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parkin counteracts symptoms in a Drosophila model of Parkinson's disease

parkin counteracts symptoms in a Drosophila model of Parkinson's disease Background: 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. Results: To investigate the role of parkin, we have generated transgenic Drosophila 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. Conclusion: 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. tem analysis reveals the selective loss of dopaminergic Background Parkinson's disease (PD) is a neurodegenerative disorder neurons from the substantia nigra region of the brain. Fil- that is characterized by muscle tremors in stationary amentous protein inclusions, known as Lewy bodies, are limbs, bradykinesia (slowed movement) and difficulty found within the neuronal cell bodies of the affected area, initiating and sustaining movements, and affects 1–2% of in most but not all PD patients [4]. Although the majority the population older than sixty years of age [1-5]. As the of PD cases appear to be sporadic, about 5–15% have disease progresses, both the sense of balance and the been determined to have an inherited basis [6,7]. memory of the affected individual deteriorate. Post-mor- Recently, mutations in a number of genes have been Page 1 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 identified as causes of PD and many of these genes are were observed in α-synuclein-expressing flies approxi- associated with the ubiquitin/proteasome protein degra- mately 20 days after eclosion. While control flies exhibit a dation pathway. strong negative geotaxis, these transgenic flies prema- turely lose their climbing ability. In addition, expression Mutations in the gene encoding the α-synuclein protein of α-synuclein in the developing eye results in precocious lead to the development of Autosomal Dominant PD degeneration of the retina. In this model expression of a (ADPD) [8,9]. The α-synuclein protein is an abundant number of genes are dysregulated prior to the onset of 140 amino acid, cytosolic protein found at the pre-synap- neurodegeneration [33]. These features recapitulate the tic region of neurons [10,11]. α-synuclein appears to be main behavioural and pathological phenotypes of PD and involved in the biosynthesis of dopamine [12,13]. Muta- provide an excellent model system to study the biological tions in the α-synuclein gene [8,9] may lead to enhanced basis of the disease. oligomerization and fibril formation of the α-synuclein protein [14-16]. The Drosophila α-synuclein based model has been used to investigate a number of aspects of PD. Pharmacological Autosomal Recessive Juvenile Parkinson's disease (ARJP), agents, such as the dopamine precursor levodopa, another inherited form of PD, has been attributed to a dopamine receptor agonists (bromocriptine, pergolide number of point mutations and deletions of the parkin and SK&F38393), and the anticholinergic atropine, were gene [17-19]. ARJP is specifically characterized by a very demonstrated to modify the age-dependent loss of climb- early age of onset, mostly before forty years of age, and the ing ability [34]. Co-expression of the molecular chaper- absence of Lewy bodies [20-22]. In humans, the parkin one HSP70 gene with α-synuclein prevented dopaminergic gene encodes a 465 amino acid protein [17] that func- neuronal degeneration [35]. Interference with endog- tions as one of a number of E3 ubiquitin protein ligases, enous chaperone activity accelerated the toxicity of α- components of the ubiquitin/proteasome degradation synuclein demonstrating a role for chaperones in the pathway [23]. Ubiquitin protein ligases act to identify pathology of the disease. Suppression of HSP90, a nega- damaged, misfolded, and short-lived proteins to mediate tive regulator of the heat shock factor 1, by feeding flies the ubiquitination (the sequential attachment of a geldanamycin prevents dopaminergic neuronal cell death number of ubiquitin monomers) of these proteins, which [36]. Recently, the expression of human parkin has been are targeted to the proteasome [24,25]. Experiments in tis- shown to suppress the loss of dopaminergic neurons sue culture have demonstrated that parkin can ubiquiti- induced by α-synuclein in Drosophila [37]. This model has nate a number of substrates including a glycosylated form proven to be an effective tool in the investigation of the of α-synuclein [26], the Pael receptor [27], CDCrel-1 [28], biological basis of PD. the α-synuclein-binding protein synphilin-1 [29], and parkin itself [28,30]. The loss of parkin may lead to an To investigate the role of parkin in the α-synuclein-based accumulation of one or a number of proteins in sufficient model of Parkinson's disease in Drosophila, we have char- quantities to cause neuronal cell death. acterized and expressed the Drosophila homologue of par- kin in this model. Our results demonstrate that parkin can The interaction of parkin with α-synuclein suggests a com- counteract the effects of α-synuclein on climbing activity mon mechanism underlying inherited forms of PD. and retinal degeneration. Indeed, elevated expression of parkin protects neuronal explants from the toxicity associated with expression of α- Results synuclein [26,31]. The disease inducing-forms of α-synu- Characterization of Drosophila melanogaster parkin clein may prevent its degradation and result in toxic accu- The Drosophila melanogaster parkin homologue was identi- mulation. In ARJP, functional parkin protein is lost along fied through a search of the Berkeley Drosophila Genome with the ability to mediate the ubiquitination of glyco- Project (BDGP) utilizing the tBlastn search algorithm. The sylated α-synuclein and may lead to the accumulation of parkin gene is located on the left arm of the third chromo- this protein [26]. Our working hypothesis is that aspects some, in the polytene chromosome section 78C within of parkin-mediated protein degradation are compromised the genomic scaffolding region AE003593 (BDGP), and in PD. consists of 6 exons over 2.2 kb (Figure 1A). A search of the genome for additional parkin homologues revealed none. The first Drosophila melanogaster model of PD was gener- Our analysis confirmed the sequence of parkin to be iden- ated by the conditional expression of human α-synuclein tical to that reported by the BDGP (AY058754.1). Two in transgenic Drosophila [32]. Flies that express α-synuclein, potential initiation codons for the parkin protein are sep- in either a pan-neural or dopaminergic neuron specific arated by 42 base pairs at the 5' region of the transcript. As manner, show a marked age-dependent loss of dorsal- the second potential start codon is preceded by CAAA, a medial dopaminergic neurons. Cytoplasmic inclusions match to the Drosophila Kozak consensus sequence (C/ Page 2 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 1 2 3 4 5 6 Genomic sequence AE 003593 244.2 243.8 242.2 Kb 244.5 243.5 243.2 242.8 242.5 Ubiquitin like domain (UBD) D. melanogaster MLELLQFGGKTLTHTLSIYVKTNTGKTLTVNLEPQWDIKNVKELVAPQLGLQPDDLKIIFAGKELSDATTIEQCDLGQQSVLHAIRLRP-------- PV 92 A. gambiae MLAIFSFGKKKLSNSLSVYVKTNTGNTLAVDLEPHMDIKDVKEMVAPRLGLEPQELKIIFAGRELSDTTTISECDLGQQSIIHVVKSRPT----AITTPQ 97 R. norvegieus ---------------MIVFVRFNSSYGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVIFAGKELQNHLTVQNCDLEQQSIVHIVQ-RPQRKSHETNASG 85 M. musculus ---------------MIVFVRFNSSYGFPVEVDSDTSILQLKEVVAKRQGVPADQLRVIFAGKELPNHLTVQNCDLEQQSIVHIVQ-RPRRRSHETNASG 85 H. sapiens ---------------MIVFVRFNSSHGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVIFAGKELRNDWTVQNCDLDQQSIVHIVQ-RPWRKGQEMNATG 85 : ::*: *:. :.*:::.. .* ::**:** : *: .::*::****:** : *:.:*** ***::* :: ** . Unique parkin domain (UPD) D. melanogaster QRQK--IQSATLEEE-EPSLSDEASKPLNETLLDLQLESEE-RLNITDEERVR----AKAHFFVHCS-QCDKLCNGKLRVRCALCKGGAFTVHRDPECWD 183 A. gambiae KRQAKPALNATISEEPSPEEQQQHNKPLSETMSELTVLDE--RNGDQSIPIGR----TKAHFFVYCS-QCEKVCTGKLRVRCGICGSGAFTVHRDPTCWD 190 R. norvegieus GDKPQSTPEGSIWEPRSLTRVDLSSHILPADSVGLAVILDTDSKSDSEAARGPAAKPTYHSFFVYCKGPCHKVQPGKLRVQCGTCRQATLTLAQGPSCWD 185 M. musculus GDEPQSTSEGSIWESRSLTRVDLSSHTLPVDSVGLAVILDTDSKRDSEAARGP-VKPTYNSFFIYCKGPCHKVQPGKLRVQCGTCKQATLTLAQGPSCWD 184 H. sapiens GDDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGRSIYNSFYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWD 185 . . * . : . * * : . *:::*. *.:: *****:*. * .::*: :.