Over-expression of BCL2 rescues muscle weakness in a mouse model of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscular dystrophy caused by a polyalanine expansion mutation in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene. In unaffected individuals, (GCG)(6) encodes the first 6 alanines in a homopolymeric stretch of 10 alanines. In most patients, this (GCG)(6) repeat is expanded to (GCG)(8-13), leading to a stretch of 12-17 alanines in mutant PABPN1, which is thought to confer a toxic gain of function. Thus, OPMD has been modelled by expressing mutant PABPN1 transgenes in the presence of endogenous copies of the gene in cells and mice. In these models, increased apoptosis is seen, but it is unclear whether this process mediates OPMD. The role of apoptosis in the pathogenesis of different muscular dystrophies is unclear. Blocking apoptosis ameliorates muscle disease in some mouse models of muscular dystrophy such as laminin α-2-deficient mice, but not in others such as dystrophin-deficient (mdx) mice. Here we demonstrate that apoptosis is not only involved in the pathology of OPMD but also is a major contributor to the muscle weakness and dysfunction in this disease. Genetically blocking apoptosis by over-expressing BCL2 ameliorates muscle weakness in our mouse model of OPMD (A17 mice). The effect of BCL2 co-expression on muscle weakness is transient, since muscle weakness is apparent in mice expressing both A17 and BCL2 transgenes at late time points. Thus, while apoptosis is a major pathway that causes muscle weakness in OPMD, other cell death pathways may also contribute to the disease when apoptosis is inhibited.     

Prevention of oculopharyngeal muscular dystrophy-associated aggregation of nuclear polyA-binding protein with a single-domain intracellular antibody

Oculopharyngeal muscular dystrophy (OPMD) belongs to the group of protein aggregation disorders and is caused by extensions of the N-terminal polyalanine stretch of the nuclear polyA-binding protein 1 (PABPN1). The presence of PABPN1-containing intranuclear aggregates in skeletal muscle is unique for OPMD and is also observed in transgenic mouse and cell models for OPMD. These models consistently support a direct role for the protein aggregation in OPMD pathogenesis. We have isolated and characterized a diverse panel of single-domain antibody reagents (VHH), recognizing different epitopes in PABPN1. The antibody reagents specifically detect endogenous PABPN1 in cell lysates on western blot and label PABPN1 in cultured cells and muscle sections. When expressed intracellularly as intrabodies in a cellular model for OPMD, aggregation of PABPN1 was prevented in a dose-dependent manner. More importantly yet, these intrabodies could also reduce the presence of already existing aggregates. Given the domain specificity of VHH-mediated aggregation interference, this approach at least allows the definition of the nucleation kernel in aggregation-prone proteins, thus facilitating etiological insight into this and other protein aggregation disorders, and ultimately, it may well provide useful therapeutic agents.     

Wild-type PABPN1 is anti-apoptotic and reduces toxicity of the oculopharyngeal muscular dystrophy mutation

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, progressive disease caused by the abnormal expansion of a polyalanine tract-encoding (GCG)(n) trinucleotide repeat in the poly-(A) binding protein nuclear 1 (PABPN1) gene. OPMD is generally inherited as an autosomal dominant disorder and the polyalanine expansion mutation is thought to confer a toxic gain-of-function on mutant PABPN1 which forms aggregates within skeletal myocyte nuclei. Here we describe a novel beneficial function of wild-type PABPN1. Wild-type PABPN1 over-expression can reduce mutant PABPN1 toxicity in both cell and mouse models of OPMD. In addition, wild-type PABPN1 provides some protection to cells against pro-apoptotic insults distinct from the OPMD mutation such as staurosporine treatment and Bax expression. Conversely, PABPN1 knockdown (which itself is not toxic) makes cells more susceptible to apoptotic stimuli. The protective effect of wild-type PABPN1 is mediated by its regulation of X-linked inhibitor of apoptosis (XIAP) protein translation. This normal activity of PABPN1 is partially lost for mutant PABPN1; elevated levels of XIAP are seen in mice expressing a wild-type but not a mutant PABPN1 transgene. This raises the possibility that a compromise of the anti-apoptotic function of PABPN1 might contribute to the disease mechanism of OPMD.     

