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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., Graphical Abstract, Alanine expansions of PABPN1 cause oculopharyngeal muscular dystrophy. Administration of a single AAV9 vector expressing a corrected version of the PABPN1 gene and two siRNAs in miRNA backbones to silence endogenous PABPN1 can restore muscle size, weight, and force in an OPMD surrogate mouse model to wild-type levels.

Background: Triple-negative breast cancer (TNBC) is an aggressive malignancy with high heterogeneity. However, the alternative polyadenylation (APA) profiles of TNBC remain unknown. Here, we aimed to define the characteristics of the APA events at post-transcription level among TNBCs. Methods: Using transcriptome microarray data, we analyzed APA profiles of 165 TNBC samples and 33 paired normal tissues. A pooled short hairpin RNA screen targeting 23 core cleavage and polyadenylation (C/P) genes was used to identify key C/P factors. Results: We established an unconventional APA subtyping system composed of four stable subtypes: 1) luminal androgen receptor (LAR), 2) mesenchymal-like immune-activated (MLIA), 3) basal-like (BL), 4) suppressed (S) subtypes. Patients in the S subtype had the worst disease-free survival comparing to other patients (log-rank p = 0.021). Enriched clinically actionable pathways and putative therapeutic APA events were analyzed among each APA subtype. Furthermore, CPSF1 and PABPN1 were identified as the master C/P factors in regulating APA events and TNBC proliferation. The depletion of CPSF1 or PABPN1 weakened cell proliferation, enhanced apoptosis, resulted in cell cycle redistribution and a reversion of APA events of genes associated with tumorigenesis, proliferation, metastasis and chemosensitivity in breast cancer. Conclusions: Our findings advance the understanding of tumor heterogeneity regulation in APA and yield new insights into therapeutic target identification in TNBC.

Several conserved nuclear RNA binding proteins (sut-1, sut-2, and parn-2) control tau aggregation and toxicity in C. elegans, mice, and human cells. MSUT2 protein normally resides in nuclear speckles, membraneless organelles composed of phase-separated RNAs and RNA-binding proteins that mediate critical steps in mRNA processing including mRNA splicing. We used human pathological tissue and transgenic mice to identify Alzheimer’s disease-specific cellular changes related to nuclear speckles. We observed that nuclear speckle constituent scaffold protein SRRM2 is mislocalized and accumulates in cytoplasmic lesions in AD brain tissue. Furthermore, progression of tauopathy in transgenic mice is accompanied by increasing mislocalization of SRRM2 from the neuronal nucleus to the soma. In AD brain tissue, SRRM2 mislocalization associates with increased severity of pathological tau deposition. These findings suggest potential mechanisms by which pathological tau impacts nuclear speckle function in diverse organisms ranging from C. elegans to mice to humans. Future translational studies aimed at restoring nuclear speckle homeostasis may provide novel candidate therapeutic targets for pharmacological intervention.

Prion diseases are caused by the propagation of PrP(Sc), the pathological conformation of the PrP(C) prion protein. The molecular mechanisms underlying PrP(Sc) propagation are still unsolved and no therapeutic solution is currently available. We thus sought to identify new anti-prion molecules and found that flunarizine inhibited PrP(Sc) propagation in cell culture and significantly prolonged survival of prion-infected mice. Using an in silico therapeutic repositioning approach based on similarities with flunarizine chemical structure, we tested azelastine, duloxetine, ebastine, loperamide and metixene and showed that they all have an anti-prion activity. Like flunarizine, these marketed drugs reduced PrP(Sc) propagation in cell culture and in mouse cerebellum organotypic slice culture, and inhibited the protein folding activity of the ribosome (PFAR). Strikingly, some of these drugs were also able to alleviate phenotypes due to PABPN1 nuclear aggregation in cell and Drosophila models of oculopharyngeal muscular dystrophy (OPMD). These data emphasize the therapeutic potential of anti-PFAR drugs for neurodegenerative and neuromuscular proteinopathies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-020-00992-6.

