Plant-based vaccines for Alzheimer's disease.

Alzheimer's disease (AD) is one of the major causes of chronic and progressive cognitive decline, with the pathological hallmarks of senile plaques and neurofibrillary tangles. Amyloid ß peptide (Aß) is the main component of senile plaques, and the pathological load of Aß in the brain has been shown to be a marker of the severity of AD. To prevent the accumulation of plaques, novel and safer plant-based vaccine strategies have been suggested. In this review, we summarize the results of plant vaccines against Aß.

Production of the herb Ruta chalepensis L. expressing amyloid ß-GFP fusion protein.

The herb Ruta chalepensis L. exhibits medical effects, such as anti-inflammatory, central nervous system depressant, and antipyretic activities. However, a genetic transformation method has not yet been developed for this species. In this paper, a simple and efficient tissue culture and genetic transformation system for R. chalepensis is reported. An amyloid ß-peptide (Aß) gene, which is considered to be a causative agent of Alzheimer's disease (AD), fused with green-fluorescent protein (GFP), was introduced into R. chalepensis. When the leaves of R. chalepensis expressing Aß-GFP were administered orally to C57BL/6J mice, serum anti-Aß antibody titers of several mice were elevated without the use of an adjuvant. These results indicated that an oral vaccine against AD using R. chalepensis may be feasible. R. chalepensis is rich in bioactive compounds that may have synergistic effects with the vaccine for AD. Plant-derived vaccines are safer and cheaper than those produced from animal cells or microbes, because plants can serve as biofactories at low cost and with high biosynthetic capacity.

Exon Skipping by Ultrasound-Enhanced Delivery of Morpholino with Bubble Liposomes for Myotonic Dystrophy Model Mice.

Abnormal splicing of the chloride channel 1 (CLCN1) gene causes myotonic dystrophy type 1 (DM1). Therefore, controlling the alternative splicing process of this gene by antisense oligonucleotides can be a promising treatment for DM1. In this study, we describe an efficient phosphorodiamidate morpholino oligomer (PMO) delivery method by ultrasound-mediated bubble liposomes, which is a known gene delivery tool with ultrasound exposure, to treat skeletal muscles in a DM1 mouse model, HSALR. Effective delivery of PMO using this technique can help control the alternative splicing of the Clcn1 gene via exon skipping and enhance the expression of Clcn1 protein in skeletal muscles and the amelioration of myotonia. Thus, exon skipping by PMO delivery with ultrasound-mediated BLs may be feasible in myotonic dystrophy model mice.

RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells.

Adenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.

Regulation of body size in Caenorhabditis elegans: effects of environmental factors and the nervous system.

Body size is one of the basic traits of animals and is regulated to adapt to the environment. Animals perceive environmental stimuli with sensory neurons, and signals from the nervous system alter the size of organs, thus regulating body size. The model animal Caenorhabditis elegans is particularly suited for genetic analysis of body size regulation, and has already contributed to the elucidation of various genetic pathways that regulate body size. In this review, we summarize the available literature regarding environmental factors that regulate body size and the role of the nervous system in such regulation. We discuss in detail a recent report on body size regulation by the neurotransmitter, dopamine.

Specific combinations of presenilins and Aph1s affect the substrate specificity and activity of γ-secretase.

The γ-secretase complex comprises presenilin (PS), nicastrin (NCT), anterior pharynx-defective 1 (Aph1), and presenilin enhancer 2 (Pen2). PS has two homologues, PS1 and PS2. Aph1 has two isoforms, Aph1a and Aph1b, with the former existing as two splice variants Aph1aL and Aph1aS. Each complex consists of one subunit each, resulting in six different γ-secretases. To better understand the functional differences among the γ-secretases, we reconstituted them using a yeast system and compared Notch1-cleavage and amyloid precursor protein (APP)-cleavage activities. Intriguingly, PS2/Aph1b had a clear substrate specificity: APP-Gal4, but not Notch-Gal4, was cleaved. In HEK cell lines expressing defined γ-secretase subunits, we showed that PS1/Aph1b, PS2/Aph1aL, PS2/Aph1aS and PS2/Aph1b γ-secretase produced amyloid ß peptide (Aß) with a higher Aß42+Aß43-to-Aß40 (Aß42(43)/Aß40) ratio than the other γ-secretases. In addition, PS2/Aph1aS γ-secretase produced less Notch intracellular domain (NICD) than did the other 5 γ-secretases. Considering that the Aß42(43)/Aß40 ratio is relevant in the pathogenesis of Alzheimer's disease (AD), and that inhibition of Notch cleavage causes severe side effect, these results suggest that the PS2/Aph1aS γ-secretase complex is a potential therapeutic target in AD.

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy.

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.

Data describing the effect of DRD4 promoter polymorphisms on promoter activity.

