OPMD ( Occulopharyngeal Muscular Dystrophy)

1. OPMD (Occulopharyngeal Muscular Dystrophy)

2. Disease characteristics. • first described by Taylor in 1915 • named by Victor et al. in 1962 • autosomal dominant disorder (OMIM 164300) • late-onset (usually after 45 years)(range 26-65years) • eyelid drooping (ptosis) • swallowing difficulty (dysphagia) • tongue atrophy and weakness (82%) • proximal lower extremity weakness (71%) • dysphonia (67%) • limitation of upward gaze (61%) • facial muscle weakness (43%) • proximal upper extremity weakness (38%) • life expectancy is not reduced.

3. Genetic • Abnormal expansion of a (GCG)6 trinucleotide repeat at the 5’-end (exon 1) of the coding region of the poly(A)-binding protein • nuclear 1 gene (PABPN1) • The (GCG)6 codes for the first 6 alanines in a homopolymeric stretch of 10 alanines. • In most patients [8], the (GCG)6 repeat is expanded to (GCG)8-13 • insertional or duplicative mutations such as (GCG)6+ GCA(GCG)2, • +GCA(GCG)3, +(GCA)2(GCG)2 or + (GCA)3(GCG)2 • rarely occur • In the latter group, mutations are secondary to the insertion of • no identical polyalanine-coding triplets due to an • unequal crossing-over mechanism. Thus, the disease • is associated with expansions of 12-17 uninterrupted • alanines located at the N-terminus of this protein. • In these mutations, the 13 alanines (A13) expansion • found in cluster populations including French Canadians

4. Genetic • Normal alleles (GCN)10 • Autosomaldominant alleles (GCN)12-17 Autosomal recessive alleles (GCN)11]

6. PROTEIN PABPN1 NB A is amino acid: alanine NB OPMD is a polyalanine myopathy

7. PABPN1 protein • Size: 306 amino acids; 32749 Da

8. Role of PABPN1 • required for progressive and efficient polymerization of poly(A) tails on the 3' ends of eukaryotic genes • stimulates poly(A) polymerase (PAPOLA) conferring processivity on the poly(A)tail elongation reaction • controls the size of the poly(A) tail to about 250 nt • present at various stages of mRNA metabolism including nucleocytoplasmic trafficking andnonsense-mediated decay (NMD) of mRNA. • Cooperates with SKIP to synergistically activate E-box-mediated transcriptionthrough MYOD1 and may regulate the expression of muscle-specific genes. • Binds to poly(A) and to poly(G) with high affinity • May protect the poly(A) tail from degradation • NB: A is nucleotide: adenosine • What is happening to the poly(A) in OPMD?

9. Role of PABPN1 protein • Binds RNA as a monomer • oligomerizes when bound to poly(A) • Identified in a mRNP granulecomplex

10. Subcellular location: • Nucleus • Shuttles between the nucleus and the cytoplasm but predominantly found in the nucleus • Its nuclear import may involve the nucleocytoplasmic transport receptor transportin • RAN-GTP-sensitive import mechanism • Is exported to the cytoplasm by a carrier-mediated pathway that is independent of mRNA traffic. • Cytoplasm • Localized in cytoplasmic mRNP granules containing untranslated mRNAs

11. Cause • small expansion of a short polyalanine tract in the poly (A) binding protein nuclear 1 protein (PABPN1) • The mechanism by which the polyalanine expansion mutation in PABPN1 causes disease is unclear • PABPN1 is a nuclear multifunctional • protein which is involved in: • pre-mRNA polyadenylation • control ofthe length of poly(A) tails • transcription regulation • mRNA nucleocytoplasmic transport

12. Pathological hallmark • filamentous intranuclear inclusions (INIs) in patient's skeletal muscle cells • The polyalanine expansion might induces a misfolding of PABPN1 and increases propensity for aggregation by conferring a toxic gain of function

13. Filamentous inclusions • in the myocytes of patients • contain PABPN1, ubiquitin, subunits of the proteasome and poly(A) RNA • the association of the expanded polyalanine mutations together with the capability to oligomerize may induce these inclusions and cell death.