Oculopharyngeal muscular dystrophy (OPMD) is caused by a small expansion of a short polyalanine (polyAla) tract in the poly(A)-binding protein nuclear 1 protein (PABPN1). Despite the monogenic nature of OPMD, no treatment is currently available. Here we report an RNA replacement strategy that has therapeutic potential in cell and C. elegans OPMD models. We develop selective microRNAs (miRNAs) against PABPN1, and we report that miRNAs and our previously developed hammerhead ribozymes (hhRzs) are capable of reducing the expression of both the mRNA and protein levels of PABPN1 by as much as 90%. Since OPMD derives from a very small expansion of GCG within the polyAla tract, our hhRz and miRNA molecules cannot distinguish between the wild-type and mutant mRNAs of PABPN1. Therefore, we designed an optimized-codon wild-type PABPN1 (opt-PABPN1) that is resistant to cleavage by hhRzs and miRNAs. Co-expression of opt-PABPN1 with either our hhRzs or miRNAs restored the level of PABPN1, concomitantly with a reduction in expanded PABPN1-associated cell death in a stable C2C12 OPMD model. Interestingly, knockdown of the PABPN1 by selective hhRzs in the C. elegans OPMD model significantly improved the motility of the PABPN1-13Ala worms. Taken together, RNA replacement therapy represents an exciting approach for OPMD treatment., Graphical Abstract

Background Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs. Objective We sought to generate a polyclonal antibody that recognizes alanine-expanded but not wild type PABPN1 with the goal of making possible analysis of expression and localization of alanine-expanded PABPN1. Methods We immunized rabbits with a GST-tagged alanine peptide and tested the resulting serum against alanine-expanded PABPN1 expressed in cell culture as well as in animal models of OPMD. Results The resulting α-alanine antibody detected PABPN1 proteins that contained 14 or more alanine residues. Importantly, the α-alanine antibody could be used to detect alanine-expanded PABPN1 in muscles from either a mouse or Drosophila model of OPMD. Conclusions This α-alanine antibody provides a new tool that will allow for more in-depth study of how alanine expansion affects aggregation, localization, and steady-state levels of alanine-expanded PABPN1 levels in vivo, providing new insight into the molecular mechanisms underlying OPMD.

// Vered Raz 1 , George Dickson 2 and Peter A. C. ’t Hoen 1 1 Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands 2 School of Biological Science, Royal Holloway University of London, Egham, Surrey, United Kingdom Correspondence to: Vered Raz, email: // Keywords : PABPN1, mRNA processing, alternative polyadenylation site, autophagy, aging muscles, Gerotarget Received : April 11, 2017 Accepted : August 03, 2017 Published : September 05, 2017 Abstract Post-transcription mRNA processing in the 3’-untranslated region (UTR) of transcripts alters mRNA landscape. Alternative polyadenylation (APA) utilization in the 3’-UTR often leads to shorter 3’-UTR affecting mRNA stability, a process that is regulated by PABPN1. In skeletal muscles PABPN1 levels reduce with age and a greater decrease in found in Oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant myopathy caused by expansion mutation in PABPN1. In OPMD models a shift from distal to proximal polyadenylation site utilization in the 3’-UTR, and PABPN1 was shown to play a prominent role in APA. Whether PABPN1-mediated APA transcripts are functional is not fully understood. We investigate nuclear export and translation efficiency of transcripts in OPMD models. We focused on autophagy-regulated genes (ATGs) with APA utilization in cell models with reduced functional PABPN1. We provide evidence that ATGs transcripts from distal PAS retain in the nucleus and thus have reduced translation efficiency in cells with reduced PABPN1. In contrast, transcripts from proximal PAS showed a higher cytoplasmic abundance but a reduced occupancy in the ribosome. We therefore suggest that in reduced PABPN1 levels ATG transcripts from APA may not effectively translate to proteins. In those conditions we found constitutive autophagosome fusion and reduced autophagy flux. Augmentation of PABPN1 restored autophagosome fusion, suggesting that PABPN1-mediated APA plays a role in autophagy in OPMD and in aging muscles.

Objective:Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant adult-onset disease characterized by progressive ptosis, dysphagia, and proximal limb weakness. The genetic cause is an expanded (GCN)n mutation in the PABPN1 gene encoding for the polyadenylate-binding protein nuclear 1. We hypothesized a potential correlation between the size of the (GCN)n expansion and the severity of the phenotype. To do this, we characterized the distribution of the genotypes as well as their correlation with age at diagnosis and phenotypical features in a large cohort of heterozygous and homozygous patients with OPMD in France with a confirmed molecular diagnosis of PABPN1.Methods:We explored 354 unrelated index cases recruited between 1999 and 2014 in several neuromuscular centers in France.Results:This cohort allowed us to characterize the frequency of mutated alleles in the French population and to demonstrate a statistical correlation between the size of the expansion and the mean age at diagnosis. We also confirmed that homozygous patients present with a more severe disease.Conclusions:It has been difficult to establish phenotype–genotype correlations because of the rare nature of this disease. Our work demonstrates that patients with OPMD with longer PABPN1 expansion are on average diagnosed at an earlier age than patients with a shorter expansion, confirming that polyalanine expansion size plays a role in OPMD, with an effect on disease severity and progression.

