Análisis de resultados

The present study provides a promising tool to investigate the biological role(s) of Parkin and the pathophysiology of PD in vivo on a molecular and cellular level. Given

the simplicity and genetic tractability of C. elegans, this model system might now be

used to uncover important aspect of PDR-1/Parkin function by a combination of biochemical, genetic and pharmacological methods.

The established model can now be used to dissect the role of PDR-1/Parkin in and its regulation by the UPR with the genetic tools available. Since many C. elegans

mutants deficient in known UPR/ERAD regulators or targets have been described, further genetic interactions can be studied directly. Double mutants of pdr-1(lg103)

h x

BR3205). Such experimental approaches would certainly reveal new genetic in

Since knock-down of pdr-1(lg103) by RNAi could not sufficiently suppress the

ypersensitivity towards proteotoxic stress conditions, an alternative strategy was

in vivo the toxic gain-of-misfunction mediated by pdr-

wit bp-1 and atf-6 loss-of-function mutants might help to further restrict PDR-

1/Parkin function to either PEK-1 and/or ATF-6 signalling. Additionally, double mutants of pdr-1(lg103) with mutants deficient in downstream genes of the UPR, like

ERAD components and chaperones, or later induced mediators of the cell death machinery, might help to identify the level of genetic interaction between IRE-1 and PDR-1/Parkin. Moreover, the system can be used to identify novel important genetic modifiers of toxicity and potential therapeutic drug targets. The severe phenotype of

pdr-1(lg103) mutants expressing α-synuclein A53T and its temperature- and dose-

dependency is perfectly suited to explore specific enhancers as well as suppressors of the PDR-1 ER-stress/α-synuclein induced proteotoxicity. Such screens could be rapidly performed in a genome-wide approach, using RNAi-feeding libraries in order to knock-down each single C. elegans gene in combination with tunicamycin

treatment or α-synuclein overexpression in the pdr-1(lg103) mutant background. For

this purpose, an appropriate strain has already been constructed into which the RNAi-hypersensitive rrf-3(pk1426) (Simmer et al., 2002) mutation has been

introduced by crossing (strain

teractions and might help to gain more insights into the affected cellular stress pathways. In addition, this might reveal further interactions and feedback-mechanism between the UPR and the cytosolic stress response that are important to completely understand the central role of PDR-1/Parkin.

h

1(lg103). Some attempts to ectopically express the corresponding in-frame deleted

ed strains (BR2789-2792 and

parkin

ORF in pdr-1 loss-of-function background, this time in order to induce proteotoxic

stress hypersensitivity, have been made. However, micro-injection of the pdr-1(lg103)

mutant genomic ORF (construct pBY1569) into pdr-1(lg101) mutant background did

not result in increased hypersensitivity against tunicamycin treatment or ectopic expression of mutant α-synuclein in the generat

BR3236-3239, respectively). Nevertheless, this is most likely caused by mosaic expression along with too low cellular levels of the corresponding protein PDR- 1(∆aa24-247), as judged by the expression of the co-injection marker sel-12::gfp. To

circumvent these experimental problems, a follow-up strategy was already initiated by generation of the constructs pBY1792 and pBY1793 (pdr-1 wild type and pdr- 1(lg103), respectively) which can be used to transform C. elegans by micro-particle

bombardment. This method should result in the isolation of strains carrying genomically integrated copies of the transgenes, thus preventing mosaic expression and ensuring sufficient cellular protein levels. Alternatively, this could also be done using a GFP-tagged mutant PDR-1 variant, in order to be able to compare intracellular localization of WT and mutant protein in vivo.

In addition, the respective pdr-1 wild type and mutant cDNAs have been

cloned into appropriate cell culture vectors in order to test hypersensitivity/resistance towards proteotoxic stress conditions. Since all pdr-1 variants are expressed in

human cells with the expected molecular weights, now, the stability of the different mutant PDR-1 proteins should be studied. Furthermore, the expressed proteins should also be used to address the question of residual E3 ligase activity of pdr-1 in-

frame deletion mutants.

In analogy, this nematode model can also be used for rapid transgenic analyses of human variants in vivo. Wild type parkin and different AR-JP

causing mutations can be introduced into different pdr-1 mutant backgrounds to

study the function of human Parkin and how mutations interfere with this. Differences in specific parkin mutations concerning their functional consequence (loss-of-function

or toxic gain-of-function) as well as their ability to form aggresomes have already been identified. This model might help to explore the pathogenic mechanisms conferred by specific parkin mutations and their contribution to the pathophysiology

of AR-JP. In addition, this might also help to understand the mechanisms of Parkin- mediated detoxification and its function in LB formation.

In order to elucidate the mechanism of PDR-1(∆aa24-247)/α-synuclein A53T mediated toxicity several experiments can be performed using the presented model. To analyze protein levels and folding of α-synuclein variant in pdr-1 mutant

background, first preliminary biochemical analyses have already been performed. However, total amounts of α-synuclein proteins variants have not been found altered in pdr-1(lg103) mutant background, compared to C. elegans wild type background.

Noticeably, separation of detergent-soluble (supernatant) and -insoluble (pellet) protein fractions showed lower levels of only α-synuclein A53T, but not WT, in the pellet fraction of pdr-1(lg103) mutants compared to N2 wild type background (Figure

47).

Figure 47. Analysis of α-synuclein Protein Expressed in pdr-1(lg103) Mutants.

Shown are Western blots of total worm lysates separated by centrifugation into detergent-soluble (supernatant) and -insoluble (pellet) fractions. Pellet fractions were solubilized using urea containing buffer. Western blots were stained with α-synuclein antibody 15G7, and GFP antibody as a loading control. The different

α-synuclein WT an

molecular weights observed for d A53T variants, arise from

Althou

different cloning strategies. Although lower protein levels for α-synuclein A53T in the pellet fraction of pdr-1(lg103) mutants can be seen, a coincident increase of α-synuclein A53T protein in the supernatant fraction cannot be determined due to protein overload.

gh these results have to be considered preliminary and thus have to be verified, they support the model of a Parkin-mediated detoxification mechanism through cytoprotective aggresome/LB formation. Additionally, these results are complete in line with the suggested neurotoxic role of specifically soluble α-synuclein species. However, aggregate formation of α-synuclein in pdr-1 mutant background

has to be studied in vivo, too. In order to characterize the nature and localization of

potential inclusions on a sub-cellular and molecular level, immuno-histological and biochemical studies should be performed. In addition, electron microscopy should be considered, to ultimately characterize aggregates.

A variety of other remaining question should be addressed using the presented model. Does co-expression of specific chaperones (e.g. Hsp70) suppress hypersensitivity of pdr-1(lg103) mutants towards proteotoxic stress? Are pdr-1(lg103)

mutants equally hypersensitive to expression of other known substrates of human Parkin? Are pdr-1 mutants or animals ectopically expressing α-synuclein affected by

specific PD-mimetics and inhibitors of protein turnover? Are mitochondria affected in

pdr-1 mutants or in animals overexpressing α-synuclein, and if so, due to a primary

or a secondary event? Are the interaction partners of PDR-1, identified from the yeast-two hybrid screen, physiologically relevant, and if so, what are their functions?

Although many questions are beyond the scope of this study and remain un- answered, the compelling model presented here will certainly help to shed light onto the molecular and cellular pathways involved into the pathophysiology of PD.

5 Experimental Procedures