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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
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.
years)
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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
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Normal alleles (GCN)10
Autosomaldominant alleles (GCN)12-17
Autosomal recessive alleles (GCN)11]
NB A is amino acid: alanine
NB OPMD is a polyalanine myopathy
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Size: 306 amino acids; 32749 Da
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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-boxmediated 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?
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Binds RNA as a monomer
oligomerizes when bound to poly(A)
Identified in a mRNP granulecomplex
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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.
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Cytoplasm
◦ Localized in cytoplasmic mRNP granules containing
untranslated mRNAs
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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
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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
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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.
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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
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Treatment for ptosis may include blepharoplasty – either
◦ resection of the levator palpebrea aponeurosis
◦ frontal suspension of the eyelids.
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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.
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Overexpression of mutant PABPN1 in COS-7
◦ formation of PABPN1 aggregates
◦ increase in cell death
 increased abnormal apoptotic nuclei
 elevated apoptotic markers
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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
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Drosophila model of OPMD.
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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
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2nd transgenic mouse (Dion et al Neurobiol Dis 2005)
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hPABPN1 (GCN)17 : under natural promoter
no myopathy phenotype even at older ages
◦ no inclusion bodies in muscles
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primarily neuronal phenotype
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inclusion bodies in neurons
presenting with coordination deficits
abnormal limb crasping
peripheral nerves alterations
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3rd transgenic mouse (Davies Nat Med 2005)
◦ hPABPN1 (GCN)17 : human skeletal actin (HSA1) promoter
◦ muscle weakness
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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
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transcriptomic studies combined
with a detailed phenotypic characterization of
this model at three time points
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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
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Knockout of PABPN1
◦ No disease phenotype
 (à véfifier dans Davies Hum Mol Genet 2008
◦ greater susceptibility to pro-apoptotic stresses
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Not understood
◦ Brais Current Neurology and Neuroscience Reports 2009
Extended poly-L-alanine segment in PABPN1 cause fibril
formation
◦ SCHEUERMANN Protein Science (2003)
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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
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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.
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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••].
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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)
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wild-type PABPN1exerts an anti-apoptotic effect associated
with an increase in aggregation
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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?
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Is introduction of the PAPBN1 protein a potential treatment?
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Intrabodies (intra-cellular antibodies)
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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.
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Expression of this intrabody affects PABPN1 aggregation and
restores muscle gene expression
◦ In a Drosophila model
◦ (Chartier Human Molecular Genetics, 2009)
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A reagent to deliver antibody into cells even in the presence
of serum is available
◦ www.biocellchallenge.comIntracellular_Antibody_Delivery_Reagent.html
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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
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Allele specific silencing using siRNA
◦ (Miller et al PNAS 2003)
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not been done so far for OPMD
◦ because we need to have a difference between the 2
alleles
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Possibility of silencing both alleles and
introducing the normal gene
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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.
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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
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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
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Silencing des mRNA avec des siRNA
◦ Le gène normal et le gène muté seront silencés
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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