Alternative polyadenylation (APA), which induces shortening of the 3′UTR, is emerging as an important phenomenon in gene regulation. APA is involved in development, cancer and cell proliferation. APA may lead to disruption of microRNA-mediated gene silencing in cancer cells via detachment of microRNA binding sites. We studied the correlation between the APA profile and the tumor aggressiveness in cases of lung cancer. We selected the top 10 genes showing significant 3′UTR shortening in lung cancer, using the package of the Bioconductor for probe-level analyses of expression microarrays. We established and evaluated the APA score by quantitative RT-PCR in 147 clinical specimens of non-small cell lung cancer and compared the results with the clinical outcomes and expression levels of APA-related genes, including PABPN1, CPEB1, E2F1 and proliferation markers (MKI67, TOP2A and MCM2). High APA scores were correlated with an advanced tumor stage and a poor prognosis (P

The genetic basis of oculopharyngeal muscular dystrophy (OPMD) is a short expansion of a polyalanine tract (normal allele: 10 alanines, mutant allele: 11-17 alanines) in the nuclear polyadenylate binding protein PABPN1 which is essential for controlling poly(A) tail length in messenger RNA. Mutant PABPN1 forms nuclear inclusions in OPMD muscle. To investigate the pathogenic role of mutant PABPN1 in vivo, we generated a ligand-inducible transgenic mouse model by using the mifepristone-inducible gene expression system. Induction of ubiquitous expression of mutant PABPN1 resulted in skeletal and cardiac myopathy. Histological changes of degenerative myopathy were preceded by nuclear inclusions of insoluble PABPN1. Downregulation of mutant PABPN1 expression attenuated the myopathy and reduced the nuclear burden of insoluble PABPN1. These results support association between mutant PABPN1 accumulation and degenerative myopathy in mice. Resolution of myopathy in mice suggests that the disease process in OPMD patients may be treatable.

Oculopharyngeal muscular dystrophy is caused by small alanine expansions in polyadenylate binding protein nuclear 1 (PABPN1) protein resulting in its intranuclear accumulation in skeletal muscle. 3F5 llama antibody specifically interferes with the PABPN1 aggregation process in vitro and in vivo. To understand the structural basis for its epitope recognition we mapped the binding interface of 3F5 with PABPN1 and provide a structural model of the 3F5-PABPN1 complex. We show that 3F5 complementarity determining regions create a cavity in which PABPN1 alpha-helix domain resides by involving critical residues previously implicated in the aggregation process. These results may increase our understanding of the PABPN1 aggregation mechanism and the therapeutic potential of 3F5. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder caused by a (GCG)n trinucleotide repeat expansion in the poly(A) binding protein nuclear-1 (PABPN1) gene, which in turn leads to an expanded polyalanine tract in the protein. We generated transgenic mice expressing either the wild type or the expanded form of human PABPN1, and transgenic animals with the expanded form showed clear signs of abnormal limb clasping, muscle weakness, coordination deficits, and peripheral nerves alterations. Analysis of mitotic and postmitotic tissues in those transgenic animals revealed ubiquitinated PABPN1-positive intranuclear inclusions (INIs) in neuronal cells. This latter observation led us to test and confirm the presence of similar INIs in postmortem brain sections from an OPMD patient. Our results indicate that expanded PABPN1, presumably via the toxic effects of its polyalanine tract, can lead to inclusion formation and neurodegeneration in both the mouse and the human.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

