Alternative splicing of myomesin 1 gene is aberrantly regulated in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a multisystemic disease caused by a CTG repeat expansion in the 3'-UTR of dystrophia myotonica-protein kinase. Aberrant regulation of alternative splicing is a characteristic feature of DM. Dozens of genes have been found to be abnormally spliced; however, few reported splicing abnormalities explain the phenotypes of DM1 patients. Thus, we hypothesized that other, unknown abnormal splicing events exist. Here, by using exon array, we identified aberrant inclusion of myomesin 1 (MYOM1) exon 17a as a novel splicing abnormality in DM1 muscle. A cellular splicing assay with a MYOM1 minigene revealed that not only MBNL1-3 but also CELF1 and 2 decreased the inclusion of MYOM1 exon 17a in HEK293T cells. Expression of expanded CUG repeat impeded MBNL1 activity but did not affect CELF1 activity on the splicing of MYOM1 minigene. Our results suggest that the downregulation of MBNL proteins should lead to the abnormal splicing of MYOM1 exon 17a in DM1 muscle.

Production of anti-amyloid ß antibodies in mice fed rice expressing amyloid ß.

The main signs of Alzheimer's disease (AD) are cognitive impairment and senile plaques composed of amyloid beta (Aß) observed in patients' brains. Therefore, therapy for AD focuses on the removal of Aß. We developed an "edible vaccine" that employs intestinal immunity with little to no side effects. Rice was utilized as an edible vaccine. It expressed GFP-Aß42. Aß rice was administered orally to wild-type (WT) mice causing production of anti-Aß antibodies. Since Aß rice was mixed with the cholera toxin B subunit (CTB), antibody against the rice seed protein was also produced. Then, mice were caused to develop immune tolerance against the rice seed protein by oral administration of Aß rice mixed with CTB. The results indicated that only anti-Aß antibodies were produced.

MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1.

The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families--muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins--are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5' end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.

ADAM19 autolysis is activated by LPS and promotes non-classical secretion of cysteine-rich protein 2.

ADAM family proteins are type I transmembrane, zinc-dependent metalloproteases. This family has multiple conserved domains, including a signal peptide, a pro-domain, a metalloprotease domain, a disintegrin (DI) domain, a cysteine-rich (Cys) domain, an EGF-like domain, a transmembrane domain, and a cytoplasmic domain. The Cys and DI domains may play active roles in regulating proteolytic activity or substrate specificity. ADAM19 has an autolytic processing activity within its Cys domain, and the processing is necessary for its proteolytic activity. To identify a new physiological function of ADAM19, we screened for associating proteins by using the extracellular domain of ADAM19 in a yeast two-hybrid system. Cysteine-rich protein 2 (CRIP2) showed an association with ADAM19 through its DI and Cys domains. Sequence analysis revealed that CRIP2 is a secretable protein without a classical signal. CRIP2 secretion was increased by overexpression of ADAM19 and decreased by suppression of ADAM19 expression. Moreover, CRIP2 secretion increased in parallel with the autolytic processing of ADAM19 stimulated by lipopolysaccharide. These findings suggest that ADAM19 autolysis is activated by lipopolysaccharide and that ADAM19 promotes the secretion of CRIP2.

Reduction of amyloid beta-peptide accumulation in Tg2576 transgenic mice by oral vaccination.

Alzheimer's disease (AD) is pathologically characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles. Amyloid beta-peptide (Abeta) is the main component of senile plaques, and the pathological load of Abeta in the brain has been shown to be a marker of the severity of AD. Abeta is produced from the amyloid precursor protein by membrane proteases and is known to aggregate. Recently, immune-mediated cerebral clearance of Abeta has been studied extensively as potential therapeutic strategy. In previous studies that used a purified Abeta challenge in a mouse model of AD, symptomatic improvement was reported. However, a clinical Alzheimer's vaccine trial in the United States was stopped because of severe side effects. Immunization with the strong adjuvant used in these trials might have activated an inflammatory Th1 response. In this study, to establish a novel, safer, lower-cost therapy for AD, we tested an oral vaccination in a wild-type and a transgenic mouse model of AD administered via green pepper leaves expressing GFP-Abeta. Anti-Abeta antibodies were effectively induced after oral immunization. We examined the immunological effects in detail and identified no inflammatory reactions. Furthermore, we demonstrated a reduction of Abeta in the immunized AD-model mice. These results suggest this edible vehicle for Abeta vaccination has a potential clinical application in the treatment of AD.