* *** Unique parkin domain (UPD) RING1 D. melanogaster DVLKSRRIPGHCESLEVACVDNAAGDPPFAEFFFKCAEHVSGGEKDFAAPLNLIKNNFKNVPCLACTDVSDTVLVFPCASQHVTCIDCFRHYCRSRLGER 283 A. gambiae DVLKRKRITGHCENYEVPCVENDEGEPPFTEFYFKCSEHSSGGEKDFAAPLSLIKTNHKNIPCIACTDTSETILVFPCVAGHVSCLDCFRQYCVTRLLER 290 R. norvegieus DVLIPNRMSGECQSPDCPGTR--------AEFFFKCGAHPTS-DKDTSVALNLITNNSRSIPCIACTDVRNPVLVFQCNHRHVICLDCFHLYCVTRLNDR 275 M. musculus DVLIPNRMSGECQSPDCPGTR--------AEFFFKCGAHPTS-DKDTSVALNLITSNRRSIPCIACTDVRSPVLVFQCNHRHVICLDCFHLYCVTRLNDR 274 H. sapiens DVLIPNRMSGECQSPHCPGTS--------AEFFFKCGAHPTS-DKETPVALHLIATNSRNITCITCTDVRSPVLVFQCNSRHVICLDCFHLYCVTRLNDR 275 *** .*:.*.*:. . . . :**:***. * :. :*: ...* ** .* :.:.*::***. ..:*** * ** *:***: ** :** :* Inbetween RING domain (IBR) D. melanogaster QFMPHPDFGYTLPCPAGCEHSFIEEIHHFKLLTREEYDRYQRFATEEYVLQAGGVLCPQPGCGMGLLVEPDCRKVTCQNG----CGYVFCRNCLQGYHIG 379 A. gambiae QFVEHPTGGYTLQCPAGCDNSFIEDVHHFKLLNKEQYERYQRFATEEFVLKNGGVLCPQPGCGMGLLVDPECRRIQCQNG----CGYVFCRSCLQGYHIG 386 R. norvegieus QFVHDAQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRYQQYGAEECVLQMGGVLCPRPGCGAGLLPEQGQRKVTCEGGNGLGCGFVFCRDCKEAYHEG 375 M. musculus QFVHDAQLGYSLPCVAGCPNSLIKELHHFRILGEEQYTRYQQYGAEECVLQMGGVLCPRPGCGAGLLPEQGQRKVTCEGGNGLGCGFVFCRDCKEAYHEG 374 H. sapiens QFVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRYQQYGAEECVLQMGGVLCPRPGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKEAYHEG 375 **: .. **:* * *** :*:*:::***::* .*:* ***::.:** **: ******:**** *** : *:: *:.* **:.***.* :.** * RING2 D. melanogaster ECLPEGTGASATNSCEYTVDPNRAAEARWDEASNVTIKVSTKPCPKCRTPTERDGGCMHMVCTRAGCGFEWCWVCQTEWTRDCMGAHWFG- 468 A. gambiae ECFETPTPSTPGNEQGYAIDPLRASEARWDEATKIAIKVTTKPCPQCRTATERDGGCMHMVCTRSGCGFEWCWVCQTPWTRDCMAAHWFG- 475 R. norvegieus ECDSMFE-ASGATSQAYRVDQRAAEQARWEEASKETIKKTTKPCPRCNVPIEKNGGCMHMKCPQPQCKLEWCWNCGCEWNRACMGDHWFDV 464 M. musculus DCDSLLE-PSGATSQAYRVDKRAAEQARWEEASKETIKKTTKPCPRCNVPIEKNGGCMHMKCPQPQCKLEWCWNCGCEWNRACMGDHWFDV 463 H. sapiens ECSAVFE-ASGTTTQAYRVDERAAEQARWEAASKETIKKTTKPCPRCHVPVEKNGGCMHMKCPQPQCRLEWCWNCGCEWNRVCMGDHWFDV 464 :* .: . * :* * :***: *:: :** :*****:*... *::****** *.:. * :**** * *.* **. ***. Dipteran a Figure 1 nd mammalian parkin proteins are well conserved Dipteran and mammalian parkin proteins are well conserved. (A) Schematic representation of the Drosophila mela- nogaster parkin transcription unit and its location in the genomic scaffolding region AE003593. (B) ClustalW alignment of the Drosophila melanogaster parkin with homologues from Anopheles gambiae, Rattus norvegicus, Mus musculus and Homo sapiens. Highlighted are the Ubiquitin-like Domain (UBL) (green box); the Unique Parkin Domain (UPD) (red box); RING1 and RING2 (blue boxes); In-Between Ring Domain (IRB) (black box). "*" and red lettering indicates amino acids that are identical in all sequences in the alignment. ":" and green lettering indicates conserved substitutions. "." and blue lettering indicates semi-con- served substitutions. A)AA(A/C)ATG) of translation initiation [38], we have ure 1B). Parkin protein homologues were identified from assigned this as the most likely start codon. Furthermore, Rattus norvegicus (NM_020093.1) and Mus musculus of the preceding fourteen potential codons only two use (AB019558.1) via the tblastn algorithm of the National preferred codons [39] (data not shown). The Drosophila Center for Biotechnology Information (NCBI). Both the parkin gene was reported by Greene and colleagues [40], R. norvegicus and M. musculus homologues were found to while the current experiments were being conducted. have 44% identity and 60% similarity to D. melanogaster parkin when analysed by the blast2 algorithm (Figure 1B). The parkin protein is well conserved In addition, we have determined that the Anopheles gam- The Drosophila melanogaster parkin protein has been biae sequence XM316606.1 [41] is a homologue of parkin. reported to be 42% identical to human parkin [40] (Fig- Like D. melanogaster, the A. gambiae transcript has two Page 3 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 potential in-frame translation start sites. The Kozak served between mammalian and dipteran species suggest- sequence prior to the first ATG is very poor, however the ing conservation of function among these species. second site closely resembles the consensus sequence and therefore it is very likely the start site. We determined that parkin suppresses degeneration of the ommaditial array in the theoretical A. gambiae parkin protein has 65% identity flies that express α-synuclein in the eye and 79% similarity to D. melanogaster parkin (Figure 1B). We generated stable transgenic flies that can conditionally The parkin protein appears to be highly conserved at the express parkin when the UAS/Gal4 expression system is amino acid sequence level. utilized [46]. In situ hybridization was used to confirm parkin expression in transgenic Drosophila (data not Alignment of D. melanogaster parkin protein sequences shown). Expression of parkin was directed to the develop- with the A. gambiae, R. norvegicus, M. musculus and H. sapi- ing eye using the GMR-Gal4 transgene resulting in no ens homologues reveals conservation of the protein obvious alteration of the eye (data not shown). In vitro throughout a number of characteristic domains, including and cell culture research suggests that parkin can prevent the Ubiquitin- like Domain (UBL), the Unique Parkin α-synuclein-induced toxicity [31,47]. Expression of human omain (UPD), the Really Interesting New Gene finger 1 D α-synuclein in the Drosophila eye causes premature deterio- (RING1) domain, the In- Between Ring (IBR) domain, ration of the retina [32]. To examine if parkin could pre- and the RING2 domain (Figure 1B). In the amino-termi- vent α-synuclein-induced degeneration, we co-expressed nal region of the proteins, the first 15 amino acids are well parkin with human α-synuclein in the developing eye. conserved between A. gambiae and D. melanogaster, but Cross-sections of the retinas of one-day-old flies that absent in the mammalian proteins. The human UBL express α-synuclein appear to be intact, as previously shows very high similarity (62%) to human ubiquitin described (Figure 2A) [32]. The retinas of one-day-old [17]. Correspondingly the Drosophila UBL (Figure 1B, flies that express both α-synuclein and parkin also appear green box) was found to have 43% identity and 67% sim- normal (Figure 2B,2C). As previously described, the reti- ilarity to Drosophila ubiquitin (AAA29007; data not nas of thirty-day-old flies that express α-synuclein show shown). The second highly conserved region is unique to signs of premature degeneration [32]. Degeneration of the parkin and has been termed the unique parkin domain normal architecture of the eye is apparent (Figure 2D, (UPD) [42] (Figure 1B, red box). D. melanogaster and A. black arrows) and reflects the disruption of the normal gambiae share a similar eight amino acid insertion in the placement and alignment of the photoreceptors and sup- UPD (Figure 1B). There are two RING-finger domains that porting cells. In contrast, thirty-day-old flies that express are defined by the consensus sequence C-X -C-X -C- α-synuclein and parkin maintain their ommatidial arrays 2 9...39 X -H-X -C/H-X -C-X -C-X -C where X can be any and morphology (Figure 2E,2F). This observation demon- 1...3 2...3 2 4...48 2 amino acid (Figure 1B, blue boxes) [43]. These RING-fin- strates that directed expression of parkin suppresses α- ger domains flank a cysteine-rich domain designated the synuclein-dependent degeneration of the ommatidial In-Between Ring (IBR) domain (Figure 1B, black box) array. [44]. These three domains are responsible for binding to specific E2 ubiquitin conjugating enzymes [23,28,30]. Retinal damage can be observed by examining an optical Between the RING1 and the IBR domains there is an eight- effect termed the pseudopupil, which is lost in aged flies een amino acid stretch of high conservation with the that express α-synuclein [32]. We examined flies that co- sequence (N/H)S(L/F)I(K/E)(E/D)(I/L)HHF(K/R)(L/ express α-synuclein and parkin, and there appeared to be I)LX(R/E)E(E/Q)Y. A 41 amino acid segment separates the some retention of this optical effect compared with flies IBR and RING2 domains, and while the first half of this that express α-synuclein alone (data not shown). Scanning segment is not well conserved the second half is highly electron microscopy of eyes revealed no obvious deterio- conserved with the sequence AX(E/Q)ARW(D/E)XA(S/ ration of the surface in flies that express α-synuclein (Fig- T)(N/K)X(T/A)IKX(S/T)TKP. The carboxy-terminus is well ure 3A and 3D) or flies that co-express α-synuclein and conserved and has the following sequence M(G/ parkin (Figure 3B,3C,3E