新倉 貴子

In this study, we review the properties of three anionic detergents, sodium dodecyl sulfate (SDS), Sarkosyl, and sodium lauroylglutamate (SLG), as they play a critical role in molecular biology research. SDS is widely used in electrophoresis and cell lysis for proteomics. Sarkosyl and, more frequently, SDS are used for the characterization of neuropathological protein fibrils and the solubilization of proteins. Many amyloid fibrils are resistant to SDS or Sarkosyl to different degrees and, thus, can be readily isolated from detergent-sensitive proteins. SLG is milder than the above two detergents and has been used in the solubilization and refolding of proteins isolated from inclusion bodies. Here, we show that both Sarkosyl and SLG have been used for protein refolding, that the effects of SLG on the native protein structure are weaker for SLG, and that SLG readily dissociates from the native proteins. We propose that SLG may be effective in cell lysis for functional proteomics due to no or weaker binding of SLG to the native proteins.

SUMOylation, a post-translational modification of lysine residues by small ubiquitin-like modifier (SUMO) proteins, has been implicated in the pathogenesis of neurodegenerative disorders including Alzheimer's disease (AD), and in neuron- and astrocyte-specific physiological functions. Global SUMOylation is increased in the AD mouse brain in the pre-plaque-forming stage but returns to wild-type levels in the plaque-bearing stage. To clarify the reason for the transient change in SUMOylation, we analyzed the alteration of global SUMOylation induced by AD-associated cytotoxic stimuli in neurons and astrocytes individually. In neurons, amyloid β42 oligomers induced some but not significant increase in levels of SUMO1-modified proteins. Both hydrogen peroxide and glutamate significantly reduced SUMO1-modified protein levels. These changes were more prominent in neurons than in astrocytes. The opposite effect of Aβ and oxidative/excitotoxic stimuli on SUMO1 modification may cause the pathological stage-associated change in the level of SUMO-modified proteins in the AD mouse brain.

Amyloid β a key molecule in the pathogenesis of Alzheimer's disease (AD), is produced from amyloid precursor protein (APP) by the cleavage of secretases. APP is SUMOylated near the cleavage site of β-secretase. SUMOylation of APP reduces amyloid β production, but its regulatory system is still unclear. SUMOylation, a modification at a lysine residue of a target protein, is mediated by activating, conjugating, and ligating enzymes and is reversed by a family of sentrin/SUMO-specific proteases (SENPs). Here, we found that both SENP1 and SENP2 induced de-SUMOylation of APP. Using quantitative PCR, we also found that expression of SENP1 but not SENP2 increased in an age-dependent manner only in female mice. The results of immunoblot analyses showed that the protein expression was consistent with the PCR results. Females, compared to males, have a higher incidence of AD in humans and show more aggressive amyloid pathology in AD mouse models. Our results provide a clue to understanding the role of SUMOylation in the sex difference in AD pathogenesis.

The molecular mechanism by which NSC number is controlled in the neurogenic regions of the adult brain is not fully understood but it has been shown that vascular niche signals regulate neural stem cell (NSC) quiescence and growth. Here, we have uncovered a role for soluble amyloid precursor protein (sAPP) as a vascular niche signal in the subventricular zone (SVZ) of the lateral ventricle of the adult mouse brain. sAPP suppresses NSC growth in culture. Further in vivo studies on the role of APP in regulating NSC number in the SVZ clearly demonstrate that endothelial deletion of App causes a significant increase in the number of BrdU label-retaining NSCs in the SVZ, whereas NSC/astrocyte deletion of App has no detectable effect on the NSC number. Taken together, these results suggest that endothelial APP functions as a vascular niche signal that negatively regulates NSC growth to control the NSC number in the SVZ.

Humanin (HN) is an endogenous 24-residue peptide. A highly potent HN derivative, SI4G-HN, which has a substitution of serine 14 to glycine, reduced amyloid burden and suppressed cognitive impairment in a mouse model of Alzheimer's disease. S14G-HN also suppressed amnesia induced by a muscarinic receptor antagonist in rodents. To understand the effects of HN on brain function, we tested the effect of S14G-HN on diazepam (DZP)-induced memory impairment and anxiety in mice using the object recognition test and zero-maze test, respectively. Intraperitoneal injection of S14G-HN reversed the DZP-induced memory deficit, whereas no significant change was observed in behavioral markers of anxiety. S14G-HN had no effect on locomotor activity in either test, indicating that S14G-HN did not affect physical functioning or motivation. These results suggest that HN preferentially influences cognitive function but not emotional function in the central nervous system. (C) 2016 Elsevier Ltd. All rights reserved.