Trehalose reduces aggregate formation and delays pathology in a transgenic mouse model of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease that presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a polyalanine tract within the coding region of polyA binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. We have previously described a transgenic mouse model of OPMD that recapitulates the human disease and develops progressive muscle weakness accompanied by the formation of aggregates in skeletal muscle nuclei. The chemical chaperone trehalose has been used effectively to alleviate symptoms in a mouse model of Huntington's disease and is thought to elicit its effect by binding and stabilizing partially folded polyglutamine proteins and inhibiting the formation of aggregates. Here, we show that trehalose reduces aggregate formation and toxicity of mutant PABPN1 in cell models. Furthermore, oral administration of trehalose attenuated muscle weakness, reduced aggregate formation and decreased the number of TUNEL-labelled nuclei in skeletal muscle in an OPMD transgenic mouse model. Thus, anti-aggregation therapy may prove effective in the treatment of human OPMD.     

Modeling oculopharyngeal muscular dystrophy in myotube cultures reveals reduced accumulation of soluble mutant PABPN1 protein

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.     

Oligomerization of polyalanine expanded PABPN1 facilitates nuclear protein aggregation that is associated with cell death.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)(6) repeat to (GCG)(8-13) in the PABPN1 gene, which results in the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of the protein. Mutated PABPN1 (mPABPN1) is able to induce nuclear protein aggregation and form filamentous nuclear inclusions, which are the pathological hallmarks of OPMD. PABPN1, when bound to poly(A) RNA, forms both linear filaments and discrete-sized, compact oligomeric particles in vitro. In the absence of poly(A) RNA, PABPN1 can form oligomers. Here we report that: (i) oligomerization of PABPN1 is mediated by two potential oligomerization domains (ODs); (ii) inactivating oligomerization of mPABPN1 by deletions of 6-8 amino acids in either of the ODs prevents nuclear protein aggregation; (iii) expression of mPABPN1 in COS-7 cells is associated with cell death; and (iv) preventing nuclear protein aggregation by inactivating oligomerization of mPABPN1 significantly reduces cell death. These findings suggest that oligomerization of PABPN1 plays a crucial role in the formation of OPMD nuclear protein aggregation, while the expanded polyalanine stretch is necessary but not sufficient to induce OPMD protein aggregation, and that the nuclear protein aggregation might be toxic and cause cell death. These observations also imply that inactivation of oligomerization of mPABPN1 might be a useful therapeutic strategy for OPMD.     

Oculopharyngeal muscular dystrophy-like nuclear inclusions are present in normal magnocellular neurosecretory neurons of the hypothalamus

Intranuclear inclusions composed of tubular filaments constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD). Autosomal dominant OPMD is caused by (GCG) repeat expansions in the gene that encodes for poly(A) binding protein nuclear 1 (PABPN1). The mutation results in the expansion of a polyalanine stretch in the N-terminus of the protein. It has been proposed that mutated PABPN1 induces protein aggregation, which in turn causes the formation of the filamentous nuclear inclusions. Here we report the presence of intranuclear inclusions composed of tubular filaments in oxytocin-producing neurons from normal rat hypothalamus. Like OPMD inclusions, the filamentous structures in neurosecretory neurons accumulate PABPN1, poly(A) RNA, ubiquitin and proteasomes. These inclusions do not contain members of Hsp40 and HDJ-2/DNAJ families of chaperones. The proportion of oxytocin-producing neurons that contain inclusions decreases during parturition and lactation (when synthesis and release of oxytocin is maximal) and increases at 1 day post-weaning (when occurs a drastic reduction in the production of the hormone). Thus, PABPN1 filaments in normal neurons are dynamic structures, the appearance of which correlate with changes in cellular activity. These data provide the first physiological evidence that polyalanine expansions are not essential to induce polymerization of PABPN1 into filamentous nuclear inclusions.     