and 3F). Although α-synuclein A)XHWF(G/D)(-/V), suggesting a possible conserved causes degeneration of the ommatidial array, the external function for the tail of the protein. As parkin undergoes structure of the eye is unaffected. self-ubiquitination [28], conserved potential ubiquitina- tion sites (lysine residues) were identified. There is a Directed-expression of parkin to dopaminergic neurons lysine residue that is completely conserved at K-42 of the does not affect climbing ability dipterans and this corresponding residue is K-27 in mam- Young wild-type adult Drosophila exhibit a strong negative mals (Figure 1B, black arrow). The mouse and rat parkin geotaxis, which is increased by mechanical stimulation homologues have been recently compared to Drosophila [48,49]. In order to measure climbing ability, flies are parkin [45], however a number of the above features were placed in a vial, gently tapped to the bottom and allowed not discussed. Overall parkin appears to be highly con- to climb up the sides [32]. When parkin is expressed in the Page 4 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Expres Figure 2 sion of parkin suppresses α-synuclein-induced retinal degeneration Expression of parkin suppresses α-synuclein-induced retinal degeneration. Flies that express α-synuclein with and without parkin were aged to 1 or 30 days old. They were fixed and embedded in epon. Tangential sections (0.5 µm thick) of the retina were cut, stained with toludine blue and examined by light microscopy. Panels A-C represent one-day-old flies and pan- els D-F represent thirty-day-old flies. Black arrows indicate degeneration of the ommatidial architecture. The genotypes are 1118 1118 1.1 1118 (A,D) w ; UAS-α-synuclein/GMR-Gal4, (B,E) w ; UAS-α-synuclein/GMR-Gal4; UAS-parkin /+, and (C,F) w ; UAS-α-synuclein/ 2.1 GMR-Gal4; UAS-parkin /+. Scale bar is 15 µm. dopaminergic neurons, these flies do not show any Median survival age for flies that express α-synuclein is change in their climbing ability over their life span when similar to flies that co-express α-synuclein with parkin (Fig- compared with controls (Figure 4A). In addition, ure 5B). This indicates that differences in climbing ability expression of parkin in dopaminergic neurons does not were not due to differences in life span. alter life span (Figure 4B). These results demonstrate that parkin expression in the dopaminergic neurons has little Discussion effect upon climbing ability or life span. Drosophila parkin has a high degree of similarity to the mammalian and A. gambiae homologues. The five charac- Parkin suppresses α-synuclein induced loss of climbing teristic domains of the parkin protein, the Ubiquitin- like ability Domain (UBL), Unique Parkin Domain (UPD), Really Flies that express α-synuclein, specifically in their Interesting New Gene finger 1 (RING1) domain, In- dopaminergic neurons through the activity of the Ddc- Between Ring (IBR) domain and RING2 all show a high Gal4 transgene, were assayed for their climbing ability degree of similarity. In addition, the two dipterans, D. over their life span, and were found to prematurely lose melanogaster and A. gambiae, have a highly conserved extra their climbing ability (Figure 5A). Co-expression of parkin segment of 15 amino acids at the amino-terminal of the with α-synuclein suppresses this premature loss of climb- protein. The regions between the three carboxy-terminus ing ability (Figure 5A). This suggests that parkin can act to domains are also highly conserved, which may indicate prevent the deleterious effects of α-synuclein expression. conservation of function. Patients with ARJP caused by mutations in the UBL domain exhibit signs of lost sub- Aging assays were carried out in tandem with the climbing strate binding [23]. The UBL domain also appears to be assays described above in order to account for changes in involved in binding the Rpn10 subunit of the 26S protea- climbing ability as a result of premature senescence. some as the R42P amino acid substitution in this domain Page 5 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Expres Figure 3 sion of α-synuclein with and without parkin does not affect the external morphology of the eye Expression of α-synuclein with and without parkin does not affect the external morphology of the eye. Scanning electron microscopy of flies that express α-synuclein with and without parkin shows no change in their external morphology over thirty days. Panels A-C represent one-day-old flies and panels D-F represent thirty-day-old flies. The genotypes are (A,D) 1118 1118 1.1 1118 w ; UAS-α-synuclein/GMR-Gal4, (B,E) w ; UAS-α-synuclein/GMR-Gal4; UAS-parkin /+, and (C,F) w ; UAS-α-synuclein/GMR- 2.1 Gal4; UAS-parkin /+. Scale bar indicates 200 µm. was identified in ARJP patients and results in impaired the RING1 domain change the subcellular localization of proteasome binding of parkin (Figure 6) [50]. Alterations parkin and enhance cytoplasmic and nuclear inclusions of the RING1, RING2 and IBR domains of parkin result in [52]. In addition, the amino acid substitutions C289G an almost complete loss of ubiquitin conjugating enzyme and C418R, which replace conserved cysteine residues in H7 (UbcH7)-binding activity, which indicates that all the RING domains, significantly decrease the solubility of three domains are functionally important in recruiting parkin in cells [53]. Ubiquitination generally occurs near specific E2 ubiquitin conjugating enzymes [51]. The the amino-terminus of proteins and ubiquitin monomers RING1 and RING2 domains are thought to collaborate to are attached to lysine residues [25]. Several lysine residues trap UbcH7 (Figure 6) [51]. Amino acid substitutions in are absolutely conserved, including one in the UBL and Page 6 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Expres Figure 4 sion of parkin does not affect climbing ability or life span Expression of parkin does not affect climbing ability or life span. Panel A – Climbing ability of flies that express parkin 1118 1.1 1118 2.1 does not differ from control flies. Genotypes are w ; UAS-parkin /Ddc-Gal4 (green open triangle), w ; UAS-parkin /Ddc- Gal4 (orange open square) and w ; Ddc-Gal4/+ (black open circles). The error bars show the standard error of the mean of twenty trials at each point. Please note that the error bars are mostly within the symbols. B – The life span of flies that express parkin does not differ from the control. The genotypes are marked the same as in panel A. The mortality of flies was examined every two days. Page 7 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Expres Figure 5 sion of parkin suppresses α-synuclein-induced loss of climbing ability Expression of parkin suppresses α-synuclein-induced loss of climbing ability. Panel A – Aged flies that express parkin and α-synuclein climb significantly better than flies that express α-synuclein (P < 0.001, one-way analysis of variance with supple- 1118 1118 mentary Newman-Keuls test). Genotypes are w ; UAS-α-synuclein/+; Ddc-Gal4/+ (green open square), w ; UAS-α-synuclein/ 1.1 1118 2.1 +; UAS-parkin /Ddc-Gal4 (red open triangle), w ; UAS-α-synuclein/+; UAS-parkin /Ddc-Gal4 (blue upside down open triangle). The error bars show the standard error of the mean of twenty trials at each point. Please note that the error bars are mostly within the symbols. B – The life span of flies that express α-synuclein with and without parkin does not differ. The genotypes are marked the same as in panel A. Page 8 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Parkin IBR UPD RING RING UBL G E2 -synuclein ubiquitination proteosome G -synuclein -synuclein o-glycosylation fibril formation? peptides ubiquitin monomer Model of par Figure 6 kin directed ubiquitination of α-synuclein Model of parkin directed ubiquitination of α-synuclein. The parkin protein consists of two functionally distinct regions. The UBL/UPD region binds target proteins such as glycosylated α-synuclein. The RING-box (RING1-IBR-RING2) region recruits specific E2 ubiquitin conjugating enzymes, which add ubiquitin monomers to the target protein. In addition to substrate binding the UBL domain interacts with the proteasome. Ubiquitin tagged α-synuclein is directed to the proteasome and degraded into polypeptides and ubiquitin monomers. UBL – Ubiquitin-like Domain, UPD – Unique Parkin Domain, RING1 or 2 – Really Interesting New Gene finger 1 or 2 domain, IBR – In-Between Ring domain. two in the UPD, and these may be targets for ubiquitina- observed when parkin is expressed in dopaminergic neu- tion. The