This data article tested whether polymorphisms within the dopamine D4 receptor (DRD4) gene promoter can lead to differences in the promoter activity. The variants, a 120-bp variable number tandem repeat (VNTR), -906 T/C, -809 G/A, -616G/C, and -521C/T, were introduced into the DRD4 promoter and the promoter activity was measured in a neural cell line using the luciferase assay. However, no differences were detected among the haplotypes investigated, and the in vitro data obtained from our protocol could not support the involvement of DRD4 promoter polymorphisms in heritable human traits.

Dopamine regulates body size in Caenorhabditis elegans.

The nervous system plays a critical role in the regulation of animal body sizes. In Caenorhabditis elegans, an amine neurotransmitter, dopamine, is required for the tactile perception of food and food-dependent behavioral changes, while its role in development is unknown. In this study, we show that dopamine negatively regulates body size through a D2-like dopamine receptor, DOP-3, in C. elegans. Dopamine alters body size without affecting food intake or developmental rate. We also found that dopamine promotes egg-laying, although the regulation of body size by dopamine was not solely caused by this effect. Furthermore, dopamine negatively regulates body size through the suppression of signaling by octopamine and Gq-coupled octopamine receptors, SER-3 and SER-6. Our results demonstrate that dopamine and octopamine regulate the body size of C. elegans and suggest a potential role for perception in addition to ingestion of food for growth.

Suppressor Mutations for Presenilin 1 Familial Alzheimer Disease Mutants Modulate γ-Secretase Activities.

γ-Secretase is a multisubunit membrane protein complex containing presenilin (PS1) as a catalytic subunit. Familial Alzheimer disease (FAD) mutations within PS1 were analyzed in yeast cells artificially expressing membrane-bound substrate, amyloid precursor protein, or Notch fused to Gal4 transcriptional activator. The FAD mutations, L166P and G384A (Leu-166 to Pro and Gly-384 to Ala substitution, respectively), were loss-of-function in yeast. We identified five amino acid substitutions that suppress the FAD mutations. The cleavage of amyloid precursor protein or Notch was recovered by the secondary mutations. We also found that secondary mutations alone activated the γ-secretase activity. FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with a missense mutation in the second or sixth loops, regained γ-secretase activity when introduced into presenilin null mouse fibroblasts. Notably, the cells with suppressor mutants produced a decreased amount of Aß42, which is responsible for Alzheimer disease. These results indicate that the yeast system is useful to screen for mutations and chemicals that modulate γ-secretase activity.

Splicing of human chloride channel 1.

Expression of chloride channel 1 (CLCN1/ClC-1) in skeletal muscle is driven by alternative splicing, a process regulated in part by RNA-binding protein families MBNL and CELF. Aberrant splicing of CLCN1 produces many mRNAs, which were translated into inactive proteins, resulting in myotonia in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. This increase in abnormal splicing variants containing exons 6B, 7A or the insertion of a TAG stop codon just before exon 7 leads to a decrease in expression of the normal splice pattern. The majority of studies examining splicing in CLCN1 have been performed using mouse Clcn1, as have investigations into the activation and suppression of normal splicing variant expression by MBNL1-3 and CELF3-6, respectively. In contrast, examinations of human CLCN1 have been less common due to the greater complexity of splicing patterns. Here, we constructed a minigene containing CLCN1 exons 5-7 and established a novel assay system to quantify the expression of the normal splicing variant of CLCN1 using real-time RT-PCR. Antisense oligonucleotides could promote normal CLCN1 alternative splicing but the effective sequence was different from that of Clcn1. This result differs from previous reports using Clcn1, highlighting the effect of differences in splicing patterns between mice and humans.

Functional analysis of SERCA1b, a highly expressed SERCA1 variant in myotonic dystrophy type 1 muscle.

Myotonic dystrophy type 1 (DM1) is a genetic disorder in which multiple genes are aberrantly spliced. Sarco/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) is one of these genes, and it encodes a P-type ATPase. SERCA1 transports Ca(2+) from the cytosol to the lumen, and is involved in muscular relaxation. It has two splice variants (SERCA1a and SERCA1b) that differ in the last eight amino acids, and the contribution of these variants to DM1 pathology is unclear. Here, we show that SERCA1b protein is highly expressed in DM1 muscle tissue, mainly localised at fast twitch fibres. Additionally, when SERCA1a and SERCA1b were overexpressed in cells, we found that the ATPase and Ca(2+) uptake activity of SERCA1a was almost double that of SERCA1b. Although the affinity for both ATP and Ca(2+) was similar between the two variants, SERCA1b was more sensitive to the inner microsomal environment. Thus, we hypothesise that aberrant expression of SERCA1b in DM1 patients is the cause of abnormal intracellular Ca(2+) homeostasis.

