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.

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.

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.

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.

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.

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.

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.

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.

Enhanced prepulse inhibition and low sensitivity to a dopamine agonist in HESR1 knockout mice.

Transcription factor Hesr family genes are important in neuronal development. We demonstrated previously that HESR1 and HESR2 modified expression of the dopamine transporter (DAT) reporter gene. HESR-family genes have been investigated in development, but their functions, especially in relation to behaviors regulated by dopamine, in adult animals remain unclear. In the present study, we investigated the effects of Hesr1 and Hesr2 on behavior. A behavioral test battery to examine spontaneous activity, anxiety-like behavior, aggressive behavior, pain sensitivity, and sensorimotor gating was conducted in Hesr1 and Hesr2 knockout (KO) mice. Enhanced prepulse inhibition (PPI), which is a form of sensorimotor gating, was observed in only Hesr1 KO mice; other behavioral traits were mostly comparable to wild-type animals in both the Hesr1 and the Hesr2 KO lines. Next, we used a dopamine agonist, apomorphine, to confirm the involvement of the dopaminergic system. Injection of apomorphine reduced the enhanced PPI in Hesr1 KO mice. Additionally, dose-dependent sensitivity to the agonist was lower in the Hesr1 KO mice than in wild-type mice, suggesting that the enhanced PPI resulted from this alteration in dopamine sensitivity. Furthermore, DAT mRNA was downregulated in Hesr1 KO mice, whereas the dopamine D1 and D2 receptors were comparable. These findings suggest Hesr1 to be a novel factor that affects dopamine sensitivity and the sensorimotor gating system.

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.

Proteome analysis of soluble nuclear proteins reveals that HMGB1/2 suppress genotoxic stress in polyglutamine diseases.

Nuclear dysfunction is a key feature of the pathology of polyglutamine (polyQ) diseases. It has been suggested that mutant polyQ proteins impair functions of nuclear factors by interacting with them directly in the nucleus. However, a systematic analysis of quantitative changes in soluble nuclear proteins in neurons expressing mutant polyQ proteins has not been performed. Here, we perform a proteome analysis of soluble nuclear proteins prepared from neurons expressing huntingtin (Htt) or ataxin-1 (AT1) protein, and show that mutant AT1 and Htt similarly reduce the concentration of soluble high mobility group B1/2 (HMGB1/2) proteins. Immunoprecipitation and pulldown assays indicate that HMGBs interact with mutant AT1 and Htt. Immunohistochemistry showed that these proteins were reduced in the nuclear region outside of inclusion bodies in affected neurons. Compensatory expression of HMGBs ameliorated polyQ-induced pathology in primary neurons and in Drosophila polyQ models. Furthermore, HMGBs repressed genotoxic stress signals induced by mutant Htt or transcriptional repression. Thus, HMGBs may be critical regulators of polyQ disease pathology and could be targets for therapy development.

Structure-activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P(2) position.

We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Aß production in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve enzymatic stability in vivo and permeability across the blood-brain barrier, so as to be practical drug. Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold at the P2 position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety.

Comparison of presenilin 1 and presenilin 2 γ-secretase activities using a yeast reconstitution system.

γ-Secretase is composed of at least four proteins, presenilin (PS), nicastrin (NCT), Aph1, and Pen2. PS is the catalytic subunit of the γ-secretase complex, having aspartic protease activity. PS has two homologs, namely, PS1 and PS2. To compare the activity of these complexes containing different PSs, we reconstituted them in yeast, which lacks γ-secretase homologs. Yeast cells were transformed with PS1 or PS2, NCT, Pen2, Aph1, and artificial substrate C55-Gal4p. After substrate cleavage, Gal4p translocates to the nucleus and activates transcription of the reporter genes ADE2, HIS3, and lacZ. γ-Secretase activity was measured based on yeast growth on selective media and ß-galactosidase activity. PS1 γ-secretase was ∼24-fold more active than PS2 γ-secretase in the ß-galactosidase assay. Using yeast microsomes containing γ-secretase and C55, we compared the concentration of Aß generated by PS1 or PS2 γ-secretase. PS1 γ-secretase produced ∼24-fold more Aß than PS2 γ-secretase. We found the optimal pH of Aß production by PS2 to be 7.0, as for PS1, and that the PS2 complex included immature NCT, unlike the PS1 complex, which included mature NCT. In this study, we compared the activity of PS1 or PS2 per one γ-secretase complex. Co-immunoprecipitation experiments using yeast microsomes showed that PS1 concentrations in the γ-secretase complex were ∼28 times higher than that of PS2. Our data suggest that the PS1 complex is only marginally less active than the PS2 complex in Aß production.

