity results in mitochondrial fragmentation, suggesting that PINK1 may promote mitochondrial fusion—exactly the opposite of the conclusion drawn from studies of the PINK1/Parkin pathway in flies. While additional work will be required to resolve these apparent conflicts, it is important to point out that the findings from studies of PINK1 and Parkin in flies have involved tissues that are profoundly affected by loss of PINK1 and Parkin activity, whereas the tissue sources of the cells that have been used in at least some of the conflicting vertebrate studies are largely unaffected by mutations in PINK1 and parkin. Thus, a possible explanation for these apparently discordant findings is that the mitochondrial fragmentation resulting from reduced PINK1 activity that has been observed in vertebrate systems involves a compensatory induction of mitochondrial fragmentation in these cells, which perhaps also explains their relative insensitivity to the loss of PINK1 activity. In potential support of this model is the finding that while the mitochondrial fragmentation seen in PINK1-deficient vertebrate cells can be rescued by inactivating Drp1, this manipulation enhances the cell death associated with PINK1 deficiency, a finding that is entirely consistent with work in flies. Future work should resolve these apparent conflicts and further clarify the influence of PINK1- and Parkin-dependent ubiquitination of dMfn on mitochondrial integrity (Poole, 2010).
Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson's disease, a neurodegenerative disorder of unknown etiology. Parkin has been linked to multiple cellular processes including protein degradation, mitochondrial homeostasis, and autophagy; however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting mitochondrial fission and/or fusion, to mediate their autophagic turnover. The precise mechanism of recruitment and the ubiquitination target are unclear. This study shows in Drosophila cells that PINK1 is required to recruit Parkin to dysfunctional mitochondria and promote their degradation. Furthermore, PINK1 and Parkin mediate the ubiquitination of the profusion factor Mfn on the outer surface of mitochondria. Loss of Drosophila PINK1 or parkin causes an increase in Mfn abundance in vivo and concomitant elongation of mitochondria. These findings provide a molecular mechanism by which the PINK1/Parkin pathway affects mitochondrial fission/fusion as suggested by previous genetic interaction studies. It is hypothesized that Mfn ubiquitination may provide a mechanism by which terminally damaged mitochondria are labeled and sequestered for degradation by autophagy (Ziviani, 2010).
Maintenance of mitochondrial homeostasis appears to be an important function of the PINK1/Parkin pathway in multiple model systems and is likely a key factor in mediating neurodegeneration. Recent studies have begun to shed light on the potential mechanism by which this pathway maintains a healthy mitochondrial population. Emerging evidence indicates that PINK1 is required to recruit Parkin to damaged or dysfunctional mitochondria, whereupon it promotes mitophagy. Regulated mitochondrial fission and fusion events are thought to contribute to a quality control mechanism to help 'sort out' terminally damaged mitochondria for degradation. Importantly, PINK1 and parkin have previously been shown to genetically interact with components of the mitochondrial fission/fusion machinery and to affect mitochondrial morphology; however, the molecular mechanisms are not known. This study provides further evidence that PINK1 is required for Parkin translocation to damaged mitochondria and that this pathway affects mitochondrial morphology. Evidence is also provided that the PINK1/Parkin pathway promotes the ubiquitination and regulates the levels of the profusion protein Mfn, thus providing a potential molecular mechanism by which PINK1/Parkin may modulate mitochondrial dynamics (Ziviani, 2010).
Consistent with recent reports, this study found that the translocation of Parkin to damaged mitochondria and their subsequent autophagy is dependent on PINK1. However, the molecular mechanisms that promote Parkin's recruitment to mitochondria are still unclear. PINK1's kinase activity, but not mitochondrial localization, appears to be necessary for Parkin translocation. Because PINK1 can be found extramitochondrially and may directly phosphorylate Parkin, this may be a mechanism to stimulate its translocation. Alternatively, it may phosphorylate a Parkin substrate, e.g., Mfn, and thereby provide a recruitment signal. Interestingly, this study found that loss of Mfn reduces but does not eliminate Parkin translocation. Recent evidence indicates that Parkin also ubiqutinates VDAC on the outer mitochondrial surface, suggesting that there may be multiple recruitment substrates. Although further work is required to elucidate these mechanisms, these findings suggest a molecular basis for the genetic hierarchy in which PINK1 acts upstream of Parkin (Ziviani, 2010).
To understand the role of Parkin translocation, this study took a candidate approach to identifying putative substrates. Because the function of Parkin and PINK1 has been linked with mitochondrial dynamics, key components of the mitochondrial fission and fusion machinery were surveyed for ubiquitin modification. Mfn, which localizes to the outer surface of mitochondria, was found to be ubiquitinated in a PINK1/Parkin-dependent manner and accumulates upon loss of PINK1 or parkin. Interestingly, the ubiquitinated isoforms do not show a typical ubiquitination 'ladder' but instead appear to reflect a pattern of one and three or four ubiquitin adducts. Although it remains to be shown that Parkin directly mediates this ubiquitination, there is evidence that Parkin can mediate monoubiquitination and K27 and K63 linkages. These modes of ubiquitination are not typically linked to proteasome degradation, and there is growing speculation that important pathogenic functions of Parkin may be proteasome independent (Ziviani, 2010).
Numerous elegant studies have demonstrated that the mitochondrial network is extremely dynamic and responds rapidly and reversibly to many physiological changes including potentially toxic challenges such as oxidative stress and calcium flux. Although mitochondrial remodeling can contribute to promoting cell death, it can also act in a protective manner by contributing to a quality control process that likely involves degradation by autophagy/lysosomes. Recent work has reported observations that, following a fission event, regulated fusion of daughter mitochondria can determine whether they rejoin the network or are sequestered for degradation. Refusion appears to be dependent upon the recovery of mitochondrial membrane potential after division and likely represents a mechanism to sort out terminally dysfunctional mitochondria. Because Mitofusins mediate the tethering and fusion of mitochondria via homo- and heterotypic interaction of their HR2 domains, it is hypothesized that Parkin-mediated Mfn ubiquitination may interfere with intermolecular interactions preventing fusion. Alternatively, Mfn ubiquitination may lead to a selective removal of Mfn from damaged mitochondria and thus reduce the refusion capacity of those mitochondria. Consistent with this, it was found that loss of parkin or PINK1, and hence loss of ubiquitination, leads to increased Mfn levels and mitochondrial elongation, presumably due to excess fusion. Thus, Mfn ubiquitination may provide a signal that simultaneously prevents the refusion of terminally damaged mitochondria and labels them for safe degradation by autophagy (Ziviani, 2010).
It is reasonable to suppose that under normal conditions the majority of mitochondria are relatively healthy, and thus mitochondrial turnover is an infrequent event. This is supported by the observation that Complex Vα levels are not significantly altered by decreased mitophagy. However, this rationale implies that Mfn accumulates and is selectively ubiquitinated on mitochondria targeted for degradation although this remains to be shown. Interestingly, the current findings provide a molecular mechanism that can explain the previously reported genetic interactions between PINK1 and parkin and the fission/fusion factors - in particular, that promoting mitochondrial fragmentation by overexpression of Drp1 or by reduction of Mfn and Opa1 is able to partially suppress the locomotor deficits, muscle degeneration, and mitochondrial abnormalities. Together these findings suggest that aberrant accumulation of Mfn may mediate the loss of mitochondrial homeostasis caused by loss of PINK1 or parkin. Although further work will be needed to determine whether this contributes to PD pathogenesis, these results support the emerging hypothesis that the PINK1/Parkin pathway acts to regulate the safe degradation of terminally damaged mitochondria as a quality control mechanism (Ziviani, 2010).
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