existence of orthologues of mammalian parkin rons is likely due to substrate specificity and to the ability in invertebrates but not plants nor fungi [54] suggest an of the parkin protein to target itself for degradation [28]. animal specific function for parkin activity. The highly Under conditions of over-expression, parkin does not conserved protein domains and sub-domains suggest the seem to target and tag essential proteins for degradation probable conservation of each domain's function, and promiscuously. This may represent an excellent fail-safe given the high degree of similarity we suggest that the mechanism the cell has developed to balance the levels of function of the Drosophila parkin protein is similar to that both parkin and its substrates. of the human parkin protein. The Drosophila model of ADPD has been used to examine We demonstrate that the directed expression of parkin in the effect of various pharmacological agents [34,36] and the dopaminergic neurons and developing eyes leads to other genetic aspects of the disease [35,37]. We expressed no obvious adverse effects. The unaltered phenotype parkin along with α-synuclein and found the suppression Page 9 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 of α-synuclein-induced retinal degeneration and prema- A. gambiae (XM316606.1) sequences individually with ture loss of climbing. These results indicate that parkin the D. melanogaster parkin protein sequence [57]. The may target α-synuclein for degradation in vivo (Figure 6). multi-alignment of the five parkin homologues from D. Although coimmuno-precipitation studies have shown melanogaster, A. gambiae, R. norvegicus, M. musculus and H. that parkin does not interact with or ubiquitinate non- sapiens was constructed by editing the results from the modified α-synuclein [29], parkin will ubiquitinate O- multialign ClustalW program from the Pôle Bio-Informa- glycosylated α-synuclein [26]. Since we show suppression tique Lyonnaise [58,59]. of the α-synuclein-induced phenotype, we believe that ectopically expressed α-synuclein is modified in Drosophila, Fly stocks and culture enabling its ubiquitination by parkin. The modification of Dr. M. Feany (Harvard Medical School) and Dr. J. Hirsh α-synuclein and subsequent ubiquitination by parkin is (University of Virginia) generously provided UAS-α-synu- 4.36 presented in Figure 6. clein [32] and Ddc-Gal4 flies [60] respectively. The GMR-Gal4 flies [61] were obtained from the Bloomington In order to select rational potential therapeutic agents, the Drosophila Stock Center at Indiana University. A BglII/ molecular mechanisms behind disease progression must XhoI fragment containing the parkin cDNA (SD01679) be characterized. Gene function studies with homologues was subcloned into the pUAST vector to generate the UAS- of disease-causing genes in model organisms have been parkin transgene. Two independent transgenic lines were made practical through the advent of genome projects. generated using heat shock π as a source of transposase Over-expression of parkin has no apparent adverse conse- and standard injection techniques into w embryos. quences and it suppresses the α-synuclein-induced PD Double transgenic lines with UAS-α-synuclein;UAS- 1.1 2.1 symptoms in Drosophila. If this relationship is conserved parkin and UAS-α-synuclein;UAS-parkin were gener- in humans, we suggest that up-regulation of parkin should ated using standard techniques. To drive expression of the be a viable treatment for PD, and the selection of thera- transgenes, Ddc-Gal4 (for expression in the dopaminergic peutic strategies should be directed towards this end. neurons) or GMR-Gal4 (for expression in the eye) homozygous females were crossed to w males (con- 1.1 Conclusions trol) or UAS-α-synuclein with or without UAS-parkin or 2.1 Our experiments demonstrate that the directed expression UAS-parkin . All flies were cultured on standard corn- of the parkin gene counteracts the PD-like symptoms in meal/yeast/molasses/agar media at 25°C. the α-synuclein-induced Drosophila model of PD. Manipu- lation of the ubiquitin/proteasome degradation pathway In situ hybridization analysis in such a specific manner apparently remedies the toxic Third instar larvae were dissected in PBS, fixed in 4% for- accumulation of α-synuclein. This study demonstrates the maldehyde and dehydrated in methanol and ethanol. The success of selective targeting of toxic proteins for degrada- carcases were probed with a DIG labelled anti-sense parkin tion as an approach to address neurodegenerative condi- RNA probe generated from a linear cut plasmid contain- tions such as Parkinson's disease. The development of ing the entire parkin cDNA using the Roche Applied Sci- therapies that regulate parkin expression or parkin protein ence DIG Northern starter kit and reduced in size with activity may be crucial in the treatment of PD. carbonate buffer treatment. To visualize parkin RNA alka- line phosphatase labelled anti-DIG anti-bodies were incu- Methods bated with the carcases and subjected to alkaline Bioinformatic and sequence analysis phosphate treatment as per the Roche Applied Science The Drosophila melanogaster homologue of parkin was DIG application manual. The eye discs were dissected out identified through a search of the Berkeley Drosophila completely and examined under light microscopy. The 1118 1118 Genome Project [55] queried with the human parkin genotypes of the larvae examined were 1) w ; 2) w ; 1.1 1118 amino acid sequence, AB009973.1. A clone of the Dro- GMR-Gal4/+; UAS-parkin /+; and 3) w ; GMR-Gal4/+; 2.1 sophila parkin cDNA (SD01679) was obtained from UAS-parkin /+ and at least ten of each genotype were Research Genetics [56], sub-cloned and sequenced examined. (Cortec DNA Service Laboratories Inc., Kingston, ON, Canada). The intron/exon map was constructed by com- Aging analysis parison of the cDNA to the corresponding genomic Adult males were collected under gaseous carbon dioxide region. Other homologues of parkin were identified with anaesthetic and aged in small groups (~10 or less per vial) the tblastn algorithm [57] of the National Center for Bio- upon standard cornmeal/yeast/molasses/agar media at technology Information (NCBI) using the theoretical 25°C in upright standard plastic shell vials. The flies were translation of SD01679 cDNA. The blast2 sequence com- scored for viability every two to three days and transferred parison program (NCBI) was used to compare the R. nor- to fresh media without anaesthesia [62]. The numbers of vegicus (NM_020093.1), M. musculus (AB019558.1) and individuals aged are as follows: UAS-α-synuclein/+; Ddc- Page 10 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 2.1 land) for help with sectioning. We thank John P. Phillips (Department of Gal4/+ = 191; UAS-α-synuclein/+; UAS-parkin /Ddc-Gal4 1.1 Molecular Biology and Genetics, University of Guelph) for advice on mat- = 292; UAS-α-synuclein/+; UAS-parkin /Ddc-Gal4 = 204; ters of longevity. We thank Jamie Kramer for advice on in situ hybridization 1118 1.1 ; Ddc-Gal4/+ = 173; UAS-parkin /Ddc-Gal4 = 262; and Peter Earle for help with citation software. We also thank Dr. Helene 2.1 and UAS-parkin /Ddc-Gal4 = 227. Volkoff, Dr. H. Dawn Marshall, Lisa Saunders and Justin Moores for com- ments on the manuscript. Locomotion assay Flies were assayed for their ability to climb as described by References Feany and Bender [32]. Every five days, forty male flies 1. Parkinson J: An essay on the Shaking Palsy. A Manual of Diseases of the Nervous System 2nd edition. Edited by: Gowers W R. Philadel- from a cohort of flies were assayed for their ability to phia, Blakiston; 1817:6366-6657. climb six centimetres in eighteen seconds in a sterile plas- 2. Spacey SD, Wood NW: The genetics of Parkinson's disease. tic vial. Twenty trials were carried out for each time point. Curr Opin Neruol 1999, 12:427-432. 3. Dawson TM: New Animal Models for Parkinson's Disease. Cell Data shown represent the results from flies tested over 2000, 101:115-118. ninety days. 4. Giasson BI, Lee VM: Parkin and the molecular pathways of Par- kinson's disease. Neuron 2001, 31:885-888. 5. Lansbury P. 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parkin counteracts symptoms in a Drosophila model of Parkinson's disease

Haywood, Annika; Staveley, Brian
BMC Neuroscience , Volume 5 (1) – Apr 16, 2004
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Springer Journals
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Copyright © 2004 by Haywood and Staveley; licensee BioMed Central Ltd.