Regulation of the alternative splicing of sarcoplasmic reticulum Ca²âº-ATPase1 (SERCA1) by phorbol 12-myristate 13-acetate (PMA) via a PKC pathway.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease with no established treatment to date. Small, cell-permeable molecules hold the potential to treat DM1. In this study, we investigated the association between protein kinase C (PKC) signaling and splicing of sarcoplasmic reticulum Ca(2+)-ATPase1 (SERCA1). Our aim was to clarify the mechanisms underlying the regulation of alternative splicing, in order to explore new therapeutic strategies for DM1. By assessing the splicing pattern of the endogenous SERCA1 gene in HEK293 cells, we found that treatment with phorbol 12-myristate 13-acetate (PMA) regulated SERCA1 splicing. Interestingly, treatment with PMA for 48 h normalized SERCA1 splicing, while treatment for 1.5h promoted aberrant splicing. These two responses showed dose dependency and were completely abolished by the PKC inhibitor Ro 31-8220. Furthermore, repression of PKCßII and PKCθ by RNAi mimicked prolonged PMA treatment. These results indicate that PKC signaling is involved in the splicing of SERCA1 and provide new evidence for a link between alternative splicing and PKC signaling.

Tumor suppressor cell adhesion molecule 1 (CADM1) is cleaved by a disintegrin and metalloprotease 10 (ADAM10) and subsequently cleaved by γ-secretase complex.

Cell adhesion molecule 1 (CADM1) is a type I transmembrane glycoprotein expressed in various tissues. CADM1 is a cell adhesion molecule with many functions, including roles in tumor suppression, apoptosis, mast cell survival, synapse formation, and spermatogenesis. CADM1 undergoes membrane-proximal cleavage called shedding, but the sheddase and mechanisms of CADM1 proteolysis have not been reported. We determined the cleavage site involved in CADM1 shedding by LC/MS/MS and showed that CADM1 shedding occurred in the membrane fraction and was inhibited by tumor necrosis factor-α protease inhibitor-1 (TAPI-1). An siRNA experiment revealed that ADAM10 mediates endogenous CADM1 shedding. In addition, the membrane-bound fragment generated by shedding was further cleaved by γ-secretase and generated CADM1-intracellular domain (ICD) in a mechanism called regulated intramembrane proteolysis (RIP). These results clarify the detailed mechanism of membrane-proximal cleavage of CADM1, suggesting the possibility of RIP-mediated CADM1 signaling.

Localization of mature neprilysin in lipid rafts.

Alzheimer's disease (AD) is characterized by senile plaques caused by amyloid-ß peptide (Aß) accumulation. It has been reported that Aß generation and accumulation occur in membrane microdomains, called lipid rafts, which are enriched in cholesterol and glycosphingolipids. Moreover, the ablation of cholesterol metabolism has been implicated in AD. Neprilysin (NEP), a neutral endopeptidase, is one of the major Aß-degrading enzymes in the brain. Activation of NEP is a possible therapeutic target. However, it remains unknown whether the activity of NEP is regulated by its association with lipid rafts. Here we show that only the mature form of NEP, which has been glycosylated in the Golgi, exists in lipid rafts, where it is directly associated with phosphatidylserine. Moreover, the localization of NEP in lipid rafts is enhanced by its dimerization, as shown using the NEP E403C homodimerization mutant. However, the protease activities of the mature form of NEP, as assessed by in vitro peptide hydrolysis, did not differ between lipid rafts and nonlipid rafts. We conclude that cholesterol and other lipids regulate the localization of mature NEP to lipid rafts, where the substrate Aß accumulates but does not modulate the protease activity of NEP.

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.