Mutations in superoxide dismutase 1 (SOD1) are a major cause of familial amyotrophic lateral sclerosis (ALS), whereby the mutant proteins misfold and aggregate to form intracellular inclusions. We report that both small ubiquitin-like modifier (SUMO) 1 and SUMO2/3 modify ALS-linked SOD1 mutant proteins at lysine 75 in a motoneuronal cell line, the cell type affected in ALS. In these cells, SUMO1 modification occurred on both lysine 75 and lysine 9 of SOD1, and modification of ALS-linked SOD1 mutant proteins by SUMO3, rather than by SUMO1, significantly increased the stability of the proteins and accelerated intracellular aggregate formation. These findings suggest the contribution of sumoylation, particularly by SUMO3, to the protein aggregation process underlying the pathogenesis of ALS.

Aquaporin-4, a predominant water channel in the brain, is specifically expressed in astrocyte endfeet and plays a central role in water homeostasis, neuronal activity, and cell migration in the brain. It has two dominant isoforms called M1 and M23, whose mRNA is driven by distinct promoters located upstream of exons 0 and 1 of the aquaporin-4 gene, respectively. To identify cis-acting elements responsible for the astrocyte-specific transcription of M1 mRNA, the promoter activity of the 5'-flanking region upstream of exon 0 in primary cultured mouse astrocytes was examined by luciferase assay, and sequences, where nuclear factors bind, were identified by electrophoretic mobility shift assay. An astrocyte-specific activity enhancing transcription from the M1 promoter was observed within 2 kb from the transcriptional start sites of M1 mRNA. At least five elements clustered within the 286-bp region were found to function as a novel astrocyte-specific enhancer. Among the five elements, a consensus sequence of Pit-1/Oct/Unc-86 (POU) transcription factors was indispensable to the astrocyte-specific enhancer since disruption of the POU motif completely abolished the enhancer activity in astrocytes. However, the POU motif alone had little activity, indicating the requirement for cooperation with other upstream elements to exert full enhancer activity.

A single mutation has resulted in large differences in neuroprotective activity of a 24 amino acid Humanin (HN). A mutation of Ser7Ala (S7A-HN) resulted in loss of activity, while a mutation of Ser14Gly (S14G-HN) resulted in about 1000-fold increase. The mechanism of the effects conferred by these mutations have been totally unclear, although our recent structure analysis suggested a possibility of the effect of mutation on the structure stability. Here, we have studied the effects of buffer and temperature on the structure of these three HN peptides. These peptides showed a similar disordered structure at 10 degrees C in 10 mM phosphate, pH 6.0. They were also similar in phosphate-buffered saline (PBS) as long as the temperature was kept low at 10 degrees C. However, a large difference was observed in both phosphate buffer and PBS between the peptides, when the temperature was raised to a physiological temperature of 37 degrees C. While S14G-HN showed small changes in both solutions at 37 degrees C, the less active HN and inactive S7A-HN showed much larger changes under the identical conditions. In addition, it appeared that structure change at 37 degrees C was faster for S7A-HN than HN. These results show that the structure stability at 37 degrees C increases in the order of S7A-HN, HN and S14G-HN, in correlation with their neuroprotective activities. (C) 2011 Elsevier B.V. All rights reserved.

We have recently shown that a 24 amino acid Humanin (HN) adopts an anti-parallel beta-sheet structure in the presence of a negatively charged 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and suggested a possibility that it interacts with lipid membranes and thereby exerts neuroprotective effects through the target cell surface receptors or the intracellular signaling molecules following membrane interaction events. The structures of two HN analogs, having either a S7A mutation or a S14G mutation, were examined under the identical conditions, as the S7A analog is inactive and the S14G analog is 1000-fold more active than the wild type HN. These analogs showed a secondary structure indistinguishable from the structure of HN in the presence of DOPG liposome, while unrelated peptides were disordered with and without DOPG. It thus appeared that HN and the analogs, regardless of the biological activities, have an ability to interact with DOPG liposome and form an anti-parallel beta-sheet structure. While the wild type HN and the S7A and S14G analogs were largely disordered in buffer, the S14G analog showed greater stability as a disordered structure in the buffer at a physiological temperature, suggesting that it maintains the disordered structure presumably required for the interaction with the DOPG liposome and thereby greater neuroprotective activity. (C) 2011 Elsevier B.V. All rights reserved.