14. Pathogenic mechanisms • mediated by polyalanine expansion mutations may be either a general disruption of cellular RNA metabolism due to the trapping by the inclusions of PABPN1, mRNAs and/or nuclear proteins, resulting in the induction of cell death • may change the normal muscle cell differentiation

15. Management • Treatment for ptosis may include blepharoplasty – either • resection of the levator palpebrea aponeurosis • frontal suspension of the eyelids. • Treatment for dysphagia may include: • Surgical intervention • in the presence of very symptomatic dysphagia • marked weight loss • near-fatal choking (which is extremely rare) • recurrent pneumonia • Cricopharyngeal myotomy alleviates symptoms in most cases.

16. Cell model • Overexpression of mutant PABPN1 in COS-7 • formation of PABPN1 aggregates • increase in cell death • increased abnormal apoptotic nuclei • elevated apoptotic markers • Y. P. Bao, L. J. Cook, D. O’Donovan, E. Uyama, D. C. Rubinsztein, Mammalian, yeast, • bacterial, and chemical chaperones reduce aggregate formation and death in a cell model • of oculopharyngeal muscular dystrophy. J. Biol. Chem. 277, 12263–12269 (2002). • 6. Y. P. Bao, S. Sarkar, E. Uyama, D. C. Rubinsztein, Congo red, doxycycline, and HSP70 overexpression • reduce aggregate formation and cell death in cell models of oculopharyngeal • muscular dystrophy. J. Med. Genet. 41, 47–51 (2004). • 7. J. E. Davies, L. Wang, L. Garcia-Oroz, L. J. Cook, C. Vacher, D. G. O’Donovan, D. C. Rubinsztein, • Doxycycline attenuates and delays toxicity of the oculopharyn

17. Animal models • Drosophila model of OPMD.

18. Animal models • Transgenic mice (UYAMA Acta Myologica 2005) • Expressing hPABPN1 under a chicken ß-actin (CAG) promoter • normal hPABPN1 (GCN)10 : • no myopathicchanges, • hPABPN1 (GCN)13 : • myopathy phenotypewith aging • more prominent in the eyelid and pharyngealmuscles • Intranuclearinclusions consisting of aggregated mutant hPABPN1 • scattered rimmed vacuoles restricted in the muscles

19. Animal models • 2nd transgenic mouse (Dion et al Neurobiol Dis 2005) • hPABPN1 (GCN)17 : under natural promoter • no myopathy phenotype even at older ages • no inclusion bodies in muscles • primarily neuronal phenotype • inclusion bodies in neurons • presenting with coordination deficits • abnormal limb crasping • peripheral nerves alterations

20. Animal models • 3rd transgenic mouse (Davies Nat Med 2005) • hPABPN1(GCN)17 : human skeletal actin (HSA1) promoter • muscle weakness • nuclear inclusions in skeletal muscles • “vacuoles” in the muscles were not rimmed vacuoles • apoptotic processes • no late-onset ptosis • no dysphagia resulting • Elevated transglutaminase 2 expression (TG2) • TG2 knockdown suppresses the toxicity and aggregation • Davies Sci Transl Med 2010 • the transgene was not expressed above endogenous levels in 5 organs

21. Animal models • transcriptomic studies combined • with a detailed phenotypic characterization of this model at three time points • Trollet Hum Mol Gen 2010 (Dickson group) • a severe and progressive muscular atrophy • associated with a reduction in muscle strength • muscle atrophy restricted to fast glycolytic fibres • containing a large number of intranuclear inclusions

22. Animal models

23. Animal models • Knockout of PABPN1 • No disease phenotype • (à véfifier dans Davies Hum Mol Genet 2008 • greater susceptibility topro-apoptotic stresses