Oculopharyngeal muscular dystrophy (OPMD) due to a small duplication in the PABPN1 gene

Oculopharyngeal muscular dystrophy (OPMD) is a late onset autosomal dominant muscle disorder. The OPMD-locus has been mapped to chromosome 14q11.2-q13. The polyadenylate binding protein nuclear 1 (PABPN1; PABP2) gene has been identified as the mutated gene. The mutation consists of a short meiotically stable trinucleotide repeat in the first exon of PABPN1 gene. We have investigated Dutch OPMD patients from four unrelated families and identified a new mutation in two of the four families. Instead of a repeat expansion we found a duplication in the first exon of the PABPN1 gene (c.27_28ins12, p.11_12insAAAA). The identification of this new mutation supports the theory of unequal crossing-over as molecular mechanism causing the mutation in the PABPN1 gene responsible for OPMD, and not the slippage model.     

Induction of HSP70 expression and recruitment of HSC70 and HSP70 in the nucleus reduce aggregation of a polyalanine expansion mutant of PABPN1 in HeLa cells

Nuclear inclusions formed by the aggregation of a polyalanine expansion mutant of the nuclear poly(A)-binding protein (PABPN1) is a hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a dominant autosomal disease in which patients exhibit progressive difficulty of swallowing and eyelid elevation, starting around the age of 50. At present, there is no specific treatment to reduce the aggregate burden in patients. However, in cell culture models of OPMD, reduction of protein aggregation can be achieved by ectopic expression of HSP70. As gene transfer may not be the most effective means to elevate HSP70 levels, we tested four pharmacological agents for their ability to induce HSP70, recruit both HSP70 and HSC70 into the cell nucleus and reduce mutant PABPN1 aggregation in a HeLa cell culture model. We show here that exposure to moderate levels of ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin produced a robust stress response resulting in the induction of HSP70 in HeLa cells expressing the mutant PABPN1 as a green fluorescent protein (GFP) fusion protein. Both HSP70 and the constitutive chaperone HSC70 localized in the nucleus of cells treated with any one of the four agents. This stress response was similar to what was observed following hyperthermia. All four agents also caused a significant reduction in the cellular burden of protein aggregates, as was judged by confocal microscopy and solubility changes of the aggregates. A concomitant reduction of cell death in drug-treated mutant PABPN1 expressing cells was also observed.     

A rheumatoid factor transgenic mouse model of autoantibody regulation

To address whether B cells expressing a disease-associated autospeclficityare regulated In normal mice, we have established a rheumatoidfactor (RF) transgenic model of autoimmunity, using V genesderived from an IgA anti-lgG2a RF isolated from an autoimmuneMRU//pr mouse. As we wished to study Induction of toleranceduring B cell development, we cloned the V_H gene Into an IgMexpression vector. The RF we chose binds only lgG2a of the ‘a’allotype (lgG2a) but not lgG2a^b allowing us to produce transgenicanimals on IgH^a and lgH^b backgrounds, which either express orlack the self-antigen. Two transgenic lines were studied. Usingmice which lack the self-antigen, we show by fluorescence activatedcell sorting and hybrldoma analysis that the H and L transgenesare expressed to the exclusion of endogenous genes in most splenicB cells.In spite of good allellc exclusion, transgenic micewhich are genetically capable of expressing lgG2a' have reducedbut significant ( {small tilde}50 _µ/tg/ml) serum levels.Nonetheless, the frequency and numbers of transgene-expressingB cells In peripheral lymphold organs of such mice which havethe self-antigen are similar to those which lack It (lgH^b mice).Thus, B cells expressing an antiself lgG2a surface receptorcan develop In this system. Whether such B cells are anerglcor otherwise regulated in autoantlgen-expressing mice Is discussed.     