Humanin (HN), a 24-residue peptide, was identified as a novel neuroprotective factor and shows anti-cell death activity against a wide spectrum of Alzheimer's disease (AD)-related cytotoxicities, including exposure to amyloid beta (Abeta), in vitro. We previously demonstrated that the injection of S14G-HN, a highly potent HN derivative, into brain ameliorated memory loss in an Abeta-injection mouse model. To fully understand HN's functions under AD-associated pathological conditions, we examined the effect of S14G-HN on triple transgenic mice harboring APP(swe), tau(P310L), and PS-1(M146V) that show the age-dependent development of multiple pathologies relating to AD. After 3 months of intranasal treatment, behavioral analyses showed that S14G-HN ameliorated cognitive impairment in male mice. Moreover, ELISA and immunohistochemical analyses showed that Abeta levels in brains were markedly lower in S14G-HN-treated male and female mice than in vehicle control mice. We also found the expression level of neprilysin, an Abeta degrading enzyme, in the outer molecular layer of hippocampal formation was increased in S14G-HN-treated mouse brains. NEP activity was also elevated by S14G-HN treatment in vitro. These findings suggest that decreased Abeta level in these mice is at least partly attributed to S14G-HN-induced increase of neprilysin level. Although HN was identified as an anti-neuronal death factor, these results indicate that HN may also have a therapeutic effect on amyloid accumulation in AD.

While neuroprotective activities of Humanin peptides have been clearly demonstrated, the functional mechanism has not been fully understood. Humanin and a majority of Humanin analogs showed a disordered structure at low peptide concentrations and aggregation at higher concentrations in aqueous solution at pH 7.0 Here we have examined the structure in lipid environments, i e., in the presence of liposome by circular dichroism Humanin underwent a large structure change into a typical beta-sheet structure at neutral pH in the presence liposome made of a negatively charged 1.2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), but not an electrically neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) As Humanin possesses a positive charge at neutral pH, the observed structure changes with DOPG suggest electrostatic binding of the peptide with the lipid No effect of NaCl on the Humanin structure was observed in neutral solution and in the presence of DOPC liposome. Increasing temperature resulted in changes in the structure due to aggregation On the other hand, the effects of temperature on the Humanin structure showed that it has a relatively stable structure in the presence of DOPG liposome independent of the presence of NaCl (C) 2010 Elsevier B.V All rights reserved

Activity-dependent neurotrophic factor 9 (ADNF9) and NAP are nine and eight amino acid peptides, which exhibit neuroprotective activity at femtomolar concentrations against cell toxic agents. We have here characterized their structures and interactions with dodecylphosphocholine (DPC) in phosphate-buffered saline (PBS). Circular dichroism analysis showed that ADNF9 and NAP are structurally disordered in PBS independent of peptide concentration and temperature, but appear to assume different secondary structure at increasing temperature. Sedimentation equilibrium analysis showed that both ADNF9 and NAP are monomeric at 37 degrees C, suggesting no self-association under physiological conditions. No secondary structure changes were observed in the presence of DPC, suggesting that ADNF9 and NAP do not interact with lipids. (C) 2009 Elsevier B.V. All rights reserved.

A novel neuroprotective peptide, Humanin (HN), has a strong tendency to aggregate in phosphate-buffered saline. Here we attempted to reduce aggregation employing an aqueous phosphate solution, without NaCl, at pH 6.0 and low peptide concentrations wherever possible. Such a condition, though not fully physiological, allowed us to determine the secondary structure and molecular weight of the peptides. Comparison of a parent HN peptide, an inactive analog (S7A-HN) and a 1000-fold more active analog (S14G-HN) showed no apparent differences in the secondary structure. These peptides were all disordered over the wide range of peptide concentration. Sedimentation analysis was done only for HN and S7A-HN and showed aggregation into soluble oligomers in 20 mM phosphate at pH 6.0. Aggregation was greatly suppressed in 5 mM phosphate at the same pH in terms of aggregate size, with the formation of smaller oligomers. Sedimentation velocity experiments at 60,000 rpm in 5 mM phosphate at pH 6.0 showed that both HN and S7A-HN distributed into soluble aggregates that sedimented to the bottom of the cell and low molecular weight species that approached sedimentation equilibrium. The mass of this low molecular weight species was determined by sedimentation equilibrium to be close to monomers for both peptides. Thus, these results clearly demonstrate that the active HN and inactive S7A-HN are identical in structure and hence there is no apparent correlation between solution structure and biological activity.