Polyalanine tracts directly induce the release of cytochrome c, independently of the mitochondrial permeability transition pore, leading to apoptosis.

In recent years, several novel types of disorder caused by the expansion of triplet repeats in specific genes have been characterized; in the "polyalanine diseases", these expanded repeats result in proteins with aberrantly elongated polyalanine tracts. In this study, we fused expanded polyalanine tracts to yellow fluorescent protein to examine their physical interaction with mitochondria. Tracts containing more than 23 alanine repeats were found to physically associate with mitochondria, strongly suggesting that an interaction between polyalanine tracts and mitochondria is a contributing factor in the pathology of polyalanine diseases. Furthermore, in in vitro experiments, polyalanine tracts induced release of cytochrome c from mitochondria and caspase-3 activation, independently of the mitochondrial permeability transition pore. These results suggest that oligomerized polyalanine tracts might induce the rupture of the mitochondrial membrane, the subsequent release of cytochrome c, and apoptosis. This novel mechanism for polyalanine tract cytotoxicity might be common to the pathogenesis of all polyalanine diseases. Further investigation of this mechanism might aid the development of therapies for these diseases.

Identification of Caenorhabditis elegans K02H8.1 (CeMBL), a functional ortholog of mammalian MBNL proteins.

The genome of the nematode Caenorhabditis elegans possesses an orthologous sequence to the Drosophila muscleblind (mbl) and mammalian muscleblind-like genes (MBNLs). This ortholog, K02H8.1, which has a high degree of homology (about 50%) to human MBNLs, encodes two zinc finger domains, as does the sequence of the Drosophila mbl gene. This distinguishes it from human MBNLs, which encode four zinc finger domains. In this study, we cloned six major isoforms of K02H8.1 using cDNA generated from C. elegans total RNA. All six of the cloned isoforms had an SL1 leader sequence at the 5'-position. Interestingly, one of the isoforms lacked a zinc finger domain-encoding sequence. To understand better the function of K02H8.1, we performed yeast three-hybrid experiments to characterize the binding of K02H8.1 to bait RNAs. K02H8.1 exhibited strong binding affinity for CUG and CCUG repeats, and the binding affinity was very similar to that of MBNLs. In addition, promoter analysis was performed using promoter-green fluorescent protein (GFP) fusion constructs. The expression of GFP driven by the K02H8.1 promoter was absent in muscle; however, significant GFP expression was detected in the neurons around the pharynx.

Inhibition by KMI-574 leads to dislocalization of BACE1 from lipid rafts.

BACE1 initiates processing of the amyloid precursor protein (APP) in the production of amyloid beta (Abeta) peptide. After beta-cleavage by BACE1, the C-terminal stub of the APP fragment is further processed by the gamma-secretase complex to produce Abeta. Because APP, Abeta, the gamma-secretase complex, and BACE1 are found in lipid raft membranes, Abeta production is widely accepted to occur in lipid rafts. However, whether BACE1 is activated within the rafts is unclear. To analyze the relationship between the activity and the localization of BACE1, we used a new BACE1 inhibitor, KMI-574, and separated raft membranes on sucrose density gradients. In the presence of KMI-574, the localization of BACE1 shifted from the rafts to nonraft membranes in HEK293 cells. We also analyzed the proteolytically inactive mutants, D93A, D289A, and D93A/D289A, of BACE1. These mutants also moved from rafts to nonrafts, and the D93A/D289A double-mutant localized exclusively to nonraft membranes. The mutants were defective in maturation by glynosylation and formed hyperoligomers, suggesting that the BACE1 oligomers could not exit from the ER and be transported to the Golgi apparatus. Our findings suggest that the activated conformation of BACE1 is important for protein transport and localization to lipid rafts.

Biochemical analysis of oligomerization of expanded polyalanine repeat proteins.

Many human proteins contain amino acid repeats that can form homopolymeric amino acid (HPAA) tracts. HPAA tract proteins that contain polyalanine sequences promote diseases, including oculopharyngeal muscular dystrophy. The pathological properties of these proteins develop when the repeats match or exceed approximately 20 residues. We analyzed the oligomerization of yellow fluorescent protein (YFP) and GST fusion proteins containing >20 alanine repeats by using sucrose density gradient centrifugation. YFP and GST fusion proteins having 23 polyalanine residues sedimented readily in sucrose density gradients, suggesting instability and oligomerization of proteins with an excess of 20 alanine repeats. Moreover, GST fusion proteins were resistant to trypsin digestion after oligomerization. Oligomerized artificial proteins with long polyalanine repeats may be suitable models for studying polyalanine-related diseases.

