poly(A) Binding Protein Nuclear 1 Research Articles

A Natural History Study of Hindlimb Physiology and Histopathology in a Heterozygous OPMD Mouse Model

ABSTRACTBackgroundOculopharyngeal muscular dystrophy (OPMD) is an adult‐onset autosomal dominant myopathy. OPMD is caused by a short alanine expansion in the gene encoding for poly(A) binding protein nuclear 1 (PABPN1) forming insoluble nuclear aggregates. OPMD patients are predominantly heterozygous, and the knock‐in Pabpn1+/A17 mouse, which expresses one copy of the expanded Pabpn1 gene under the PABPN1 promoter genetically, mimics OPMD. Insights into the A17/+ mouse model are necessary to evaluate its preclinical value and test therapeutics for OPMD. Here, we performed a natural disease history study for the A17/+ model.MethodsWe combined muscle force measurements of the tibialis anterior with magnetic resonance imaging (MRI) measurements of the calf muscles made in 4‐, 8‐ and 12‐month‐old wild‐type and A17/+ mice. These measures were complemented by muscle histopathology staining and image quantification to detect PABPN1 aggregates and to assess muscle wasting. Statistical significance between genotypes over the three time points was assessed using ANOVA or Student's t test.ResultsPABPN1 nuclear aggregates were found in the 12‐month‐old A17/+ mice at similar quantities of ~2% across hindlimb muscles. We did not observe changes in muscle strength of the tibialis anterior in A17/+ mice. MRI analyses of hindlimb muscles revealed no metabolic difference, no fatty infiltration and limited muscle atrophy between A17/+ and +/+ mice. The plantaris muscle in A17/+ showed 30% atrophy at 12 months of age, which was corroborated by a 30% myofiber shift in the myosin heavy chain −2A to −2B ratio. Histopathologic staining did not reveal muscle wasting in the hindlimb muscles.ConclusionsDespite the presence of PABPN1 insoluble aggregates in hindlimb muscles, muscle involvement in the 12‐month‐old A17/+ mice was limited. Our results query the usefulness of A17/+ hindlimb muscles for preclinical studies.

Dysregulation of RNA-Exosome machinery is directly linked to major cancer hallmarks in prostate cancer: Oncogenic role of PABPN1

Novel biomarkers and therapeutic strategies for prostate-cancer (PCa) are required to overcome its lethal progression. The dysregulation/implication of the RNA-Exosome-complex (REC; cellular machinery controlling the 3′-5′processing/degradation of most RNAs) in different cancer-types, including PCa, is poorly known. Herein, different cellular/molecular/preclinical approaches with human PCa-samples (tissues and/or plasma of 7 independent cohorts), and in-vitro/in-vivo PCa-models were used to comprehensively characterize the REC-profile and explore its role in PCa. Moreover, isoginkgetin (REC-inhibitor) effects were evaluated on PCa-cells. We demonstrated a specific dysregulation of the REC-components in PCa-tissues, identifying the Poly(A)-Binding-Protein-Nuclear 1 (PABPN1) factor as a critical regulator of major cancer hallmarks. PABPN1 is consistently overexpressed in different human PCa-cohorts and associated with poor-progression, invasion and metastasis. PABPN1 silencing decreased relevant cancer hallmarks in multiple PCa-models (proliferation/migration/tumourspheres/colonies, etc.) through the modulation of key cancer-related lncRNAs (PCA3/FALEC/DLEU2) and mRNAs (CDK2/CDK6/CDKN1A). Plasma PABPN1 levels were altered in patients with metastatic and tumour-relapse. Finally, pharmacological inhibition of REC-activity drastically inhibited PCa-cell aggressiveness. Altogether, the REC is drastically dysregulated in PCa, wherein this novel molecular event/mechanism, especially PABPN1 alteration, may be potentially exploited as a novel prognostic and therapeutic tool for PCa.

Oculopharyngeal muscular dystrophy mutations link the RNA-binding protein HNRNPQ to autophagosome biogenesis.