A 24-amino acid peptide, Humanin (HN), is a novel peptide that protects neuronal cells in vitro and in vivo from Alzheimer's disease-related toxicities. We have shown before that the structures of HN and a 1000-fold more active analog, HNG, with a Ser14Gly mutation are largely disordered. During additional mutational analysis, a shorter 17-amino acid form, AGA-(C8R)HNG17, was accidentally discovered to have a 100-fold higher activity than HNG. Here we have characterized the structural properties of the AGA-(C8R)HNG17 analog by circular dichroism (CD) and sedimentation equilibrium analysis. First, the structure in water was characterized, since these peptides have been dissolved in water prior to biological analysis. The AGA-(C8R)HNG17 peptide exhibited extensive beta-sheet structure in water, completely different from the aqueous HN and HNG structures. The beta-sheet structure was converted to a disordered structure upon dilution into phosphate-buffered saline (PBS) at low peptide concentration (e.g., below 0.2 mg/ml), which was similar to the structure of HN and HNG, observed under similar conditions. Sedimentation equilibrium analysis showed that the AGA-(C8R)HNG 17 analog was essentially monomeric in PBS, while HNG showed extensive aggregation. Such aggregation of HNG was observed when the peptide was added to the serum-containing cell culture media. Thus, the mutations introduced into the AGA-(C8R)HNG17 analog generated a peptide different from the parent HNG and HN peptides in the self-association properties and hence the solubility, which most likely contributed to the increased biological activity of the AGA-(C8R)HNG17 analog. (C) 2008 Elsevier B.V. All rights reserved.

Epidemiological studies indicate that women have a higher risk of Alzheimer's disease (AD) even after adjustment for age. Though transgenic mouse models of AD develop AD-related amyloid beta (Abeta) and/or tau pathology, gender differences have not been well documented in these models. In this study, we found that female 3xTg-AD transgenic mice expressing mutant APP, presenilin-1 and tau have significantly more aggressive Abeta pathology. We also found an increase in beta-secretase activity and a reduction of neprilysin in female mice compared to males; this suggests that a combination of increased Abeta production and decreased Abeta degradation may contribute to higher risk of AD in females. In contrast to significantly more aggressive Abeta pathology in females, gender did not affect the levels of phosphorylated tau in 3xTg-AD mice. These results point to the involvement of Abeta pathways in the higher risk of AD in women. In addition to comparison of pathology between genders at 9, 16 and 23 months of age, we examined the progression of Abeta pathology at additional age points; i.e., brain Abeta load, intraneuronal oligomeric Abeta distribution and plaque load, in male 3xTg-AD mice at 3,6,9,12,16,20 and 23 months of age. These findings confirm progressive Abeta pathology in 3xTg-AD transgenic mice, and provide guidance for their use in therapeutic research. (C) 2008 Elsevier B.V. All rights reserved.

We have examined the structure of two Humanin (HN) analog peptides, HNG and AGA-(C8R) HNG17, in the presence of sodium dodecylsulfate (SDS) and trifluoroethanol (TFE) using CD and sedimentation velocity. Both HNG and AGA-(C8R) HNG17 underwent complex conformational changes with increasing concentrations of SDS and TFE, in contrast to general trend of increasing helix with their concentration. To our surprise, both peptides appear to converge into a similar structure in SDS and TFE at higher concentrations; e. g., above 0.05% SDS or 30-40% TFE. Sedimentation velocity analysis showed extensive aggregation of HNG at 0.1 mg/ml in PBS in the absence of SDS, but a highly homogeneous solution in 0.1% SDS, indicating formation of a uniform structure by SDS. These two peptides also formed an intermediate structure both in SDS and TFE at lower concentrations, which appeared to be associated with extensive aggregation. It is interesting that the structure changes of these peptides occur well below the critical micelle concentration of SDS, suggesting that conformational changes are mediated through molecular, not micellar, interactions with SDS.