1,3-Capryloyl-2-arachidonoyl glycerol activates alpha-secretase activity and suppresses Abeta40 secretion in A172 cells.

Alzheimer's disease (AD) is characterized by the deposition of amyloid beta-peptide (Abeta), derived from amyloid precursor protein (APP). Membrane states, such as lipid components or membrane fluidity, are important for enzymes related to APP processing in meeting their substrates efficiently. We analyzed the effects of triglycerides combined with polyunsaturated fatty acids (PUFAs) and/or caprylic acids on APP proteolysis. Our results showed that 1,3-capryloyl-2-arachidonoyl glycerol (8A8) moderately increased alpha-secretase activity (18%) in A172 cells. beta-Secretase activity was not statistically significantly changed in HEK293 cells stably expressing BACE1. However, Abeta40 secretion decreased by 22%. Thus, we conclude that 8A8 is a useful lipid compound for activating alpha-secretase activity and suppressing Abeta40 secretion.

Csk-homologous kinase interacts with SHPS-1 and enhances neurite outgrowth of PC12 cells.

SHPS-1 is an immunoglobulin superfamily protein with four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic region. Various neurotrophic factors induce the tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with the protein tyrosine phosphatase SHP-2. Using a yeast two-hybrid screen, we identified a protein tyrosine kinase, Csk-homologous kinase (CHK), as an SHPS-1-interacting protein. Immunoprecipitation and pull-down assays using glutathione S-transferase (GST) fusion proteins containing the Src homology 2 (SH2) domain of CHK revealed that CHK associates with tyrosine-phosphorylated SHPS-1 via its SH2 domain. HIS3 assay in a yeast two-hybrid system using the tyrosine-to-phenylalanine mutants of SHPS-1 indicated that the first and second ITIMs of SHPS-1 are required to bind CHK. Over-expression of wild-type CHK, but not a kinase-inactive CHK mutant, enhanced the phosphorylation of SHPS-1 and its subsequent association with SHP-2. CHK phosphorylated each of four tyrosines in the cytoplasmic region of SHPS-1 in vitro. Co-expression of SHPS-1 and CHK enhanced neurite outgrowth in PC12 cells. Thus, CHK phosphorylates and associates with SHPS-1 and is involved in neural differentiation via SHP-2 activation.

Expression of polyalanine stretches induces mitochondrial dysfunction.

In recent years, several novel types of disorders have been characterized, including what have been termed polyalanine diseases, in which patients have expanded triplet repeats in specific genes, resulting in the translation of aberrantly elongated polyalanine stretches. In this study, we showed that yellow fluorescent protein (YFP)-fused elongated polyalanine stretches localized exclusively to the cytoplasm and formed aggregates. Additionally, the polyalanine stretches themselves were toxic. We sought to identify proteins that bound directly to the polyalanine stretches, as factors that might be involved in triggering cell death. Many mitochondrial proteins were identified as polyalanine-binding proteins. We showed that one of the identified proteins, succinate dehydrogenase subunit A, was decreased in the mitochondria of cells expressing polyalanine stretches; as a result, succinate oxidative activity was decreased. Furthermore, the polyalanine stretches also associated directly with mitochondria. This suggests that polya-lanine stretches might directly induce cell death. Additionally, the mitochondrial membrane potential was reduced in cells expressing polyalanine stretches. We propose a novel mechanism by which polyalanine stretches may cause cytotoxicity through mitochondrial dysfunction. This may be a common mechanism underlying the pathogenesis of all polyalanine diseases.

MBNL1 associates with YB-1 in cytoplasmic stress granules.

The muscleblind-like (MBNL) protein family is thought to be involved in the molecular mechanism of myotonic dystrophy (DM). Although it has been shown to have splicing activity, a broader function in cellular RNA metabolism has been implicated. In this study, we attempted to find the binding proteins of MBNL1 in order to elucidate its physiological function. First, we performed a GST pull-down assay using GST-MBNL1-6xHis as bait. Several proteins were identified, including YB-1, a multifunctional DNA/RNA-binding protein, and DDX1, a DEAD box RNA helicase. MBNL1 formed an RNP complex with YB-1 and DDX1 in binding assays. YB-1 also showed a weak but significant effect on alpha-actinin splice site selection. Interestingly, in response to stress, MBNL1 moved to cytoplasmic stress granules, where it colocalized with YB-1, which was previously reported to be a component of stress granules. We found that DDX1 also colocalized with MBNL1 at stress granules. These results provide new insight into the dynamics of MBNL1 in response to stress, and they suggest a role for MBNL1 in mRNA metabolism in the cytoplasm.