Symptoms in late-onset neuromuscular disorders initiate only from midlife onwards and progress with age. These disorders are primarily determined by identified hereditable mutations, but their late-onset symptom manifestation is not fully understood. Here, we review recent research developments on the late-onset autosomal dominant oculopharyngeal muscular dystrophy (OPMD). OPMD is caused by an expansion mutation in the gene encoding for poly-adenylate RNA binding protein1 (PABPN1). The molecular pathogenesis for the disease is still poorly understood. Despite a ubiquitous expression of PABPN1, symptoms in OPMD are limited to skeletal muscles. We discuss recent studies showing that PABPN1 levels in skeletal muscles are lower compared with other tissues, and specifically in skeletal muscles, PABPN1 expression declines from midlife onwards. In OPMD, aggregation of expanded PABPN1 causes an additional decline in the level of the functional protein, which is associated with severe muscle weakness in OPMD. Reduced PABNPN1 expression in muscle cell culture causes myogenic defects, suggesting that PABPN1 loss-of-function causes muscle weakness in OPMD and in the elderly.Molecular signatures of OPMD muscles are similar to these of normal muscle aging, although expression trends progress faster in OPMD. We discuss a working hypothesis that aging-associated factors trigger late-onset symptoms in OPMD, and contribute to accelerated muscle weakness in OPMD. We focus on the pharyngeal and eyelid muscles, which are often affected in OPMD patients. We suggest that muscle weakness in OPMD is a paradigm for muscle aging.

BackgroundThe nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed proteinthat plays critical roles at multiple steps in post-transcriptional regulation ofgene expression. Short expansions of the polyalanine tract in the N-terminus ofPABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onsetdisease characterized by eyelid drooping, difficulty in swallowing, and weaknessin the proximal limb muscles. Why alanine-expanded PABPN1 leads to muscle-specificpathology is unknown. Given the general function of PABPN1 in RNA metabolism,intrinsic characteristics of skeletal muscle may make this tissue susceptible tothe effects of mutant PABPN1.MethodsTo begin to understand the muscle specificity of OPMD, we investigated thesteady-state levels of PABPN1 in different tissues of humans and mice.Additionally, we analyzed the levels of PABPN1 during muscle regeneration afterinjury in mice. Furthermore, we assessed the dynamics of PABPN1 mRNA decay inskeletal muscle compared to kidney.ResultsHere, we show that the steady-state levels of both PABPN1 mRNA and protein aredrastically lower in mouse and human skeletal muscle, particularly those impactedin OPMD, compared to other tissues. In contrast, PABPN1 levels are increasedduring muscle regeneration, suggesting a greater requirement for PABPN1 functionduring tissue repair. Further analysis indicates that modulation of PABPN1expression is likely due to post-transcriptional mechanisms acting at the level ofmRNA stability.ConclusionsOur results demonstrate that PABPN1 steady-state levels and likely control ofexpression differ significantly in skeletal muscle as compared to other tissues,which could have important implications for understanding the muscle-specificnature of OPMD.

Contains fulltext : 181501.pdf (Publisher’s version ) (Open Access) BACKGROUND: Muscle fibrosis characterizes degenerated muscles in muscular dystrophies and in late onset myopathies. Fibrotic muscles often exhibit thickening of the extracellular matrix (ECM). The molecular regulation of this process is not fully understood. In oculopharyngeal muscular dystrophy (OPMD), an expansion of an alanine tract at the N-terminus of poly(A)-binding protein nuclear 1 (PABPN1) causes muscle symptoms. OPMD patient muscle degeneration initiates after midlife, while at an earlier age carriers of alanine expansion mutant PABPN1 (expPABPN1) are clinically pre-symptomatic. OPMD is characterized by fibrosis in skeletal muscles but the causative molecular mechanisms are not fully understood. METHODS: We studied the molecular processes that are involved in OPMD pathology using cross-species mRNA expression profiles in muscles from patients and model systems. We identified significant dysregulation of the ECM functional group, among which the procollagen C-endopeptidase enhancer 1 gene (PCOLCE) was consistently down-regulated across species. We investigated PCOLCE subcellular localization in OPMD muscle samples and OPMD model systems to investigate any functional relevance of PCOLCE down-regulation in this disease. RESULTS: We found that muscle degeneration in OPMD is associated with PCOLCE down-regulation. In addition to its known presence at the ECM, we also found PCOLCE within the nucleus of muscle cells. PCOLCE sub-cellular localization changes during myoblast cell fusion and is disrupted in cells expressing mutant expPABPN1. Our results show that PCOLCE binds to soluble PABPN1 and co-localizes with aggregated PABPN1 with a preference for the mutant protein. In muscle biopsies from OPMD patients we find that extracellular PCOLCE is depleted with its concomitant enrichment within the nuclear compartment. CONCLUSIONS: PCOLCE regulates collagen processing at the ECM. Depletion of extracellular PCOLCE is associated with the expression of expPABPN1 in OPMD patient muscles. PCOLCE is also localized within the nucleus where it binds to PABPN1, suggesting that PCOLCE shuttles between the ECM and the nucleus. PCOLCE preferentially binds to expPABPN1. Nuclear-localized PCOLCE is enriched in muscle cells expressing expPABPN1. We suggest that nuclear entrapment of PCOLCE and its extracellular depletion represents a novel molecular mechanism in late-onset muscle fibrosis.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of specific muscles. OPMD is caused by extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Insoluble nuclear inclusions form in diseased muscles. We have generated a Drosophila model of OPMD that recapitulates the features of the disorder. Here, we show that the antiprion drugs 6-aminophenanthridine (6AP) and guanabenz acetate (GA), which prevent formation of amyloid fibers by prion proteins in cell models, alleviate OPMD phenotypes in Drosophila, including muscle degeneration and nuclear inclusion formation. The large ribosomal RNA and its activity in protein folding were recently identified as a specific cellular target of 6AP and GA. We show that deletions of the ribosomal DNA locus reduce OPMD phenotypes and act synergistically with sub-effective doses of 6AP. In a complementary approach, we demonstrate that ribosomal RNA accelerates in vitro fibril formation of PABPN1 N-terminal domain. These results reveal the conserved role of ribosomal RNA in different protein aggregation disorders and identify 6AP and GA as general anti-aggregation molecules.