A 24-amino acid long peptide, Humanin, protects neurons from Alzheimer's disease (AD)-related cell toxicities at sub-nM-uM concentrations. Activity-dependent neurotrophic factor (ADNF) is a glia-derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and AD-related and amyotrophic lateral sclerosis-related insults at fM concentrations. An attempt was made to further improve the activity of Humanin by fusing this peptide to ADNF9, a 9-amino acid long core peptide of the ADNF. This fusion resulted in a novel molecule, termed Colivelin, with the neuroprotective activity at fM range, which is similar to 10(3)-10(7) fold higher than the activity of Humanin and Humanin analogs and follows the activity profile of fM-active ADNF9. We have characterized the structural properties of Colivelin and compared with those of ADNF9 and Humanin in water and phosphate-buffered saline (PBS). The secondary structure of Colivelin was similar to that of ADNF9, but not that of Humanin, and hence was not the average of the contributions of the two peptides fused. Colivelin was stable and monomeric in PBS, consistent with the monomeric property of ADNF9, while Humanin showed strong tendency to self-associate. Thus, it is evident that the structural properties of Colivelin resemble those of ADNF9, rather than those of Humanin. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

The knowledge of tumor-associated antigens is required for most types of immunotherapy and can substantially facilitate diagnosis. To identify potential tumor-associated genes expressed in cutaneous T-cell lymphoma (CTCL), we used three complementary strategies: antigens which elicit a humoral immune response in CTCL patients were detected by serological analysis of a recombinant cDNA expression library. cDNAs differentially expressed in CTCL but not peripheral blood monocytes were identified by comparative cDNA hybridization and suppression subtractive hybridization. We identified 43 genes selectively expressed by CTCL cells, that have not yet been described in the context of CTCL development, but most of which had been reported to be associated with cancer. Expression analysis by database mining and subsequently RT-PCR on selected clones confirmed their selective expression in CTCL tissues. Serological tests showed that 15 clones were recognized by sera of CTCL patients but not of healthy donors. Analysis of serological tests for 11 clones using serum antibody detection array (SADA) and 100 sera of controls and CTCL patients each revealed up to 5% reactive sera in the tumor group. The expression pattern of the detected clones and their immunogenicity demonstrates that they might be relevant for the understanding of CTCL and suggests particularly three clones, HD-CL-41 (DRAK2), HD-CL-49 (nudC) and HD-CL-12 (ZNF195) for further analysis with respect to their prognostic and therapeutic value for CTCL.

Activity-dependent neurotrophic factor (ADNF) is a glia-derived neurotrophic peptide, which protects neurons from tetrodoxin treatment and Alzheimer's disease-related and amyotrophic-lateral-sclerosis-related insults at femto-molar concentrations. However, the mechanism of the femto-molar neuroprotection by the peptide has not been elucidated. The characterization of the peptide structure in solution at molecular level should shed light in the mechanism of such extremely high biological activity. From that point of view, the secondary and quaternary structure analysis of ADNF9, an active core fragment peptide of ADNF, was performed by circular dichroism (CD) and sedimentation equilibrium. ADNF9 has also been shown to exhibit a neurotrophic activity in femto-molar concentrations; in this study it showed sub-pM neuro-protective activity against V6421-APP-induced cytotoxity in the mouse primary cortical neuron. CD analysis showed that the secondary structure of ADNF9 is identical in water and phosphate-buffered saline (PBS) and is independent of the peptide concentration. The CD spectra appear to be characterized most likely as disordered. The sedimentation equilibrium experiments demonstrated monomeric structure of the protein over the wide range of peptide concentration. There is a slight enhancement of CD intensity at 37 degrees C relative to 20 degrees C, suggesting a possible hydrophobic association of the peptide. There is no change in the secondary structure in PBS upon freeze-thaw treatment, which has previously been suggested to cause activity loss.

The structure of a highly potent Ser14Gly analog of antiAlzheimer peptide, Humanin, was examined by circular dichroism (CD). The secondary structure is more disordered in water than in phosphate-buffered saline (PBS). The peptide structure in water is little dependent on both peptide concentration and temperature. On the contrary, the peptide structure was significantly different in PBS from the structure in water, which is more apparent at a higher peptide concentration and temperature. The observed different structure in PBS appears to be due to self-association of the peptide, which is enhanced by elevated temperature and, hence, via hydrophobic interactions. The wild-type Humanin also behaved similarly, i.e., it assumed a disordered structure in water but underwent conformational changes in PBS. Although high peptide concentrations for CD measurements are not encountered in vivo, the results suggest the tendency of the peptide to interact hydrophobically with other structures as well as with itself. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