Subject
Biomedicine; Neurosciences; Neurobiology; Animal Models
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1471-2202
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10.1186/1471-2202-5-14
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15090075
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Abstract

Background: 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. Results: To investigate the role of parkin, we have generated transgenic Drosophila 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. Conclusion: 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. tem analysis reveals the selective loss of dopaminergic Background Parkinson's disease (PD) is a neurodegenerative disorder neurons from the substantia nigra region of the brain. Fil- that is characterized by muscle tremors in stationary amentous protein inclusions, known as Lewy bodies, are limbs, bradykinesia (slowed movement) and difficulty found within the neuronal cell bodies of the affected area, initiating and sustaining movements, and affects 1–2% of in most but not all PD patients [4]. Although the majority the population older than sixty years of age [1-5]. As the of PD cases appear to be sporadic, about 5–15% have disease progresses, both the sense of balance and the been determined to have an inherited basis [6,7]. memory of the affected individual deteriorate. Post-mor- Recently, mutations in a number of genes have been Page 1 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 identified as causes of PD and many of these genes are were observed in α-synuclein-expressing flies approxi- associated with the ubiquitin/proteasome protein degra- mately 20 days after eclosion. While control flies exhibit a dation pathway. strong negative geotaxis, these transgenic flies prema- turely lose their climbing ability. In addition, expression Mutations in the gene encoding the α-synuclein protein of α-synuclein in the developing eye results in precocious lead to the development of Autosomal Dominant PD degeneration of the retina. In this model expression of a (ADPD) [8,9]. The α-synuclein protein is an abundant number of genes are dysregulated prior to the onset of 140 amino acid, cytosolic protein found at the pre-synap- neurodegeneration [33]. These features recapitulate the tic region of neurons [10,11]. α-synuclein appears to be main behavioural and pathological phenotypes of PD and involved in the biosynthesis of dopamine [12,13]. Muta- provide an excellent model system to study the biological tions in the α-synuclein gene [8,9] may lead to enhanced basis of the disease. oligomerization and fibril formation of the α-synuclein protein [14-16]. The Drosophila α-synuclein based model has been used to investigate a number of aspects of PD. Pharmacological Autosomal Recessive Juvenile Parkinson's disease (ARJP), agents, such as the dopamine precursor levodopa, another inherited form of PD, has been attributed to a dopamine receptor agonists (bromocriptine, pergolide number of point mutations and deletions of the parkin and SK&F38393), and the anticholinergic atropine, were gene [17-19]. ARJP is specifically characterized by a very demonstrated to modify the age-dependent loss of climb- early age of onset, mostly before forty years of age, and the ing ability [34]. Co-expression of the molecular chaper- absence of Lewy bodies [20-22]. In humans, the parkin one HSP70 gene with α-synuclein prevented dopaminergic gene encodes a 465 amino acid protein [17] that func- neuronal degeneration [35]. Interference with endog- tions as one of a number of E3 ubiquitin protein ligases, enous chaperone activity accelerated the toxicity of α- components of the ubiquitin/proteasome degradation synuclein demonstrating a role for chaperones in the pathway [23]. Ubiquitin protein ligases act to identify pathology of the disease. Suppression of HSP90, a nega- damaged, misfolded, and short-lived proteins to mediate tive regulator of the heat shock factor 1, by feeding flies the ubiquitination (the sequential attachment of a geldanamycin prevents dopaminergic neuronal cell death number of ubiquitin monomers) of these proteins, which [36]. Recently, the expression of human parkin has been are targeted to the proteasome [24,25]. Experiments in tis- shown to suppress the loss of dopaminergic neurons sue culture have demonstrated that parkin can ubiquiti- induced by α-synuclein in Drosophila [37]. This model has nate a number of substrates including a glycosylated form proven to be an effective tool in the investigation of the of α-synuclein [26], the Pael receptor [27], CDCrel-1 [28], biological basis of PD. the α-synuclein-binding protein synphilin-1 [29], and parkin itself [28,30]. The loss of parkin may lead to an To investigate the role of parkin in the α-synuclein-based accumulation of one or a number of proteins in sufficient model of Parkinson's disease in Drosophila, we have char- quantities to cause neuronal cell death. acterized and expressed the Drosophila homologue of par- kin in this model. Our results demonstrate that parkin can The interaction of parkin with α-synuclein suggests a com- counteract the effects of α-synuclein on climbing activity mon mechanism underlying inherited forms of PD. and retinal degeneration. Indeed, elevated expression of parkin protects neuronal explants from the toxicity associated with expression of α- Results synuclein [26,31]. The disease inducing-forms of α-synu- Characterization of Drosophila melanogaster parkin clein may prevent its degradation and result in toxic accu- The Drosophila melanogaster parkin homologue was identi- mulation. In ARJP, functional parkin protein is lost along fied through a search of the Berkeley Drosophila Genome with the ability to mediate the ubiquitination of glyco- Project (BDGP) utilizing the tBlastn search algorithm. The sylated α-synuclein and may lead to the accumulation of parkin gene is located on the left arm of the third chromo- this protein [26]. Our working hypothesis is that aspects some, in the polytene chromosome section 78C within of parkin-mediated protein degradation are compromised the genomic scaffolding region AE003593 (BDGP), and in PD. consists of 6 exons over 2.2 kb (Figure 1A). A search of the genome for additional parkin homologues revealed none. The first Drosophila melanogaster model of PD was gener- Our analysis confirmed the sequence of parkin to be iden- ated by the conditional expression of human α-synuclein tical to that reported by the BDGP (AY058754.1). Two in transgenic Drosophila [32]. Flies that express α-synuclein, potential initiation codons for the parkin protein are sep- in either a pan-neural or dopaminergic neuron specific arated by 42 base pairs at the 5' region of the transcript. As manner, show a marked age-dependent loss of dorsal- the second potential start codon is preceded by CAAA, a medial dopaminergic neurons. Cytoplasmic inclusions match to the Drosophila Kozak consensus sequence (C/ Page 2 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 1 2 3 4 5 6 Genomic sequence AE 003593 244.2 243.8 242.2 Kb 244.5 243.5 243.2 242.8 242.5 Ubiquitin like domain (UBD) D. melanogaster MLELLQFGGKTLTHTLSIYVKTNTGKTLTVNLEPQWDIKNVKELVAPQLGLQPDDLKIIFAGKELSDATTIEQCDLGQQSVLHAIRLRP-------- PV 92 A. gambiae MLAIFSFGKKKLSNSLSVYVKTNTGNTLAVDLEPHMDIKDVKEMVAPRLGLEPQELKIIFAGRELSDTTTISECDLGQQSIIHVVKSRPT----AITTPQ 97 R. norvegieus ---------------MIVFVRFNSSYGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVIFAGKELQNHLTVQNCDLEQQSIVHIVQ-RPQRKSHETNASG 85 M. musculus ---------------MIVFVRFNSSYGFPVEVDSDTSILQLKEVVAKRQGVPADQLRVIFAGKELPNHLTVQNCDLEQQSIVHIVQ-RPRRRSHETNASG 85 H. sapiens ---------------MIVFVRFNSSHGFPVEVDSDTSIFQLKEVVAKRQGVPADQLRVIFAGKELRNDWTVQNCDLDQQSIVHIVQ-RPWRKGQEMNATG 85 : ::*: *:. :.*:::.. .* ::**:** : *: .::*::****:** : *:.:*** ***::* :: ** . Unique parkin domain (UPD) D. melanogaster QRQK--IQSATLEEE-EPSLSDEASKPLNETLLDLQLESEE-RLNITDEERVR----AKAHFFVHCS-QCDKLCNGKLRVRCALCKGGAFTVHRDPECWD 183 A. gambiae KRQAKPALNATISEEPSPEEQQQHNKPLSETMSELTVLDE--RNGDQSIPIGR----TKAHFFVYCS-QCEKVCTGKLRVRCGICGSGAFTVHRDPTCWD 190 R. norvegieus GDKPQSTPEGSIWEPRSLTRVDLSSHILPADSVGLAVILDTDSKSDSEAARGPAAKPTYHSFFVYCKGPCHKVQPGKLRVQCGTCRQATLTLAQGPSCWD 185 M. musculus GDEPQSTSEGSIWESRSLTRVDLSSHTLPVDSVGLAVILDTDSKRDSEAARGP-VKPTYNSFFIYCKGPCHKVQPGKLRVQCGTCKQATLTLAQGPSCWD 184 H. sapiens GDDPRNAAGGCEREPQSLTRVDLSSSVLPGDSVGLAVILHTDSRKDSPPAGSPAGRSIYNSFYVYCKGPCQRVQPGKLRVQCSTCRQATLTLTQGPSCWD 185 . . * . : . * * : . *:::*. *.:: *****:*. * .::*: :.