Antisense oligonucleotide-mediated exon skipping of CHRNA1 pre-mRNA as potential therapy for Congenital Myasthenic Syndromes.

CHRNA1 encodes the α subunit of nicotinic acetylcholine receptors (nAChRs) and is expressed at the neuromuscular junction. Moreover, it is one of the causative genes of Congenital Myasthenic Syndromes (CMS). CHRNA1 undergoes alternative splicing to produce two splice variants: P3A(-), without exon P3A, and P3A(+), with the exon P3A. Only P3A(-) forms functional nAChR. Aberrant alternative splicing caused by intronic or exonic point mutations in patients leads to an extraordinary increase in P3A(+) and a concomitant decrease in P3A(-). Consequently this resulted in a shortage of functional receptors. Aiming to restore the imbalance between the two splice products, antisense oligonucleotides (AONs) were employed to induce exon P3A skipping. Three AON sequences were designed to sterically block the putative binding sequences for splicing factors necessary for exon recognition. Herein, we show that AON complementary to the 5' splice site of the exon was the most effective at exon skipping of the minigene with causative mutations, as well as endogenous wild-type CHRNA1. We conclude that single administration of the AON against the 5' splice site is a promising therapeutic approach for patients based on the dose-dependent effect of the AON and the additive effect of combined AONs. This conclusion is favorable to patients with inherited diseases of uncertain etiology that arise from aberrant splicing leading to a subsequent loss of functional translation products because our findings encourage the option of AON treatment as a therapeutic for these prospectively identified diseases.

Aß induces oxidative stress in senescence-accelerated (SAMP8) mice.

According to the amyloid hypothesis, amyloid ß accumulates in brains with Alzheimer's disease (AD) and triggers cell death and memory deficit. Previously, we developed a rice Aß vaccine expressing Aß, which reduced brain Aß levels in the Tg2576 mouse model of familial AD. We used senescence-accelerated SAMP8 mice as a model of sporadic AD and investigated the relationship between Aß and oxidative stress. Insoluble Aß and 4-hydroxynonenal (4-HNE) levels tended to be reduced in SAMP8 mice-fed the rice Aß vaccine. We attempted to clarify the relationship between oxidative stress and Aß in vitro. Addition of Aß peptide to the culture medium resulted in an increase in 4-HNE levels in SH-SY5Y cells. Tg2576 mice, which express large amounts of Aß in their brain, also exhibited increased 4-HNE levels; this increase was inhibited by the Aß vaccine. These results indicate that Aß induces oxidative stress in cultured cells and in the mouse brain.

ABLIM1 splicing is abnormal in skeletal muscle of patients with DM1 and regulated by MBNL, CELF and PTBP1.

Myotonic dystrophy type 1 (DM1) is an RNA-mediated disorder characterized by muscle weakness, cardiac defects and multiple symptoms and is caused by expanded CTG repeats within the 3' untranslated region of the DMPK gene. In this study, we found abnormal splicing of actin-binding LIM protein 1 (ABLIM1) in skeletal muscles of patients with DM1 and a DM1 mouse model (HSA(LR) ). An exon 11 inclusion isoform is expressed in skeletal muscle and heart of non-DM1 individuals, but not in skeletal muscle of patients with DM1 or other adult human tissues. Moreover, we determined that ABLIM1 splicing is regulated by several splice factors, including MBNL family proteins, CELF1, 2 and 6, and PTBP1, using a cellular splicing assay. MBNL proteins promoted the inclusion of ABLIM1 exon 11, but other proteins and expanded CUG repeats repressed exon 11 of ABLIM1. This result is consistent with the hypothesis that MBNL proteins are trapped by expanded CUG repeats and inactivated in DM1 and that CELF1 is activated in DM1. However, activation of PTBP1 has not been reported in DM1. Our results suggest that the exon 11 inclusion isoform of ABLIM1 may have a muscle-specific function, and its abnormal splicing could be related to muscle symptoms of DM1.

Nuclear localization of MBNL1: splicing-mediated autoregulation and repression of repeat-derived aberrant proteins.

In some neurological diseases caused by repeat expansions such as myotonic dystrophy, the RNA-binding protein muscleblind-like 1 (MBNL1) accumulates in intranuclear inclusions containing mutant repeat RNA. The interaction between MBNL1 and mutant RNA in the nucleus is a key event leading to loss of MBNL function, yet the details of this effect have been elusive. Here, we investigated the mechanism and significance of MBNL1 nuclear localization. We found that MBNL1 contains two classes of nuclear localization signal (NLS), a classical bipartite NLS and a novel conformational NLS. Alternative splicing of exon 7 acts as a switch between these NLS types and couples MBNL1 activity and intracellular localization. Depending on its nuclear localization, MBNL1 promoted nuclear accumulation of mutant RNA containing a CUG or CAG repeat, some of which produced proteins containing homopolymeric tracts such as polyglutamine. Furthermore, MBNL1 repressed the expression of these homopolymeric proteins including those presumably produced through repeat-associated non-ATG (RAN) translation. These results suggest that nuclear retention of expanded RNA reflects a novel role of MBNL proteins in repressing aberrant protein expression and may provide pathological and therapeutic implications for a wide range of repeat expansion diseases associated with nuclear RNA retention and/or RAN translation.

