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 26-65 years)

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.

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

Normal alleles (GCN)10 Autosomaldominant alleles (GCN)12-17

Autosomal recessive alleles (GCN)11]

NB A is amino acid: alanineNB OPMD is a polyalanine myopathy

Size: 306 amino acids; 32749 Da

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?

Binds RNA as a monomer oligomerizes when bound to poly(A) Identified in a mRNP granulecomplex

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

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 of the length of poly(A) tails ◦ transcription regulation◦ mRNA nucleocytoplasmic transport

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

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.

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

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.

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

Drosophila model of OPMD.

Transgenic mice (UYAMA Acta Myologica 2005)

◦ Expressing hPABPN1 under a chicken ß-actin (CAG) promoter ◦ normal hPABPN1 (GCN)10 :

no myopathic changes,

◦ hPABPN1 (GCN)13 : myopathy phenotype with aging

more prominent in the eyelid and pharyngeal muscles Intranuclear inclusions consisting of aggregated mutant hPABPN1 scattered rimmed vacuoles restricted in the muscles

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

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

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

Knockout of PABPN1◦ No disease phenotype

(à véfifier dans Davies Hum Mol Genet 2008

◦ greater susceptibility to pro-apoptotic stresses

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

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.

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••].

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?

 

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

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

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

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.

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

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

In the promoter of PABPN1 there is binding sites for the following transcription factors MyoD   p53   Sp1   POU3F1   Arnt   AhR   HNF-4alpha2   COUP-TF1   NRSF form 1   NRSF form 2