Quantitative analysis of CUG-BP1 binding to RNA repeats.

CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-3-like factors (CELF) family of RNA-binding proteins, and is involved in myotonic dystrophy type 1 (DM1). Several mRNA targets of CUG-BP1 have been identified, including the insulin receptor, muscle chloride channel, and cardiac troponin T. On the other hand, CUG-BP1 has only a weak affinity for CUG repeats. We conducted quantitative-binding assays to assess CUG-BP1 affinities for several repeat RNAs by surface plasmon resonance (SPR). Although we detected interactions between CUG-BP1 and CUG repeats, other UG-rich sequences actually showed stronger interactions. Binding constants of CUG-BP1 for RNAs indicated that the affinity for UG repeats was far stronger than for CUG repeats. We also found that N-terminal deletion mutant of CUG-BP1 has UG repeat-binding activity in a yeast three-hybrid system, although C-terminal deletion mutant does not. Our data indicates that CUG-BP1 specifically recognized UG repeats, probably through cooperative binding of RNA recognition motifs at both ends of the protein. This is the first report of a binding constant for CUG-BP1 calculated in vitro.

Methylmercury activates ASK1/JNK signaling pathways, leading to apoptosis due to both mitochondria- and endoplasmic reticulum (ER)-generated processes in myogenic cell lines.

Cellular stress responses following exposure to methylmercury (MeHg) were investigated using myogenic cell lines that showed different susceptibilities to MeHg. The susceptible cell line showed apoptosis within 24h after exposure to low levels of MeHg. The activation of caspase 12, 9, and 3 was detected in the apoptotic cells at 14-16 h after MeHg exposure, suggesting that MeHg causes apoptosis via both mitochondria- and endoplasmic reticulum (ER)-generated processes. An early increase in the level of intracellular reactive oxygen species (ROS) was quantitatively recognized since 2-3h after exposure to MeHg in both MeHg-susceptible and non-susceptible cell lines; however, the increase was lower in the latter cell line. The phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) was also recognized in both cell lines, with the increase in intracellular ROS. However, the activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathways was observed only in the MeHg-susceptible cell line. In contrast, the non-susceptible cell line exhibited activation of the cell survival ERK pathway. Up-regulation of metallothioneine I and Hic-5 mRNAs encoding proteins induced by oxidative stress was recognized during the early stage of MeHg cytotoxicity in the MeHg-susceptible cell line. Quantitative real-time PCR and western blot analyses confirmed that ER stress is a late event during MeHg cytotoxicity. Coaddition of the antioxidant Trolox dramatically suppressed the increase in the level of ROS, activation of caspases and, finally, apoptosis. However, later treatment with Trolox attenuated its protective effect against MeHg cytotoxicity. The results indicate that failure to protect cells against the early oxidative stress triggers ER stress and apoptosis processes. Combined treatment with protective factors against oxidative and ER stresses is necessary, especially in the later stages of MeHg cytotoxicity.

A freeze-and-thaw method to reuse agarose gels for DNA electrophoresis.

A novel protocol to reuse agarose following agarose gel electrophoresis was established in this study. By repeated freeze-and-thaw of the agarose gel, ethidium bromide and other buffer components in the gel were safely removed from the gel without generation of any toxic fume. The agarose recovered using this method can be used for further electrophoretic experiments without any issues.

Interactions between homopolymeric amino acids (HPAAs).