* *** Unique parkin domain (UPD) RING1 D. melanogaster DVLKSRRIPGHCESLEVACVDNAAGDPPFAEFFFKCAEHVSGGEKDFAAPLNLIKNNFKNVPCLACTDVSDTVLVFPCASQHVTCIDCFRHYCRSRLGER 283 A. gambiae DVLKRKRITGHCENYEVPCVENDEGEPPFTEFYFKCSEHSSGGEKDFAAPLSLIKTNHKNIPCIACTDTSETILVFPCVAGHVSCLDCFRQYCVTRLLER 290 R. norvegieus DVLIPNRMSGECQSPDCPGTR--------AEFFFKCGAHPTS-DKDTSVALNLITNNSRSIPCIACTDVRNPVLVFQCNHRHVICLDCFHLYCVTRLNDR 275 M. musculus DVLIPNRMSGECQSPDCPGTR--------AEFFFKCGAHPTS-DKDTSVALNLITSNRRSIPCIACTDVRSPVLVFQCNHRHVICLDCFHLYCVTRLNDR 274 H. sapiens DVLIPNRMSGECQSPHCPGTS--------AEFFFKCGAHPTS-DKETPVALHLIATNSRNITCITCTDVRSPVLVFQCNSRHVICLDCFHLYCVTRLNDR 275 *** .*:.*.*:. . . . :**:***. * :. :*: ...* ** .* :.:.*::***. ..:*** * ** *:***: ** :** :* Inbetween RING domain (IBR) D. melanogaster QFMPHPDFGYTLPCPAGCEHSFIEEIHHFKLLTREEYDRYQRFATEEYVLQAGGVLCPQPGCGMGLLVEPDCRKVTCQNG----CGYVFCRNCLQGYHIG 379 A. gambiae QFVEHPTGGYTLQCPAGCDNSFIEDVHHFKLLNKEQYERYQRFATEEFVLKNGGVLCPQPGCGMGLLVDPECRRIQCQNG----CGYVFCRSCLQGYHIG 386 R. norvegieus QFVHDAQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRYQQYGAEECVLQMGGVLCPRPGCGAGLLPEQGQRKVTCEGGNGLGCGFVFCRDCKEAYHEG 375 M. musculus QFVHDAQLGYSLPCVAGCPNSLIKELHHFRILGEEQYTRYQQYGAEECVLQMGGVLCPRPGCGAGLLPEQGQRKVTCEGGNGLGCGFVFCRDCKEAYHEG 374 H. sapiens QFVHDPQLGYSLPCVAGCPNSLIKELHHFRILGEEQYNRYQQYGAEECVLQMGGVLCPRPGCGAGLLPEPDQRKVTCEGGNGLGCGFAFCRECKEAYHEG 375 **: .. **:* * *** :*:*:::***::* .*:* ***::.:** **: ******:**** *** : *:: *:.* **:.***.* :.** * RING2 D. melanogaster ECLPEGTGASATNSCEYTVDPNRAAEARWDEASNVTIKVSTKPCPKCRTPTERDGGCMHMVCTRAGCGFEWCWVCQTEWTRDCMGAHWFG- 468 A. gambiae ECFETPTPSTPGNEQGYAIDPLRASEARWDEATKIAIKVTTKPCPQCRTATERDGGCMHMVCTRSGCGFEWCWVCQTPWTRDCMAAHWFG- 475 R. norvegieus ECDSMFE-ASGATSQAYRVDQRAAEQARWEEASKETIKKTTKPCPRCNVPIEKNGGCMHMKCPQPQCKLEWCWNCGCEWNRACMGDHWFDV 464 M. musculus DCDSLLE-PSGATSQAYRVDKRAAEQARWEEASKETIKKTTKPCPRCNVPIEKNGGCMHMKCPQPQCKLEWCWNCGCEWNRACMGDHWFDV 463 H. sapiens ECSAVFE-ASGTTTQAYRVDERAAEQARWEAASKETIKKTTKPCPRCHVPVEKNGGCMHMKCPQPQCRLEWCWNCGCEWNRVCMGDHWFDV 464 :* .: . * :* * :***: *:: :** :*****:*... *::****** *.:. * :**** * *.* **. ***. Dipteran a Figure 1 nd mammalian parkin proteins are well conserved Dipteran and mammalian parkin proteins are well conserved. (A) Schematic representation of the Drosophila mela- nogaster parkin transcription unit and its location in the genomic scaffolding region AE003593. (B) ClustalW alignment of the Drosophila melanogaster parkin with homologues from Anopheles gambiae, Rattus norvegicus, Mus musculus and Homo sapiens. Highlighted are the Ubiquitin-like Domain (UBL) (green box); the Unique Parkin Domain (UPD) (red box); RING1 and RING2 (blue boxes); In-Between Ring Domain (IRB) (black box). "*" and red lettering indicates amino acids that are identical in all sequences in the alignment. ":" and green lettering indicates conserved substitutions. "." and blue lettering indicates semi-con- served substitutions. A)AA(A/C)ATG) of translation initiation [38], we have ure 1B). Parkin protein homologues were identified from assigned this as the most likely start codon. Furthermore, Rattus norvegicus (NM_020093.1) and Mus musculus of the preceding fourteen potential codons only two use (AB019558.1) via the tblastn algorithm of the National preferred codons [39] (data not shown). The Drosophila Center for Biotechnology Information (NCBI). Both the parkin gene was reported by Greene and colleagues [40], R. norvegicus and M. musculus homologues were found to while the current experiments were being conducted. have 44% identity and 60% similarity to D. melanogaster parkin when analysed by the blast2 algorithm (Figure 1B). The parkin protein is well conserved In addition, we have determined that the Anopheles gam- The Drosophila melanogaster parkin protein has been biae sequence XM316606.1 [41] is a homologue of parkin. reported to be 42% identical to human parkin [40] (Fig- Like D. melanogaster, the A. gambiae transcript has two Page 3 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 potential in-frame translation start sites. The Kozak served between mammalian and dipteran species suggest- sequence prior to the first ATG is very poor, however the ing conservation of function among these species. second site closely resembles the consensus sequence and therefore it is very likely the start site. We determined that parkin suppresses degeneration of the ommaditial array in the theoretical A. gambiae parkin protein has 65% identity flies that express α-synuclein in the eye and 79% similarity to D. melanogaster parkin (Figure 1B). We generated stable transgenic flies that can conditionally The parkin protein appears to be highly conserved at the express parkin when the UAS/Gal4 expression system is amino acid sequence level. utilized [46]. In situ hybridization was used to confirm parkin expression in transgenic Drosophila (data not Alignment of D. melanogaster parkin protein sequences shown). Expression of parkin was directed to the develop- with the A. gambiae, R. norvegicus, M. musculus and H. sapi- ing eye using the GMR-Gal4 transgene resulting in no ens homologues reveals conservation of the protein obvious alteration of the eye (data not shown). In vitro throughout a number of characteristic domains, including and cell culture research suggests that parkin can prevent the Ubiquitin- like Domain (UBL), the Unique Parkin α-synuclein-induced toxicity [31,47]. Expression of human omain (UPD), the Really Interesting New Gene finger 1 D α-synuclein in the Drosophila eye causes premature deterio- (RING1) domain, the In- Between Ring (IBR) domain, ration of the retina [32]. To examine if parkin could pre- and the RING2 domain (Figure 1B). In the amino-termi- vent α-synuclein-induced degeneration, we co-expressed nal region of the proteins, the first 15 amino acids are well parkin with human α-synuclein in the developing eye. conserved between A. gambiae and D. melanogaster, but Cross-sections of the retinas of one-day-old flies that absent in the mammalian proteins. The human UBL express α-synuclein appear to be intact, as previously shows very high similarity (62%) to human ubiquitin described (Figure 2A) [32]. The retinas of one-day-old [17]. Correspondingly the Drosophila UBL (Figure 1B, flies that express both α-synuclein and parkin also appear green box) was found to have 43% identity and 67% sim- normal (Figure 2B,2C). As previously described, the reti- ilarity to Drosophila ubiquitin (AAA29007; data not nas of thirty-day-old flies that express α-synuclein show shown). The second highly conserved region is unique to signs of premature degeneration [32]. Degeneration of the parkin and has been termed the unique parkin domain normal architecture of the eye is apparent (Figure 2D, (UPD) [42] (Figure 1B, red box). D. melanogaster and A. black arrows) and reflects the disruption of the normal gambiae share a similar eight amino acid insertion in the placement and alignment of the photoreceptors and sup- UPD (Figure 1B). There are two RING-finger domains that porting cells. In contrast, thirty-day-old flies that express are defined by the consensus sequence C-X -C-X -C- α-synuclein and parkin maintain their ommatidial arrays 2 9...39 X -H-X -C/H-X -C-X -C-X -C where X can be any and morphology (Figure 2E,2F). This observation demon- 1...3 2...3 2 4...48 2 amino acid (Figure 1B, blue boxes) [43]. These RING-fin- strates that directed expression of parkin suppresses α- ger domains flank a cysteine-rich domain designated the synuclein-dependent degeneration of the ommatidial In-Between Ring (IBR) domain (Figure 1B, black box) array. [44]. These three domains are responsible for binding to specific E2 ubiquitin conjugating enzymes [23,28,30]. Retinal damage can be observed by examining an optical Between the RING1 and the IBR domains there is an eight- effect termed the pseudopupil, which is lost in aged flies een amino acid stretch of high conservation with the that express α-synuclein [32]. We examined flies that co- sequence (N/H)S(L/F)I(K/E)(E/D)(I/L)HHF(K/R)(L/ express α-synuclein and