Autophagy is an intracellular degradative process with an important role in cellular homeostasis. Here, we show that the RNA binding protein (RBP), heterogeneous nuclear ribonucleoprotein Q (HNRNPQ)/SYNCRIP is required to stimulate early events in autophagosome biogenesis, in particular the induction of VPS34 kinase by ULK1-mediated beclin 1 phosphorylation. The RBPs HNRNPQ and poly(A) binding protein nuclear 1 (PABPN1) form a regulatory network that controls the turnover of distinct autophagy-related (ATG) proteins. We also show that oculopharyngeal muscular dystrophy (OPMD) mutations engender a switch from autophagosome stimulation to autophagosome inhibition by impairing PABPN1 and HNRNPQ control of the level of ULK1. The overexpression of HNRNPQ in OPMD patient-derived cells rescues the defective autophagy in these cells. Our data reveal a regulatory mechanism of autophagy induction that is compromised by PABPN1 disease mutations, and may thus further contribute to their deleterious effects.

PABPN1 promotes clear cell renal cell carcinoma progression by suppressing the alternative polyadenylation of SGPL1 and CREG1.

Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism in cancer development and progression. Poly(A) binding protein nuclear 1 (PABPN1) is a gene that encodes abundant nuclear protein, binds with high affinity to nascent poly(A) tails, and is crucial for 3'-UTR (3'-untranslated region) APA. Although PABPN1 has been recently reported as a dominant master APA regulator in clear cell renal cell carcinoma (ccRCC), the underlying functional mechanism remain unclear and the genes subject to PABPN1 regulation that contribute to ccRCC progression have not been identified. Here, we found that PABPN1 is upregulated in ccRCC, and its expression is highly associated with the clinical prognosis of ccRCC patients. PABPN1 promotes ccRCC cell proliferation, migration, invasion, and exerts an influence on sphingolipid metabolism and cell cycle. Moreover, PABPN1 depletion significantly suppressed cancer cell growth via induction of cell cycle arrest and apoptosis. In particular, we characterized PABPN1-regulated 3'-UTR APA of sphingosine-1-phosphate lyase 1 (SGPL1) and cellular repressor of E1A stimulated genes 1 (CREG1), which contribute to ccRCC progression. Collectively, our data revealed that PABPN1 promotes ccRCC progression at least in part, by suppressing SGPL1 and CREG1. Thus, PABPN1 may be a potential therapeutic target in ccRCC.

PABPN1 aggregation is driven by Ala expansion and poly(A)-RNA binding, leading to CFIm25 sequestration that impairs alternative polyadenylation

Poly(A)-binding protein nuclear 1 (PABPN1) is an RNA-binding protein localized in nuclear speckles, while its alanine (Ala)-expanded variants accumulate as intranuclear aggregates in oculopharyngeal muscular dystrophy. The factors that drive PABPN1 aggregation and its cellular consequences remain largely unknown. Here, we investigated the roles of Ala stretch and poly(A) RNA in the phase transition of PABPN1 using biochemical and molecular cell biology methods. We have revealed that the Ala stretch controls its mobility in nuclear speckles, and Ala expansion leads to aggregation from the dynamic speckles. Poly(A) nucleotide is essential to the early-stage condensation that thereby facilitates speckle formation and transition to solid-like aggregates. Moreover, the PABPN1 aggregates can sequester CFIm25, a component of the pre-mRNA 3'-UTR processing complex, in an mRNA-dependent manner and consequently impair the function of CFIm25 in alternative polyadenylation. In conclusion, our study elucidates a molecular mechanism underlying PABPN1 aggregation and sequestration, which will be beneficial for understanding PABPN1 proteinopathy.

The small compound Icerguastat reduces muscle defects in oculopharyngeal muscular dystrophy through the PERK pathway of the unfolded protein response

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. Here, using Drosophila OPMD models, we show that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. We show that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD.

TERRA stability is regulated by RALY and polyadenylation in a telomere-specific manner

Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from telomeres that plays key roles in telomere maintenance. A fraction of TERRA is polyadenylated, and the presence of the poly(A) tail influences TERRA localization and stability. However, the mechanisms of TERRA biogenesis remain mostly elusive. Here, we show that the stability of TERRA transcripts is regulated by the RNA-binding protein associated with lethal yellow mutation (RALY). RALY depletion results in lower TERRA levels, impaired localization of TERRA at telomeres, and ultimately telomere damage. Importantly, we show that TERRA polyadenylation is telomere specific and that RALY preferentially stabilizes non-polyadenylated TERRA transcripts. Finally, we report that TERRA interacts with the poly(A)-binding protein nuclear 1 (PABPN1). Altogether, our results indicate that TERRA stability is regulated by the interplay between RALY and PABPN1, defined by the TERRA polyadenylation state. Our findings also suggest that different telomeres may trigger distinct TERRA-mediated responses.