Dysbindin-1, a schizophrenia-related protein, interacts with HDAC3.

DTNBP1 is a key candidate gene associated with schizophrenia. The expression of its protein product, dysbindin-1, is altered in the brains of schizophrenic patients; however, the physiological functions of dysbindin-1 in the central nervous system are unclear. Several studies have shown that both dysbindin-1 and histone deacetylase 3 (HDAC3) can be phosphorylated by the DNA-dependent protein kinase complex. In this study, we investigated the relationship between dysbindin-1 and HDAC3. We found that dysbindin-1 formed a protein complex with HDAC3 in human neuroblastoma cells and in mouse brain. The interaction between dysbindin-1 and HDAC3 occurred in an isoform-specific manner: HDAC3 coupled with dysbindin-1A and -1B, but not -1C. We also found that dysbindin-1B expression was increased in the nucleus in the presence of HDAC3, and, conversely, that the phosphorylation level of HDAC3 increased in the presence of dysbindin-1B. Taken together, these results identify a novel binding partner for dysbindin-1, which may potentially provide a new avenue for research into the neurological mechanisms of schizophrenia.

Usefulness of monitoring γ-H2AX and cell cycle arrest in HepG2 cells for estimating genotoxicity using a high-content analysis system.

Formation of the phosphorylated protein γ-H2AX is a well-established marker of DNA strand breakage induced by DNA-damaging compounds. Many of these genotoxic compounds also inhibit cell division, leading to arrest at specific points in the cell cycle. Detection of γ-H2AX in combination with cell cycle arrest may therefore be useful for estimating the genotoxicity of experimental compounds. In this study, we examined γ-H2AX formation and cell cycle arrest using high-content screening (HCS) as a method for determining genotoxicity. HepG2 cells were treated with a panel of compounds and then stained with Hoechst 33342 and anti-γ-H2AX, anti-phospho-histone H3, and anti-tubulin antibodies. In total, 19 genotoxic and 7 nongenotoxic compounds were tested in this study. γ-H2AX production was observed within 1 h posttreatment for the majority of Ames-positive compounds, topoisomerase inhibitors, and DNA polymerase inhibitors. Cell cycle arrest in either the S or G2 phase was detected for all DNA-damaging compounds 24 h posttreatment, whereas tubulin-targeting compounds were shown to induce cell cycle arrest in the mitotic phase. Together, these results show that HCS is a simple, rapid, and effective tool for estimating the genotoxicity of compounds through detection of γ-H2AX production and cell cycle arrest.

[Current studies in myotonic dystrophy].

Myotonic dystrophy (DM) is a genetic, progressive, multisystemic disease with muscular disorder as its primary symptom. There are two types of DM (DM1 and DM2) caused by mutations in different genes, and in Japan, DM occurs with an incidence of approximately 1 in 20,000. The pathogenic mechanism underlying the disease is RNA toxicity caused by transcripts of aberrantly elongated CTG or CCTG repeats located in the 3' untranslated region or in the intron. The current treatments for DM is limited to symptomatic care. In this review, we will discuss several new therapeutic strategies based on recent studies of RNA toxicity.

Nonredundant function of two highly homologous octopamine receptors in food-deprivation-mediated signaling in Caenorhabditis elegans.

It is common for neurotransmitters to possess multiple receptors that couple to the same intracellular signaling molecules. This study analyzes two highly homologous G-protein-coupled octopamine receptors using the model animal Caenorhabditis elegans. In C. elegans, the amine neurotransmitter octopamine induces activation of cAMP response element-binding protein (CREB) in the cholinergic SIA neurons in the absence of food through activation of the Gq-coupled octopamine receptor SER-3 in these neurons. We also analyzed another Gq-coupled octopamine receptor, SER-6, that is highly homologous to SER-3. As seen in ser-3 deletion mutants, octopamine- and food-deprivation-mediated CREB activation was decreased in ser-6 deletion mutants compared with wild-type animals, suggesting that both SER-3 and SER-6 are required for signal transduction. Cell-specific expression of SER-6 in the SIA neurons was sufficient to restore CREB activation in the ser-6 mutants, indicating that SER-6, like SER-3, functions in these neurons. Taken together, these results demonstrate that two similar G-protein-coupled receptors, SER-3 and SER-6, function in the same cells in a nonredundant manner.