Expression of human CD81 in transgenic mice does not confer susceptibility to hepatitis C virus infection

We previously demonstrated that hepatitis C virus (HCV) binds to human CD81 through the E2 glycoprotein. Therefore, expression of the human CD81 molecule in transgenic mice was expected to provide a new tool to study HCV infection in vivo, as the chimpanzee is the only species currently available as a laboratory animal model that can be infected with HCV. We produced transgenic mice expressing the human CD81 protein in a wide variety of tissues. We confirmed binding of recombinant E2 glycoprotein to the liver tissue as well as to thymocytes and splenic lymphocytes in the transgenic mice. We inoculated chimpanzee plasma infected with HCV into these animals. None of these transgenic animals showed evidence of viral replication. Furthermore, human CD81 transgenic mice that lack expression of endogenous mouse CD81 were also resistant to HCV infection. We conclude that expression of human CD81 alone is insufficient to confer susceptibility to HCV infection in the mouse. The presence of additional possible factors for HCV infection is discussed.     

高复制HBV转基因小鼠模型对抗乙型肝炎病毒药物的效应研究

 目的：研究高复制HBV转基因小鼠模型对抗乙肝病毒药物的效应评价。 方法： 选用抗乙肝药物拉咪呋啶、大剂量重组乙肝蛋白疫苗、α-1b型干扰素和RNA干扰在转基因小鼠进行药效及作用机制评价。 结果：拉咪呋啶、重组乙肝疫苗、α-1b型干扰素均可使HBV转基因小鼠血清中HBV DNA滴度显著降低。其中后两者还可提高机体脾细胞IL-2和IFN-γ的水平及使分泌IFN-γ脾细胞Elispot斑点数明显增加。将RNA干扰表达载体pU6-siHBV质粒尾静脉注入小鼠体内。注射后5 d血清HBsAg下降56.7%,抑制作用持续14 d。肝脏免疫组化显示HBcAg阳性细胞明显减少，但血清HBV DNA定量无明显降低。 结论： 本近交系高复制HBV转基因小鼠模型对抗乙肝药物药效学评估是可靠、可行的。     

Development of VH81X transgene-bearing B cells in fetus and adult: sites for expansion and deletion in conventional and CD5/B1 cells

The most D-proximal functional VH gene, VH81X, is preferentially expressed in the mouse fetal B cell repertoire; however, it is expressed in few B cells in the adult. To determine when VH81X gene expression affects size and phenotype of particular stages in B cell differentiation, transgenic mice have been developed expressing a germline fetal liver-derived VH81X-mu rearrangement. Comparative analysis of B lymphopoiesis reveals similarities and differences between fetal liver and adult bone marrow which pinpoint developmental stages in mice during which VH81X-expressing B cell progenitors expand or deplete compartment sizes. These include a similar reduction in c- kitR+ and establishment of a predominant CD43low/HSAhigh phenotype within the B220+ CD43+ compartment which is dependent on the association of the transgene with lambda 5. In contrast, the CD43- pre- B and immature B cell compartments are expanded in the fetus but not in the adult. In addition, there are other factors that later disfavor the survival of VH81X-expressing B1 and B2 cells. Thus the failure to detect VH81X-bearing B cells in the adult is the result of a multistep selection process occurring at all stages during B repertoire expansion.      

Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy

Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.     

Transgenic Mice Over-Expressing the C-99 Fragment of βPP with an α-Secretase Site Mutation Develop a Myopathy Similar to Human Inclusion Body Myositis

Inclusion body myositis (IBM) is the most common muscle disease in the elderly. Amyloid-β protein (Aβ) has been shown to accumulate abnormally in the vacuolated fibers and to localize to amyloid-like fibrils in muscles from IBM patients. We studied the skeletal muscles from a line of transgenic mice over-expressing the carboxyl-terminal 99 amino acids (C99) of the β-amyloid precursor protein (βPP) with a substitution of lysine-612 to valine (K612V), intended to abolish α-secretase recognition and to preserve the Aβ domain of C99. The majority (87%) of the 24-month-old transgenic mice showed myopathic changes, and approximately one-third of them had degenerating fibers with sarcoplasmic vacuoles and thioflavin-S-positive deposits. Ultrastructurally, the inclusions were aggregates of short thin amyloid-like fibrils, 6 to 8 nm in diameter. These features are similar to those of human IBM. Immunocytochemistry using an antibody against Aβ showed membranous staining in most muscle fibers of transgenic mice, as well as granular or vacuolar cytoplasmic staining in the atrophic fibers. Western blots showed a high level of accumulation of carboxyl-terminal fragments of βPP in the muscles of the transgenic mice with the most severe IBM-like lesions. The expression of IBM-like lesions was age dependent. These transgenic mice provide a model for the study of IBM and for the peripheral expression of a key element in the pathogenesis of Alzheimer disease.     

