Publishable Activity Report Polyala LSHM-CT-2005-018675

Publishable Activity Report PolyALA LSHM-CT-2005-018675

Workpackage 1:

In sum, we have established a competitive European Network on OPMD. We have created a communication structure that facilitated the exchange of information, resources and reagents between researchers in the field, and strengthened interactions with patients and patient organizations. Through our website ( ) queries have already been received from OPMD patients and their families, from all over the world.

To maintain and extend the community created by this Network and expand its activities beyond the current funding period, Partners 4 and 5 applied jointly to the e-Rare Call for proposals 2009 - European Research Projects on Rare Disease.

Meetings of the Consortium:

1. Institute of Molecular Medicine (IMM), Lisbon, 21-22 July 2006.

The meeting was hosted by Partner 1. Those present were Prof. M. Carmo-Fonseca as chairman of the meeting as well as Patricia Calado, João Paulo Tavanez, Francisco Enguita and Ana Grosso from the IMM (Partner 1); Dr Michael Antoniou and Rafael Yanez from King’s College London (Partner 2); Prof. G Dickson from RHUL (Partner 3); Dr Silvère van der Maarel, Ellen Sterrenburg and Baziel van Engelen from LEIDEN Partner 4); Dr Martine Simonelig and Aymeric Chartier from CNRS (Partner 5); Prof. Elmar Wahle and Knut Kölbel from MLUH (Partner 6).

2. King’s College London, 13-14 July 2007.

The meeting was hosted and chaired by Partners 2 and 3. Those present were Prof. M. Carmo-Fonseca and Dr. Francisco Enguita from IMM (Partner 1); Dr Michael Antoniou from King’s College London (Partner 2); Prof. G Dickson and Dr. Capucine Trollet from RHUL (Partner 3); Dr Silvère van der Maarel and Dr. Ellen Sterrenburg from LEIDEN Partner 4); Dr Martine Simonelig, Dr Yannick Bidet and Dr. Aymeric Chartier from CNRS (Partner 5); Prof. Elmar Wahle and Knut Kölbel from MLUH (Partner 6).

3. LeidenUniversityMedicalCenter, 16-17 May 2008.

The meeting was hosted by Partner 4. Those present were Prof. M. Carmo-Fonseca from IMM (Partner 1); Dr Michael Antoniou and Dr. Olivia Bales from King’s College London (Partner 2); Prof. G Dickson and Dr. Capucine Trollet from RHUL (Partner 3); Dr Silvère van der Maarel, Dr. Baziel van Engelen, Dr. Vered Raz, Dr. Sabine Baars, Karlien van der Wees, and Tsion Abraham from LEIDEN Partner 4); Dr Martine Simonelig, Dr Nicolas Barbezier and Dr. Aymeric Chartier from CNRS (Partner 5); Prof. Elmar Wahle and Knut Kölbel from MLUH (Partner 6).

4. Martin-LutherUniversity of Halle, 19-20 June, 2009.

The meeting was hosted by Partner 6. Those present were Prof. M. Carmo-Fonseca from IMM (Partner 1); Dr Michael Antoniou and Dr. Olivia Bales from King’s College London (Partner 2); Prof. G Dickson and Dr. Capucine Trollet from RHUL (Partner 3); Dr Silvère van der Maarel and Dr. Sabine Baars from LEIDEN Partner 4); Dr Martine Simonelig, Dr Nicolas Barbezier and Dr. Aymeric Chartier from CNRS (Partner 5); Prof. Elmar Wahle, Knut Kölbel, K Fronz and S Otto from MLUH (Partner 6).

Main scientific achievements of Workpackage 2:

Determine whether arginine methylation of PABPN1 is involved in OPMD

PABPN1 carries thirteen arginine residues in its C-terminal domain which are all quantitatively asymmetrically dimethylated. Therefore, the protein must be a substrate for type II protein arginine methyl transferases, PRMTs, the type of enzyme generating this kind of posttranslational modification. Partner 6 determined the steady state kinetic parameters for the methylation of PABPN1 by PRMT1, -3 and -6 and the results show that PRMT1 is the most efficient enzyme acting on PABPN1. The efficiencies of methylation of wild-type and alanine-expanded PABPN1 by PRMT1 were also compared. The results show that the mutant protein is methylated with a two-fold reduced apparent affinity for the enzyme and with slightly reduced maximal velocity (Fronz et al 2008). We consider it extremely unlikely that these minor differences are biologically relevant, since even a genetic knock-out of PRMT1 has only a mild effect on the extent of PABPN1 methylation. Partner 6 also determined, by a number of different methods, that PRMT1 and -3 catalyze arginine dimethylation in a distributive manner, i.e. with intermittent release of the substrate after each methyl group addition. This is true both for the transition from monomethyl- to dimethylarginine and for methylation of multiple arginine residues in a single substrate molecule (Kölbel et al 2009). A paper dealing with arginine methylation of the S. pombe orthologue of PABPN1 suggested that lack of methylation enhanced the tendency of the protein to self-interact. However, the biochemical results obtained by Partner 6 do not support the view that lack of methylation favours PABPN1 aggregation. Thus, absence of methylation is unlikely to contribute to the development of OPMD. In a test of candidate genes to identify mutant genes that suppress OPMD phenotypes in Drososphila, Partner 5 found that a deletion overlapping PRMT1 encoding gene is a good suppressor of the wing position defects observed in the Drosophila model of OPMD. This preliminary result suggests that absence of methylation may actually be able to counteract the development of OPMD and is thus in agreement with the biochemical data. Moreover, these results also suggest that a potential sequestration of PRMTs in the PABPN1 aggregates causing undermethylation of other cellular proteins is unlikely to be involved in OPMD. Therefore,methylation experiments were not pursued.

Determine whether expansion of PABPN1 affects polyadenylation

A genetic approach has been undertaken by Partner 5 to determine whether genes involved in mRNA polyadenylation could have a role in OPMD. A total of 5 genes involved in polyadenylation, deadenylation, poly(A) tail regulation or other steps of mRNA metabolism were identified having a strong suppressor effect, and mutants in the gene hrg, encoding the Drosophila poly(A) polymerase has a strong enhancer effect. These results raise the possibility that in OPMD cells, poly(A) tails might be shorter than wild-type, or mRNAs more unstable. Consistent with this prediction, the transcriptomic analysis performed by Partner 4 identified mRNAs involved in mitochondrial biogenesis and function down-regulated in the Drosophila OPMD model. Partner 5 showed that a subset of these down-regulated mRNAs have shorter poly(A) tails.

Determine whether the alanine expansion of PABPN1 affects a putative involvement of the protein in pre-mRNA splicing

In order to examine if polyalanine tract expansion affects the binding of cellular proteins to PABPN1, Partner 1 performed pull-down experiments from cell extracts with immobilized PABPN1 variants. Although we identified association of PABPN1 with several splicing factors including SKIP, U2AF65 and RNA helicase p68 (Tavanez et al in preparation), these interactions were not affected by the alanine expansion of PABPN1. However, we did observe that PRMT1, PRMT3, Hsp70 and Hsp90 are more abundantly associated with expanded PABPN1 relative to normal PABPN1. Immunofluorescence microscopy revealed accumulation of these proteins at intranuclear inclusions in muscle from OPMD patients. Recombinant PABPN1 with expanded polyalanine stretches binds Hsp70 with higher affinity, and data from molecular simulations suggest that expansions of the PABPN1 polyalanine tract result in transition from a disordered, flexible conformation to a stable helical secondary structure. Taken together, these results suggest that the pathological mutation in the PABPN1 gene alters the protein conformation and induces a preferential interaction with type I PRMTs and Hsp70 chaperones. This in turn causes sequestration in intranuclear inclusions, possibly leading to a progressive cellular defect in arginine methylation and chaperone activity (Tavanez et al 2009).

Main scientific achievements of Workpackage 3:

Identification and characterisation of cellular pathways affected in OPMD during progression of the disease

Partner 1 found that expression of a PABPN1 variant containing the most common OPMD mutation specifically stimulates the production of Evi3 transcripts in a mouse skeletal muscle cell line, in the absence of microscopically visible intranuclear inclusions. Murine Evi3 and its human homolog ZNF521 act as modulators of BMP signaling, and we show that mouse cells expressing mutant PABPN1 fail to activate BMP-dependent transcription. Importantly, we also detected Evi3 up-regulated in the OPMD mouse model (Calado et al in preparation).

Partner 4 performed atranscriptome analysis of the Immortomouse myoblast OPMD cell model constructed by Partner 2 and found evidence for downregulation of extracellular matrix genes. The product of one these genes, PCOLCE, was shown to accumulate in OPMD intranuclear inclusions. PCOLCE is an enzyme that enhances the function of procollagen C-proteinase which cleaves the collagen propeptides. Furthermore, it also possesses putative RNA binding domains via which it has been proposed to bind and stabilize mRNA involved in the synthesis of non-collagenous as well as collagen proteins. A reduction of PCOLCE mRNA was also found in OPMD patients (Raz et al, under revision).