parkin, and there appeared to be I)LX(R/E)E(E/Q)Y. A 41 amino acid segment separates the some retention of this optical effect compared with flies IBR and RING2 domains, and while the first half of this that express α-synuclein alone (data not shown). Scanning segment is not well conserved the second half is highly electron microscopy of eyes revealed no obvious deterio- conserved with the sequence AX(E/Q)ARW(D/E)XA(S/ ration of the surface in flies that express α-synuclein (Fig- T)(N/K)X(T/A)IKX(S/T)TKP. The carboxy-terminus is well ure 3A and 3D) or flies that co-express α-synuclein and conserved and has the following sequence M(G/ parkin (Figure 3B,3C,3E and 3F). Although α-synuclein A)XHWF(G/D)(-/V), suggesting a possible conserved causes degeneration of the ommatidial array, the external function for the tail of the protein. As parkin undergoes structure of the eye is unaffected. self-ubiquitination [28], conserved potential ubiquitina- tion sites (lysine residues) were identified. There is a Directed-expression of parkin to dopaminergic neurons lysine residue that is completely conserved at K-42 of the does not affect climbing ability dipterans and this corresponding residue is K-27 in mam- Young wild-type adult Drosophila exhibit a strong negative mals (Figure 1B, black arrow). The mouse and rat parkin geotaxis, which is increased by mechanical stimulation homologues have been recently compared to Drosophila [48,49]. In order to measure climbing ability, flies are parkin [45], however a number of the above features were placed in a vial, gently tapped to the bottom and allowed not discussed. Overall parkin appears to be highly con- to climb up the sides [32]. When parkin is expressed in the Page 4 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Expres Figure 2 sion of parkin suppresses α-synuclein-induced retinal degeneration Expression of parkin suppresses α-synuclein-induced retinal degeneration. Flies that express α-synuclein with and without parkin were aged to 1 or 30 days old. They were fixed and embedded in epon. Tangential sections (0.5 µm thick) of the retina were cut, stained with toludine blue and examined by light microscopy. Panels A-C represent one-day-old flies and pan- els D-F represent thirty-day-old flies. Black arrows indicate degeneration of the ommatidial architecture. The genotypes are 1118 1118 1.1 1118 (A,D) w ; UAS-α-synuclein/GMR-Gal4, (B,E) w ; UAS-α-synuclein/GMR-Gal4; UAS-parkin /+, and (C,F) w ; UAS-α-synuclein/ 2.1 GMR-Gal4; UAS-parkin /+. Scale bar is 15 µm. dopaminergic neurons, these flies do not show any Median survival age for flies that express α-synuclein is change in their climbing ability over their life span when similar to flies that co-express α-synuclein with parkin (Fig- compared with controls (Figure 4A). In addition, ure 5B). This indicates that differences in climbing ability expression of parkin in dopaminergic neurons does not were not due to differences in life span. alter life span (Figure 4B). These results demonstrate that parkin expression in the dopaminergic neurons has little Discussion effect upon climbing ability or life span. Drosophila parkin has a high degree of similarity to the mammalian and A. gambiae homologues. The five charac- Parkin suppresses α-synuclein induced loss of climbing teristic domains of the parkin protein, the Ubiquitin- like ability Domain (UBL), Unique Parkin Domain (UPD), Really Flies that express α-synuclein, specifically in their Interesting New Gene finger 1 (RING1) domain, In- dopaminergic neurons through the activity of the Ddc- Between Ring (IBR) domain and RING2 all show a high Gal4 transgene, were assayed for their climbing ability degree of similarity. In addition, the two dipterans, D. over their life span, and were found to prematurely lose melanogaster and A. gambiae, have a highly conserved extra their climbing ability (Figure 5A). Co-expression of parkin segment of 15 amino acids at the amino-terminal of the with α-synuclein suppresses this premature loss of climb- protein. The regions between the three carboxy-terminus ing ability (Figure 5A). This suggests that parkin can act to domains are also highly conserved, which may indicate prevent the deleterious effects of α-synuclein expression. conservation of function. Patients with ARJP caused by mutations in the UBL domain exhibit signs of lost sub- Aging assays were carried out in tandem with the climbing strate binding [23]. The UBL domain also appears to be assays described above in order to account for changes in involved in binding the Rpn10 subunit of the 26S protea- climbing ability as a result of premature senescence. some as the R42P amino acid substitution in this domain Page 5 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Expres Figure 3 sion of α-synuclein with and without parkin does not affect the external morphology of the eye Expression of α-synuclein with and without parkin does not affect the external morphology of the eye. Scanning electron microscopy of flies that express α-synuclein with and without parkin shows no change in their external morphology over thirty days. Panels A-C represent one-day-old flies and panels D-F represent thirty-day-old flies. The genotypes are (A,D) 1118 1118 1.1 1118 w ; UAS-α-synuclein/GMR-Gal4, (B,E) w ; UAS-α-synuclein/GMR-Gal4; UAS-parkin /+, and (C,F) w ; UAS-α-synuclein/GMR- 2.1 Gal4; UAS-parkin /+. Scale bar indicates 200 µm. was identified in ARJP patients and results in impaired the RING1 domain change the subcellular localization of proteasome binding of parkin (Figure 6) [50]. Alterations parkin and enhance cytoplasmic and nuclear inclusions of the RING1, RING2 and IBR domains of parkin result in [52]. In addition, the amino acid substitutions C289G an almost complete loss of ubiquitin conjugating enzyme and C418R, which replace conserved cysteine residues in H7 (UbcH7)-binding activity, which indicates that all the RING domains, significantly decrease the solubility of three domains are functionally important in recruiting parkin in cells [53]. Ubiquitination generally occurs near specific E2 ubiquitin conjugating enzymes [51]. The the amino-terminus of proteins and ubiquitin monomers RING1 and RING2 domains are thought to collaborate to are attached to lysine residues [25]. Several lysine residues trap UbcH7 (Figure 6) [51]. Amino acid substitutions in are absolutely conserved, including one in the UBL and Page 6 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Expres Figure 4 sion of parkin does not affect climbing ability or life span Expression of parkin does not affect climbing ability or life span. Panel A – Climbing ability of flies that express parkin 1118 1.1 1118 2.1 does not differ from control flies. Genotypes are w ; UAS-parkin /Ddc-Gal4 (green open triangle), w ; UAS-parkin /Ddc- Gal4 (orange open square) and w ; Ddc-Gal4/+ (black open circles). The error bars show the standard error of the mean of twenty trials at each point. Please note that the error bars are mostly within the symbols. B – The life span of flies that express parkin does not differ from the control. The genotypes are marked the same as in panel A. The mortality of flies was examined every two days. Page 7 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Expres Figure 5 sion of parkin suppresses α-synuclein-induced loss of climbing ability Expression of parkin suppresses α-synuclein-induced loss of climbing ability. Panel A – Aged flies that express parkin and α-synuclein climb significantly better than flies that express α-synuclein (P < 0.001, one-way analysis of variance with supple- 1118 1118 mentary Newman-Keuls test). Genotypes are w ; UAS-α-synuclein/+; Ddc-Gal4/+ (green open square), w ; UAS-α-synuclein/ 1.1 1118 2.1 +; UAS-parkin /Ddc-Gal4 (red open triangle), w ; UAS-α-synuclein/+; UAS-parkin /Ddc-Gal4 (blue upside down open triangle). The error bars show the standard error of the mean of twenty trials at each point. Please note that the error bars are mostly within the symbols. B – The life span of flies that express α-synuclein with and without parkin does not differ. The genotypes are marked the same as in panel A. Page 8 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 Parkin IBR UPD RING RING UBL G E2 -synuclein