PABPN1 regulates mRNA alternative polyadenylation to inhibit bladder cancer progression

BackgroundAbout 10–20% of patients with bladder cancer (BC) progress to muscle-invasive diseases, of which the underlying key molecular events have yet to be addressed.ResultsHere, we identified poly(A) binding protein nuclear 1 (PABPN1), a general factor of alternative polyadenylation (APA), was downregulated in BC. Overexpression and knockdown of PABPN1 significantly decreased and increased BC aggressiveness, respectively. Mechanistically, we provide evidence that the preference of PABPN1-bound polyadenylation signals (PASs) depends on the relative location between canonical and non-canonical PASs. PABPN1 shapes inputs converging on Wnt signaling, cell cycle, and lipid biosynthesis.ConclusionsTogether, these findings provide insights into how PABPN1-mediated APA regulation contributes to BC progression, and suggest that pharmacological targeting PABPN1 might have therapeutic potential in patients with BC.

Assessment of PABPN1 nuclear inclusions on a large cohort of patients and in a human xenograft model of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a rare muscle disease characterized by an onset of weakness in the pharyngeal and eyelid muscles. The disease is caused by the extension of a polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) protein leading to the formation of intranuclear inclusions or aggregates in the muscle of OPMD patients. Despite numerous studies stressing the deleterious role of nuclear inclusions in cellular and animal OPMD models, their exact contribution to human disease is still unclear. In this study, we used a large and unique collection of human muscle biopsy samples to perform an in-depth analysis of PABPN1 aggregates in relation to age, genotype and muscle status with the final aim to improve our understanding of OPMD physiopathology. Here we demonstrate that age and genotype influence PABPN1 aggregates: the percentage of myonuclei containing PABPN1 aggregates increases with age and the chaperone HSP70 co-localize more frequently with PABPN1 aggregates with a larger polyalanine tract. In addition to the previously described PRMT1 and HSP70 co-factors, we identified new components of PABPN1 aggregates including GRP78/BiP, RPL24 and p62. We also observed that myonuclei containing aggregates are larger than myonuclei without. When comparing two muscles from the same patient, a similar amount of aggregates is observed in different muscles, except for the pharyngeal muscle where fewer aggregates are observed. This could be due to the peculiar nature of this muscle which has a low level of PAPBN1 and contains regenerating fibers. To confirm the fate of PABPN1 aggregates in a regenerating muscle, we generated a xenograft model by transplanting human OPMD muscle biopsy samples into the hindlimb of an immunodeficient mouse. Xenografts from subjects with OPMD displayed regeneration of human myofibers and PABPN1 aggregates were rapidly present—although to a lower extent-after muscle fiber regeneration. Our data obtained on human OPMD samples add support to the dual non-exclusive models in OPMD combining toxic PABPN1 intranuclear inclusions together with PABPN1 loss of function which altogether result in this late-onset and muscle selective disease.

CRISPR-Cas9 mediated generation of a conditional poly(A) binding protein nuclear 1 (Pabpn1) mouse model reveals an essential role for hematopoietic stem cells

Poly(A) binding protein nuclear 1 (PABPN1) is known for its role in poly(A) tail addition and regulation of poly(A) tail length. In addition, it has been shown to be involved in alternative polyadenylation (APA). APA is a process regulating differential selection of polyadenylation sites, thereby influencing protein isoform expression and 3ʹ-UTR make-up. In this study, we generated an inducible Pabpn1flox/flox mouse model using crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, in combination with a long single-stranded DNA (ssDNA) template. We performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency for in vivo application. Pabpn1flox/flox mice were generated and crossed to MxCre mice for validation experiments, allowing the induction of Cre expression in the bone marrow (BM) by poly(I:C) (pIC) injections. Validation experiments revealed successful deletion of Pabpn1 and absence of PABPN1 protein. Functionally, knockout (KO) of Pabpn1 led to a rapid and robust depletion of hematopoietic stem and progenitor cells (HSPCs) as well as myeloid cells, suggesting an essential role of Pabpn1 in the hematopoietic lineage. Overall, the mouse model allows an inducible in-depth in vivo analysis of the role of PABPN1 and APA regulation in different tissues and disease settings.

Exosome-Mediated Transfer of miR-1323 from Cancer-Associated Fibroblasts Confers Radioresistance of C33A Cells by Targeting PABPN1 and Activating Wnt/β-Catenin Signaling Pathway in Cervical Cancer.