Many human proteins contain consecutive amino acid repeats, known as homopolymeric amino acid (HPAA) tracts. Some inherited diseases are caused by proteins in which HPAAs are expanded to an excessive length. To this day, nine polyglutamine-related diseases and nine polyalanine-related diseases have been reported, including Huntington's disease and oculopharyngeal muscular dystrophy. In this study, potential HPAA-HPAA interactions were examined by yeast two-hybrid assays using HPAAs of approximately 30 residues in length. The results indicate that hydrophobic HPAAs interact with themselves and with other hydrophobic HPAAs. Previously, we reported that hydrophobic HPAAs formed large aggregates in COS-7 cells. Here, those HPAAs were shown to have significant interactions with each other, suggesting that hydrophobicity plays an important role in aggregation. Among the observed HPAA-HPAA interactions, the Ala28-Ala29 interaction was notable because polyalanine tracts of these lengths have been established to be pathogenic in several polyalanine-related diseases. By testing several constructs of different lengths, we clarified that polyalanine self-interacts at longer lengths (>23 residues) but not at shorter lengths (six to approximately 23 residues) in a yeast two-hybrid assay and a GST pulldown assay. This self-interaction was found to be SDS sensitive in SDS-PAGE and native-PAGE assays. Moreover, the intracellular localization of these long polyalanine tracts was also observed to be disturbed. Our results suggest that long tracts of polyalanine acquire SDS-sensitive self-association properties, which may be a prerequisite event for their abnormal folding. The misfolding of these tracts is thought to be a common molecular aspect underlying the pathogenesis of polyalanine-related diseases.

Endoplasmic reticulum stress caused by aggregate-prone proteins containing homopolymeric amino acids.

Many human proteins have homopolymeric amino acid (HPAA) tracts, but their physiological functions or cellular effects are not well understood. Previously, we expressed 20 HPAAs in mammalian cells and showed characteristic intracellular localization, in that hydrophobic HPAAs aggregated strongly and caused high cytotoxicity in proportion to their hydrophobicity. In the present study, we investigated the cytotoxicity of these aggregate-prone hydrophobic HPAAs, assuming that the ubiquitin proteasome system is impaired in the same manner as other well-known aggregate-prone polyglutamine-containing proteins. Some highly hydrophobic HPAAs caused a deficiency in the ubiquitin proteasome system and excess endoplasmic reticulum stress, leading to apoptosis. These results indicate that the property of causing excess endoplasmic reticulum stress by proteasome impairment may contribute to the strong cytotoxicity of highly hydrophobic HPAAs, and proteasome impairment and the resulting excess endoplasmic reticulum stress is not a common cytotoxic effect of aggregate-prone proteins such as polyglutamine.

Identification of an estrogenic hormone receptor in Caenorhabditis elegans.

Changes in both behavior and gene expression occur in Caenorhabditis elegans following exposure to sex hormones such as estrogen and progesterone, and to bisphenol A (BPA), an estrogenic endocrine-disrupting compound. However, only one steroid hormone receptor has been identified. Of the 284 known nuclear hormone receptors (NHRs) in C. elegans, we selected nhr-14, nhr-69, and nhr-121 for analysis as potential estrogenic hormone receptors, because they share sequence similarity with the human estrogen receptor. First, the genes were cloned and expressed in Escherichia coli, and then the affinity of each protein for estrogen was determined using a surface plasmon resonance (SPR) biosensor. All three NHRs bound estrogen in a dose-dependent fashion. To evaluate the specificity of the binding, we performed a solution competition assay using an SPR biosensor. According to our results, only NHR-14 was able to interact with estrogen. Therefore, we next examined whether nhr-14 regulates estrogen signaling in vivo. To investigate whether these interactions actually control the response of C. elegans to hormones, we investigated the expression of vitellogenin, an estrogen responsive gene, in an nhr-14 mutant. Semi-quantitative RT-PCR showed that vitellogenin expression was significantly reduced in the mutant. This suggests that NHR-14 is a C. elegans estrogenic hormone receptor and that it controls gene expression in response to estrogen.

Expression of MBNL and CELF mRNA transcripts in muscles with myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder that causes muscle wasting, myotonia, cardiac conduction abnormalities, and other multi-systemic symptoms. Current evidence supports a pathogenic mechanism involving aberrantly expanded CTG repeats in the 3'-untranslated region of the DM protein kinase (DMPK) gene. The repeats are thought to recruit various RNA-binding proteins such as muscleblind-like (MBNL) proteins into foci in the nuclei of DM cells, resulting in loss of function. However, aberrant regulation of transcription or subsequent RNA processing of MBNL-family mRNAs might also be part of the pathogenic mechanism of DM. We used real-time RT-PCR analysis to examine the possibility that MBNL mRNA expression is altered in DM1 patients. We also examined mRNA expression for members of the CUG-BP and ETR-3-like factor (CELF) family of RNA-binding proteins given that CELF proteins regulate alternative splicing and are also implicated in DM. We found that DM1 muscles displayed aberrant regulation of alternative splicing as reported previously; however, the levels of MBNL and CELF mRNA expression did not show any significant changes. Our results suggest that the expression and stability of the mRNA for these RNA-binding proteins are unaffected in DM1.