24. Pathological mechanism • Not understood • Brais Current Neurology and Neuroscience Reports 2009 • Extended poly-L-alanine segment in PABPN1 cause fibril formation • SCHEUERMANN Protein Science (2003) • Phenotypes observed in homozygotes and compound heterozygotes favor a gene dosage effect • Mutated PABPN1 forms aggregates in nuclei • However aggregation may not be pathologic per se

25. Pathological mechanism • PABPN1 is an ubiquitous polyadenylation factor essential for the formation of poly(A) tails of eukaryotic mRNA • PABPN1shuttles between the nucleus and the cytoplasm • Despite the strong evidence that the expanded polyalanine domain influences PABPN1 aggregation and toxicity, there is mounting evidence suggesting that the larger aggregates may not play a key role in the underlining pathology but, rather, might even be protective.

26. Pathological mechanism • With more extreme over-expression of PABPN1 harboring very large expansions, the toxicity was greatest with the largest constructs in which aggregates were not formed • the soluble mutated PABPN1 is the true culprit, while aggregates that arise through over-expression are visible bystanders of a • molecular toxicity caused by the soluble PABPN1 • cells able to produce the inclusion may in fact • be protected against the toxicity [24••].

27. Potential therapies • Gene therapy • Wild-type PABPN1 over-expression can reduce mutant PABPN1 toxicity in both cell and mouse models of OPMD • (Davies Hum Mol Gen 2008) • wild-type PABPN1exerts an anti-apoptotic effect associated with an increase in aggregation • Brais Current Neurology and Neuroscience Reports 2009 • Over-expression of either the wild type (wt) or mutant (mut) PABPN1 slowed down the cell proliferation • Thus AAV PAPBN1 is a possibility • How much over expression is required? • Is there some toxicity? • Is introduction of the PAPBN1 protein a potential treatment?

28. Potential therapies • Intrabodies (intra-cellular antibodies) • Verheesen Human Molecular Genetics, 2006 • Llama intrabodies • natural single-chain antibodies are produced in Camelids • when engineered, combined the advantages of • being single-chain • small sized and very stable. • Expression of this intrabody affects PABPN1 aggregation and restores muscle gene expression • In a Drosophila model • (Chartier Human Molecular Genetics, 2009) • A reagent to deliver antibody into cells even in the presence of serum is available • www.biocellchallenge.comIntracellular_Antibody_Delivery_Reagent.html

29. Potential therapies • Myoblast transplantation • provokes premature senescence in dividing myoblasts • may be due to intranuclear toxic aggregates • Transplantation of autologous myoblasts may increase myogenic capacity • Phase 1 clinical trial done by Butler-Browne and Mouly • Done with a cricopharyngeal myotomy • Results not published

30. Potential therapies • Allele specific silencing using siRNA • (Miller et al PNAS 2003) • not been done so far for OPMD • because we need to have a difference between the 2 alleles • Possibility of silencing both alleles and introducing the normal gene

31. Potential therapies • Inhibitors of transglutaminase 2 • Cystamine • protects against the toxicity of mutant PABPN1 • inhibition of transglutaminase 2 (in 3rd mouse model) • reduces the aggregation and toxicity of mutant PABPN1 in human cells • attenuated muscle weakness • decreased apoptotic markers in muscle.

32. doxycycline and trehalose • diminish mutated PABPN1 toxicity in a mouse transgenic model • interfere with protein aggregation, • Davies Nat Med 2005 • Davies Hum Mol Genet 2006

33. Thérapiesque je propose • Livrer la protéine PABPN1 normale • Muté (KO) le gène anormal avec des ZFNs • Ceci va possiblement muté aussi le gène normal mais ce n’est pasible pas un problème puisque les animaux KO n’ont pas de symptomes • Silencing des mRNA avec des siRNA • Le gène normal et le gène muté seront silencés

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36. PABPN1 promoter • In the promoter of PABPN1 there is binding sites for the following transcription factors MyoDp53Sp1POU3F1ArntAhRHNF-4alpha2COUP-TF1NRSF form 1NRSF form 2