Plenty of pieces of evidence suggest that the resistance to radiotherapy greatly influences the therapeutic effect in cervical cancer (CCa). MicroRNAs (miRNAs) have been reported to regulate cellular processes by acting as tumor suppressors or promoters, thereby driving radioresistance or radiosensitivity. Meanwhile, it has been reported that microRNA-1323 (miR-1323) widely participates in cancer progression and radiotherapy effects. However, the role of miR-1323 is still not clear in CCa. Hence, in this study, we are going to investigate the molecular mechanism of miR-1323 in CCa cells. In the beginning, miR-1323 was found aberrantly upregulated in CCa cells via RT-qPCR assay. Functional assays indicated that miR-1323 was transferred by cancer-associated fibroblasts-secreted (CAFs-secreted) exosomes and miR-1323 downregulation suppressed cell proliferation, migration, invasion, and increased cell radiosensitivity in CCa. Mechanism assays demonstrated that miR-1323 targeted poly(A)-binding protein nuclear 1 (PABPN1). Besides, PABPN1 recruited insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to regulate glycogen synthase kinase 3 beta (GSK-3β) and influenced Wnt/β-catenin signaling pathway. Therefore, rescue experiments were implemented to validate that PABPN1 overexpression rescued the inhibited cancer development and radioresistance induced by the miR-1323 inhibitor. In conclusion, miR-1323 was involved in CCa progression and radioresistance which might provide a novel insight for CCa treatment.

Alternative Polyadenylation Utilization Results in Ribosome Assembly and mRNA Translation Deficiencies in a Model for Muscle Aging.

Aging-associated muscle wasting is regulated by multiple molecular processes, whereby aberrant mRNA processing regulation induces muscle wasting. The poly(A)-binding protein nuclear 1 (PABPN1) regulates polyadenylation site (PAS) utilization, in the absence of PABPN1 the alternative polyadenylation (APA) is utilized. Reduced PABPN1 levels induce muscle wasting where the expression of cellular processes regulating protein homeostasis, the ubiquitin-proteasome system, and translation, are robustly dysregulated. Translation is affected by mRNA levels, but PABPN1 impact on translation is not fully understood. Here we show that a persistent reduction in PABPN1 levels led to a significant loss of translation efficiency. RNA-sequencing of rRNA-depleted libraries from polysome traces revealed reduced mRNA abundance across ribosomal fractions, as well as reduced levels of small RNAs. We show that the abundance of translated mRNAs in the polysomes correlated with PAS switches at the 3′-UTR. Those mRNAs are enriched in cellular processes that are essential for proper muscle function. This study suggests that the effect of PABPN1 on translation efficiency impacts protein homeostasis in aging-associated muscle atrophy.

Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.

BB-301: a silence and replace AAV-based vector for the treatment of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering “silence and replace” sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codon-optimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.

FSHD / OPMD / MYOTONIC DYSTROPHY: P.240 Targeting unfolded protein response to resolve aggresomes accumulation in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a rare late-onset genetic myopathy, affecting mainly eyelid and pharyngeal muscles leading to ptosis and dysphagia, respectively. OPMD is caused by a mutation in Poly-A Binding Protein Nuclear 1 (PABPN1) gene, that codes for a protein essential for proper mRNA biogenesis in nucleus. Accordingly, mutated PABPN1 in OPMD results in PABPN1 intranuclear aggregates in muscle that sequestrate mRNA and proteins and thus induce a wide range of deleterious consequences on muscle homeostasis (mitochondrial dysfunctions, calcium homeostasis disruption, fibrosis, force impairment, atrophy). We previously demonstrated that mouse model of OPMD presents muscle Endoplasmic Reticulum (ER) stress, i.e. an accumulation of misfolded proteins in ER lumen, and that restoring the physiological and resolutive Unfolded Protein Response (UPR) through guanabenz acetate treatment is efficient to decrease PABPN1 nuclear aggregates and improve muscle function in OPMD mouse (Malerba et al Hum Mol Genet 2019). To validate these results in human, we now analyze muscle biopsies from OPMD patients compared to healthy donors and observe a disruption of both key genes and proteins of UPR signalling in OPMD muscles. Interestingly, we find that the alteration is positively correlated with the clinical state of the muscle. Furthermore, we detect GRP78 (BiP), the most important ER chaperone, trapped in PABPN1 nuclear aggregates, suggesting a reduced folding capacity in ER. As a consequence, OPMD muscles present an accumulation of large beta-sheet protein aggregates called aggresomes. Consistently, we observe that human OPMD myoblasts are more sensible to develop aggresomes than healthy myoblasts in response to ER stress. Overall, our results unravel an impairment of ER folding and UPR as well as an aggresomes accumulation in human OPMD muscle. Thus, restoring the protein folding in ER as well as improving the aggresomes clearance could be a promising therapeutic strategy against OPMD.

Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100 000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11-18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV `silence' and `replace' vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the `silence' and `replace' cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.

Deacetylation Inhibition Reverses PABPN1-Dependent Muscle Wasting

SummaryReduced poly(A)-binding protein nuclear 1 (PABPN1) levels cause aging-associated muscle wasting. PABPN1 is a multifunctional regulator of mRNA processing. To elucidate the molecular mechanisms causing PABPN1-mediated muscle wasting, we compared the transcriptome with the proteome in mouse muscles expressing short hairpin RNA to PABPN1 (shPab). We found greater variations in the proteome than in mRNA expression profiles. Protein accumulation in the shPab proteome was concomitant with reduced proteasomal activity. Notably, protein acetylation appeared to be decreased in shPab versus control proteomes (63%). Acetylome profiling in shPab muscles revealed prominent peptide deacetylation associated with elevated sirtuin-1 (SIRT1) deacetylase. We show that SIRT1 mRNA levels are controlled by PABPN1 via alternative polyadenylation site utilization. Most importantly, SIRT1 deacetylase inhibition by sirtinol increased PABPN1 levels and reversed muscle wasting. We suggest that perturbation of a multifactorial regulatory loop involving PABPN1 and SIRT1 plays an imperative role in aging-associated muscle wasting.Video

Pharmacological modulation of the ER stress response ameliorates oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease leading to ptosis, dysphagia and proximal limb muscles at later stages. A short abnormal (GCN) triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. Here we demonstrate that treating mice with guanabenz acetate (GA), an FDA-approved antihypertensive drug, reduces the size and number of nuclear aggregates, improves muscle force, protects myofibers from the pathology-derived turnover and decreases fibrosis. GA targets various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. We demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiation factor 2α subunit and the splicing of Xbp1, key components of the UPR. Altogether these data show that modulation of protein folding regulation is beneficial for OPMD and promote the further development of GA or its derivatives for treatment of OPMD in humans. Furthermore, they support the recent evidences that treating ER stress could be therapeutically relevant in other more common proteinopathies.

P.354 - Nuclear PABPN1 aggregates in OPMD: correlation study and therapy

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease manifested primarily by progressive weakness of the eyelid and pharyngeal muscles, leading to ptosis and dysphagia. A short abnormal triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to the accumulation of nuclear insoluble aggregates in muscles of OPMD patients. While the roles of PABPN1 in post-transcriptional gene regulation are established, the molecular basis of OPMD and the exact contribution of these PABPN1 aggregates remain poorly understood. We are currently investigating the role they might have in the pathology, searching triggers that induce their formation, and testing drugs to remove them. Using a large collection of OPMD patient's muscle biopsies, we are evaluating the percentage, size and characteristics of these aggregates using immunofluorescent and electronic microscopy. Any potential correlation of these parameters with age and genotype is carefully monitored. Results obtained so far suggest an increase in the percentage of nuclear aggregates together with age. Although there are controversies regarding the role of aggregates in OPMD, it is commonly accepted that aggregates and/or the early oligomeric species are toxic (Abu-Baker et al., 2013; Davies et al., 2006). Decreasing their number in OPMD models greatly ameliorate the muscle phenotype. Within the frame of an European OPMD network, we are testing drugs to reduce aggregate formation. Among them, guanabenz acetate (GA) has been identified as a good candidate in drosophila OPMD model (Barbezier et al., 2011). Here we show that GA is also efficient in cell and mouse OPMD models: treatment with GA decreases the percentage of nuclear aggregates and ameliorates muscle strength and atrophy in mice. Altogether these studies will shed light on the exact contribution of aggregate in OPMD and help design potential therapeutic targets.

Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology.

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (Pabpn1+/A17) that contains one alanine-expanded Pabpn1 allele under the control of the native promoter and one wild-type Pabpn1 allele. This mouse is the closest available genocopy of OPMD patients. We show that Pabpn1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous Pabpn1 knock-out mouse model (Pabpn1+/Δ). Like the Pabpn1+/A17 mice, Pabpn1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in Pabpn1+/Δ mice only partially overlap with those detected in Pabpn1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in Pabpn1+/A17 mice.