Alzheimer-associated APP+1 transgenic mice: frameshift beta-amyloid precursor protein is secreted in cerebrospinal fluid without inducing neuropathology

Biomarkers present in the cerebrospinal fluid (CSF) of Alzheimer Disease patients could be instrumental in guiding diagnosis and monitoring of progression of the disease. We have previously reported on the secretion of a frameshifted form of amyloid-beta precursor protein, APP+1, into the CSF of Alzheimer patients and controls. APP+1 is secreted efficiently in controls, but during the progression of Alzheimer Disease, its secretion is reduced and APP+1 accumulates in tangle-bearing neurons. Here we describe the generation of a transgenic mouse line expressing APP+1 in the brain. These mice do not suffer from overt pathology or neurodegeneration, suggesting that APP+1 is not neurotoxic. To measure APP+1 levels in the CSF, we serially sampled CSF from the cisterna magna in the same mouse over a period of months. Indeed, APP+1 is secreted into the CSF of the transgenic mice, and APP+1 levels are stable over 1 year. This mouse model may guide the study of secretion deficits as found in Alzheimer Disease.     

Evidence that intestinal IgA plasma cells in {micro},{varkappa} transgenic mice are derived from B-1 (Ly-1 B) cells

B6-Sp6 transgenic mice carry fully rearranged (BALB/c-derived,Igh-C^a allotype) µ. heavy chain and light chain transgenes,specific for trinitrophenyl, on a C57BL background (Igh-C^b allotype).FACS analyses show that the majority of B cells in peripherallymphoid organs and bone marrow(BM) express transgenic IgM exclusively.A small proportion of the B cells, however, express endogenousIgM, usually concomitant with transgenic IgM. Three criteriaestablish that the endogenous IgM expressing B cells belongto the B-1 cell lineage. (I) Endogenous IgM expressing B cellsin B6-Sp6 mice have the same localization pattern as B-1 cellsfrom normal animals: they are enriched in the peritoneal cavity.(II) The endogenous IgM⁺ B cells have the phenotype of B-1 cells:the endogenous IgM⁺ peritoneal B cells express Mac-1 (CD11b)and low levels of IgD, and most also express CD5 (L-1). (III)B6-Sp6 BM poorly reconstitutes endogenous IgM⁺ B cells, justas adult BM from normal mice poorly reconstitutes B-1 cells.In contrast, B cells which only express the transgene are readilyreconstituted by B6-Sp6 BM. The few endogenous IgM⁺ cells inthe B6-Sp6 BM recipients are located in the peritoneal cavityand have the phenotype of B-1b cells (previously the Ly-1 Bsister population), which are known to be reconstituted by adultBM.Two-color immunofluorescence staining of tissue sectionsfrom the gut and from isolated gut lamina propria cells showsthe presence of many IgA containing cells, about one-third ofwhich simultaneously express cytoplasmic (transgenic) IgM. TheC-region of this IgA is produced by endogenous C a genes, becausethe transgene encodes only for C_µ. Furthermore, the majorityof gut IgA containing cells do not express the Idiotype of thetransgene, indicating that most of the gut IgA cells are encodedby endogenous V_H genes and thus the result of an isotype switchfrom endogenous IgM expressing B cells. Since the endogenousIgM⁺ cells are B-1 cells (both B-1a and B-1b), the data stronglyindicate that the intestinal IgA plasma cells also belong tothe B-1 cell lineage.