Partner 4 further performed atranscriptome analysis of the Drosophila model of OPMD generated and characterized by Partner 5. This study has identified 817 genes that are deregulated in OPMD flies, at least at one time point. GO enrichment indicates that two major pathways are deregulated in OPMD: i) mitochondrion biogenesis and function, genes in this category are down-regulated at all three time points, and ii) ribosome biogenesis and translation, genes in this pathway are up regulated, starting at the second time point.

Given the diversity of results obtained from the different transcriptomic analysis, we have not yet identified common pathways affected in the Drosophila and mouse models, and in muscles from OPMD patients. Therefore, we have not yet reached the stage of discovery of novel biomarkers for intervention strategies in human patients.

Main scientific achievements of Workpackage 4:

Identify anti-PABPN1 intrabodies with potential therapeutic interest

Intrabodies are antibodies that are modified to be expressed intracellularly and target specific antigens in subcellular locations. They are commonly generated by artificially linking the variable domains of antibody heavy and light chains. However, natural single-chain antibodies are produced in Camelids and, when engineered, combined the advantages of being single-chain, small sized and very stable. Here, we determine the in vivo efficiency of Llama intrabodies against PABPN1, using the established Drosophila model of OPMD. Among six anti-PABPN1 intrabodies expressed in muscle nuclei, we identify one as a strong suppressor of OPMD muscle degeneration in Drosophila, leading to nearly complete rescue. Expression of this intrabody affects PABPN1 aggregation and restores muscle gene expression. This approach promotes the identification of intrabodies with high therapeutic value and highlights the potential of natural single-chain intrabodies in treating protein aggregation diseases (Chartier et al 2009; collaboration between Partners 4 and 5).

Identify drugs that revert the muscle phenotype in the Drosophila model

An assay as been developed by Partner 5 to screen for molecules that could alleviate OPMD-like phenotypes in Drosophila. A molecule was identified that reduces the percentage of abnormal winged flies to 50-60% when larvae are fed with it. This molecule (called 6AP) is an anti-aggregation molecule previously identified in models of prion disease. The cellular target of 6AP has been identified as being the large ribosomal RNA subunit. We, therefore tried to determine whether the 6AP molecule was active on OPMD through the same cellular pathway. We used the genetic approach to show that the 6AP molecule acts synergistically with a decrease level of ribosomal RNA. The model from these experiments proposes that the ribosomal RNA would be involved in PABPN1 folding leading to aggregation. The 6AP molecule which binds the ribosomal RNA would prevent its function in protein folding and would reduce the aggregation load. These results identify the 6AP as a good suppressor of OPMD in Drosophila. They are of interest because the 6AP appears to have similar molecular effect as other less toxic molecules that should be more useful for future OPMD therapeutic approaches.

Identify suppressor genes of the muscle phenotype in the Drosophila model

Partner 5 identified 18 suppressor genes. These genes are involved in the HSP70 pathway (3 genes), protein degradation via the proteasome (2 genes), apoptosis (1 gene), poly(A) tail length regulation (6 genes), translation (1 gene) and unknown functions (5 genes). The identification of genes known to be involved in OPMD such as the HSP70 pathway or apoptosis validates the screen. This genetic screen allows to identify new pathways involved in OPMD, in particular poly(A) tail length regulation. Genetics and molecular studies will be undertaken to identify the function of the suppressor genes whose function is unknown at the moment.

Identify human equivalent of Drosophila OPMD suppressor genes

Partner 5 found that all the genes identified as suppressors in the genetic screen have a human homologue. The Drosophilahrg gene encoding poly(A) polymerase was identified as enhancer of OPMD. It also has a human homologue, PAP-, that encodes the canonical poly(A) polymerase (PAPOLA).

Main scientific achievements of Workpackage 5:

Partners 2 and 3 constructed a range of plasmid, adeno-associated viral and lentiviral vectors to deliver therapeutic genes in OPMD, including wt or codon optimised PABPN1, shRNA constructs and potential suppressors of mutant PABPN1 function. The relative efficiency of different muscle-specific expression cassettes for delivery of therapeutic genes was assessed in cells and in mice (Foster K et al 2009; Talbot G et al, submitted). A lentiviral vector expressing PABPN1-specific intrabody 3F5 was developed, which effectively reduces PABPN1-aggregate formation in muscle cell culture models of OPMD. Lentiviral vectors expressing shRNA against PABPN1 were developed, which effectively knock down PABPN1 expression in human myoblasts. Partner 3 has further tested the viral vectors constructed for the knockdown/replacement strategy in the transgenic mouse model of OPMD. The results show good transduction efficiency, but small reduction (30%) of the hPABPN1 mRNA. However, this was not sufficient to induce a reduction of the aggregates.