Humanin (HN) inhibits neuronal cell death induced by various Alzheimer's disease (AD)-related insults. It has been proposed that HN binds to a putative receptor on the cell membrane and triggers a signal transduction cascade linked to neuroprotection. Recently, it was shown that HN binds to pertussis toxin (PTX)-sensitive G protein-coupled formylpeptide receptor-like-1 molecule (FPRL-1), reduces A beta(1-42) aggregation and fibril formation, and suppresses the A (1-42) toxicity on monomiclear phagocytic cells [Ying, G., Iribarren, P., Zhou, Y., Gong, W., Zhang, N., Yu, ZX, Le, Y., Cui, Y., Wang, J.M., 2004. Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor. Journal of Immunology 172 (11), 7078-7085.]. We here show that siRNA-mediated disruption of expression of the mouse counterpart of FPRL-1, FPR2, did not result in attenuation of HN-mediated rescue of neuronal cell death induced by AD-related insults. We simultaneously provide evidence that neuroprotection by HN in F11 cells is mediated by the STAT3 transcription factor as well as by certain tyrosine kinases. Altogether, we speculate that a receptor other than FPR2 exists that mediates HN neuroprotection in I'll neurohybrid cells. (c) 2005 Elsevier Inc. All rights reserved.

Amyotrophic lateral sclerosis (ALS) is the most common fatal motor neuron disease. It has been generally accepted that the proapoptotic property of the familial AILS (FALS)-linked mutant SOD1 genes plays an important role in the pathogenesis of some FALS cases. We found here that expression of N19S-SOD1, a novel SOD1 mutant originally found in a sporadic ALS patient, induces lower grade death in NSC34 cells than FALS-linked mutant SOD1. In agreement, intracytoplasmic aggregate formation and SOD1 polymerization are less prominently induced by ectopic expression of N19S-SOD1 than FALS-linked mutant SOD1. We further found that additional cell stresses, such as inhibition of proteasomal activity or up-regulation of intracellular oxidative stress, enhance N19S-SOD1-induced aggregate formation and polymerization of N19S-SOD1. Such analysis of the intracellular polymerization and the ubiquitination of N19S-SOD1 have further suggested that it is recognized as a misfolded protein, like FALS-linked mutant SOD1, whereas wild-type SOD1 is not. Altogether, it is speculated that the N19S mutation of SOD1 in cooperation with associated cell stresses contributes to the onset of ALS as a risk factor. (c) 2005 Wiley-Liss Inc.

Amyotrophic lateral sclerosis (ALS) is the most common fatal motor neuron disease, affecting mostly middle-aged people. There are no curative therapies for ALS. Several lines of evidence have supported the notion that the proapoptotic property of familial ALS (FALS)-linked mutant Cu/Zn -superoxide dismutase-1 (SOD1) genes may play an important role in the pathogenesis of some FALS cases. Here we found that activity-dependent neurotrophic factor (ADNF), a neurotrophic factor originally identified to have the anti-Alzheimer's disease (AD) activity, protected against neuronal cell death caused by FALS-linked A4T-, G85R- and G93R-SOD1 in a dose-responsive fashion. Notably, ADNF-mediated complete suppression of SOD1 mutant-induced neuronal cell death occurs at concentrations as low as 100 fM. ADNF maintains the neuroprotective activity even at concentrations of more than 1 nM. This is in clear contrast to the previous finding that ADNF loses its protective activity against neurotoxicity induced by AD-relevant insults, including some familial AD genes and amyloid beta peptide at concentrations of more than 1 nM. Characterization of the neuroprotective activity of ADNF against cell death caused by SOD1 mutants revealed that CaMKIV and certain tyrosine kinases are involved in ADNF-mediated neuroprotection. Moreover, in vivo studies showed that intracerebroventricularly administered ADNF significantly improved motor performance of G93A-SOD1 transgenic mice, a widely used model of FALS, although survival was extended only marginally. Thus, the neuroprotective activity of ADNF provides a novel insight into the development of curative drugs for ALS. (C) 2004 Wiley-Liss, Inc.