ubiquitination proteosome G -synuclein -synuclein o-glycosylation fibril formation? peptides ubiquitin monomer Model of par Figure 6 kin directed ubiquitination of α-synuclein Model of parkin directed ubiquitination of α-synuclein. The parkin protein consists of two functionally distinct regions. The UBL/UPD region binds target proteins such as glycosylated α-synuclein. The RING-box (RING1-IBR-RING2) region recruits specific E2 ubiquitin conjugating enzymes, which add ubiquitin monomers to the target protein. In addition to substrate binding the UBL domain interacts with the proteasome. Ubiquitin tagged α-synuclein is directed to the proteasome and degraded into polypeptides and ubiquitin monomers. UBL – Ubiquitin-like Domain, UPD – Unique Parkin Domain, RING1 or 2 – Really Interesting New Gene finger 1 or 2 domain, IBR – In-Between Ring domain. two in the UPD, and these may be targets for ubiquitina- observed when parkin is expressed in dopaminergic neu- tion. The existence of orthologues of mammalian parkin rons is likely due to substrate specificity and to the ability in invertebrates but not plants nor fungi [54] suggest an of the parkin protein to target itself for degradation [28]. animal specific function for parkin activity. The highly Under conditions of over-expression, parkin does not conserved protein domains and sub-domains suggest the seem to target and tag essential proteins for degradation probable conservation of each domain's function, and promiscuously. This may represent an excellent fail-safe given the high degree of similarity we suggest that the mechanism the cell has developed to balance the levels of function of the Drosophila parkin protein is similar to that both parkin and its substrates. of the human parkin protein. The Drosophila model of ADPD has been used to examine We demonstrate that the directed expression of parkin in the effect of various pharmacological agents [34,36] and the dopaminergic neurons and developing eyes leads to other genetic aspects of the disease [35,37]. We expressed no obvious adverse effects. The unaltered phenotype parkin along with α-synuclein and found the suppression Page 9 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 of α-synuclein-induced retinal degeneration and prema- A. gambiae (XM316606.1) sequences individually with ture loss of climbing. These results indicate that parkin the D. melanogaster parkin protein sequence [57]. The may target α-synuclein for degradation in vivo (Figure 6). multi-alignment of the five parkin homologues from D. Although coimmuno-precipitation studies have shown melanogaster, A. gambiae, R. norvegicus, M. musculus and H. that parkin does not interact with or ubiquitinate non- sapiens was constructed by editing the results from the modified α-synuclein [29], parkin will ubiquitinate O- multialign ClustalW program from the Pôle Bio-Informa- glycosylated α-synuclein [26]. Since we show suppression tique Lyonnaise [58,59]. of the α-synuclein-induced phenotype, we believe that ectopically expressed α-synuclein is modified in Drosophila, Fly stocks and culture enabling its ubiquitination by parkin. The modification of Dr. M. Feany (Harvard Medical School) and Dr. J. Hirsh α-synuclein and subsequent ubiquitination by parkin is (University of Virginia) generously provided UAS-α-synu- 4.36 presented in Figure 6. clein [32] and Ddc-Gal4 flies [60] respectively. The GMR-Gal4 flies [61] were obtained from the Bloomington In order to select rational potential therapeutic agents, the Drosophila Stock Center at Indiana University. A BglII/ molecular mechanisms behind disease progression must XhoI fragment containing the parkin cDNA (SD01679) be characterized. Gene function studies with homologues was subcloned into the pUAST vector to generate the UAS- of disease-causing genes in model organisms have been parkin transgene. Two independent transgenic lines were made practical through the advent of genome projects. generated using heat shock π as a source of transposase Over-expression of parkin has no apparent adverse conse- and standard injection techniques into w embryos. quences and it suppresses the α-synuclein-induced PD Double transgenic lines with UAS-α-synuclein;UAS- 1.1 2.1 symptoms in Drosophila. If this relationship is conserved parkin and UAS-α-synuclein;UAS-parkin were gener- in humans, we suggest that up-regulation of parkin should ated using standard techniques. To drive expression of the be a viable treatment for PD, and the selection of thera- transgenes, Ddc-Gal4 (for expression in the dopaminergic peutic strategies should be directed towards this end. neurons) or GMR-Gal4 (for expression in the eye) homozygous females were crossed to w males (con- 1.1 Conclusions trol) or UAS-α-synuclein with or without UAS-parkin or 2.1 Our experiments demonstrate that the directed expression UAS-parkin . All flies were cultured on standard corn- of the parkin gene counteracts the PD-like symptoms in meal/yeast/molasses/agar media at 25°C. the α-synuclein-induced Drosophila model of PD. Manipu- lation of the ubiquitin/proteasome degradation pathway In situ hybridization analysis in such a specific manner apparently remedies the toxic Third instar larvae were dissected in PBS, fixed in 4% for- accumulation of α-synuclein. This study demonstrates the maldehyde and dehydrated in methanol and ethanol. The success of selective targeting of toxic proteins for degrada- carcases were probed with a DIG labelled anti-sense parkin tion as an approach to address neurodegenerative condi- RNA probe generated from a linear cut plasmid contain- tions such as Parkinson's disease. The development of ing the entire parkin cDNA using the Roche Applied Sci- therapies that regulate parkin expression or parkin protein ence DIG Northern starter kit and reduced in size with activity may be crucial in the treatment of PD. carbonate buffer treatment. To visualize parkin RNA alka- line phosphatase labelled anti-DIG anti-bodies were incu- Methods bated with the carcases and subjected to alkaline Bioinformatic and sequence analysis phosphate treatment as per the Roche Applied Science The Drosophila melanogaster homologue of parkin was DIG application manual. The eye discs were dissected out identified through a search of the Berkeley Drosophila completely and examined under light microscopy. The 1118 1118 Genome Project [55] queried with the human parkin genotypes of the larvae examined were 1) w ; 2) w ; 1.1 1118 amino acid sequence, AB009973.1. A clone of the Dro- GMR-Gal4/+; UAS-parkin /+; and 3) w ; GMR-Gal4/+; 2.1 sophila parkin cDNA (SD01679) was obtained from UAS-parkin /+ and at least ten of each genotype were Research Genetics [56], sub-cloned and sequenced examined. (Cortec DNA Service Laboratories Inc., Kingston, ON, Canada). The intron/exon map was constructed by com- Aging analysis parison of the cDNA to the corresponding genomic Adult males were collected under gaseous carbon dioxide region. Other homologues of parkin were identified with anaesthetic and aged in small groups (~10 or less per vial) the tblastn algorithm [57] of the National Center for Bio- upon standard cornmeal/yeast/molasses/agar media at technology Information (NCBI) using the theoretical 25°C in upright standard plastic shell vials. The flies were translation of SD01679 cDNA. The blast2 sequence com- scored for viability every two to three days and transferred parison program (NCBI) was used to compare the R. nor- to fresh media without anaesthesia [62]. The numbers of vegicus (NM_020093.1), M. musculus (AB019558.1) and individuals aged are as follows: UAS-α-synuclein/+; Ddc- Page 10 of 12 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/14 2.1 land) for help with sectioning. We thank John P. Phillips (Department of Gal4/+ = 191; UAS-α-synuclein/+; UAS-parkin /Ddc-Gal4 1.1 Molecular Biology and Genetics, University of Guelph) for advice on mat- = 292; UAS-α-synuclein/+; UAS-parkin /Ddc-Gal4 = 204; ters of longevity. We thank Jamie Kramer for advice on in situ hybridization 1118 1.1 ; Ddc-Gal4/+ = 173; UAS-parkin /Ddc-Gal4 = 262; and Peter Earle for help with citation software. We also thank Dr. Helene 2.1 and UAS-parkin /Ddc-Gal4 = 227. Volkoff, Dr. H. 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