Developmental pathways may be play a role in adult cell survival. However, whether they interact with longevity/cell survival pathways to confer protection against disease-associated proteotoxicity remains largely unknown. We previously reported that the inhibition of key longevity modulators such as the deacetylase sir-2.1 /SIRT1 (Sir2) and its target daf-16 /FoxO protects transgenics nematodes from muscle cell decline and abnormal motility produced by the expression of mutant (polyalanine-expanded) PABPN1, the oculopharyngeal muscular dystrophy (OPMD) protein. Here, we report that canonical Wnt signaling ( i ) modulates muscular pathology in mutant PABPN1 nematodes, and ( ii ) cooperates with the Sir2-FoxO longevity pathway to confer protection against mutant PABPN1 toxicity at the cellular and behavioral levels. Mutant PABPN1 toxicity was modified by genes along the canonical Wnt pathway, several of which depend on daf-16 for activity. s-catenin and pop-1 /TCF RNAi suppressed the protection from mutant PABPN1 confered by loss-of-function mutations in sir-2.1 and daf-16 . Moreover, the aggravation of muscle cell pathology by increased sir-2.1 dosage was reversed by s-catenin and pop-1 RNAi. The chemical inhibition of GSK-3s, a repressor of s-catenin activity, protected against mutant PABPN1 toxicity in a daf-16 -dependent manner, which is consistent with a cross-talk between s-catenin signaling and Sir2-FoxO signaling in protecting from mutant PABPN1 toxicity. Our data reveal that canonical Wnt signaling and Sir2-FoxO signaling interact to modulate diseased muscle survival, and indicate that GSK-3s inhibitors and sirtuin inhibitors both have therapeutic potential for muscle protection in OPMD.

Here we report the case of a 73-year-old Italian woman affected by genetically confirmed oculopharyngeal muscular dystrophy (OPMD) with a negative family history. As OPMD is usually transmitted as an autosomal-dominant meiotically stable trait, this case allows us to suggest that putative de novo OPMD mutations might occur more frequently than previously thought; moreover, when compatible with a proper clinical phenotype, OPMD might be included in the differential diagnosis even in the absence of a positive family history.

Oculopharyngeal muscular dystrophy (OPMD) is caused by expansion of a (GCN)10 to a (GCN)11–17 repeat coding for a polyalanine domain at the N-terminal part of poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by the presence of intranuclear inclusions (INIs) in skeletal muscle fibers of patients. The formation of GFP-b13AlaPABPN1 INIs and their fate through the cell cycle were followed by time-lapse imaging. Our observations demonstrated that the GFP-b13AlaPABPN1 INIs are dynamic structures that can disassemble during mitosis. However, their presence in cells occasionally led to apoptosis. The length of the polyalanine tail or the overexpression of PABPN1 did not significantly affect the percentage of soluble PABPN1 in vitro. Moreover, overexpression of either the wild type (wt) or mutant (mut) forms of PABPN1 slowed down the cell proliferation. The slowing down of proliferation together with the occasional occurrence of apoptosis could contribute in vivo to the late onset of this disease.