Although neurotoxic functions are well characterized in familial Alzheimer's disease (FAD)-linked N141I mutant of presenilin (PS)2, little has been known about M239V-PS2, another established FAD-causative mutant. We found that expression of M239V-PS2 caused neuronal cytotoxicity. M239V-PS2 exerted three forms of cytotoxicity: one was sensitive to both an antioxidant glutathione-ethyl-ester (GEE) and a caspase inhibitor Ac-DEVD-CHO (DEVD); the second was sensitive to GEE but resistant to DEVD; and the third was resistant to both. The GEE/DEVD-sensitive cytotoxicity by M239V-PS2 was likely through NADPH oxidase and the GEE-sensitive/DEVD- resistant cytotoxicity through xanthine oxidase (XO). Both mechanisms by M239V-PS2 were suppressed by pertussis toxin (PTX) and were mediated by Galpha(o), but not by Galpha(i). Although Abeta1-43 itself induced no cytotoxicity, Abeta1-43 potentiated all three components of M239V-PS2 cytotoxicity. As these cytotoxic mechanisms by M239V-PS2 are fully shared with N14I-PS2, they are most likely implicated in the pathomechanism of FAD by PS2 mutations. Notably, cytotoxicity by M239V-PS2 could be inhibited by the combination of two clinically usable inhibitors of superoxide-generating enzymes, apocynin and oxypurinol. (C) 2004 Wiley-Liss, Inc.

Amyloid precursor protein (APPP), a precursor of amyloid beta (Abeta) peptide, is one of the molecules involved in the pathogenesis of Alzheimer's disease (AD). Specific mutations in APPP have been found in patients inheriting familial AD (FAD). These mutant APPP proteins cause cell death in neuronal cell lines in vitro, but the molecular mechanism of cytotoxicity has not yet been clarified completely. We analyzed the cytotoxic mechanisms of the London-type AbetaPP mutant, V642I-AbetaPP, in primary cortical neurons utilizing an adenovirus-mediated gene transfer system. Expression of V642I-AbetaPP protein induced degeneration of the primary neurons. This cytotoxicity was blocked by pertussis toxin, a specific inhibitor for heterotrimeric G proteins, Go/i, and was suppressed by an inhibitor of caspase-3/7 and an antioxidant, glutathione ethyl ester. A specific inhibitor for NADPH oxidase, apocynin, but not a xanthine oxidase inhibitor or a nitric oxide inhibitor, blocked V642I-AbetaPP-induced cytotoxicity. Among mitogen-activated protein kinase (MAPK) family proteins, c-Jun N-terminal kinase (JNK) and p38MAPK, but not extracellular regulated kinase (ERK), were involved in this cytotoxic pathway. The V642I-AbetaPP-induced cytotoxicity was not suppressed by two secretase inhibitors, suggesting that Abeta does not play a major role in this cytotoxicity. Two neuroprotective factors, insulin-like growth factor I (IGF-I) and Humanin, protected these primary neurons from V642I-AbetaPP-induced cytotoxicity. Furthermore, interleukin-6 and -11 also attenuated this cytotoxicity. This study demonstrated that the signaling pathway activated by mutated AbetaPP in the primary neurons is the same as that by the other artificial insults such as antibody binding to APPP and the artificial dimerization of cytoplasmic domain of AbetaPP. The potential of neurotrophic factors and cytokines in AD therapy is also indicated. (C) 2004 Wiley-Liss, Inc.

The 24-residue peptide Humanin (HN) protects neuronal cells from insults of various Alzheimer's disease (AD) genes and Abeta by forming a homodimer. We have previously shown that P3A, S7A, C8A, L9A, L12A, T13A, S14A and P19A mutations nullify the neuroprotective function of HN [Yamagishi, Y., Hashimoto, Y., Niikura, T. & Nishimoto, I. (2003) Peptides, 24, 585-595]. Here we examined whether any of these 'null' mutants could function as dominant-negative mutants. Homodimerization-defective mutants, P3A-, L12A-, S14A- and P19A-HN, specifically blocked neuroprotection by HN, but not by activity-dependent neurotrophic factor. Furthermore, insertion of S7A, the mutation that blocks the homodimerization of HN, but not insertion of G5A abolished the antagonizing function of L12A-HN. While L12A-HN and G5A/L12A-HN actually inhibited HN homodimerization, S7A/L12A-HN had no effect. These data indicate that P3A-, L12A-, S14A- and P19A-HN function as HN antagonists by forming an inactive dimer with HN. This study provides a novel insight into the understanding of the in vivo function of HN, as well as into the development of clinically applicable HN neutralizers.

Certain cases of familial Alzheimer's disease are caused by mutants of amyloid-beta precursor protein (AbetaPP), including V642I-AbetaPP, K595N/M596L-AbetaPP (NL-AbetaPP), A617G-AbetaPP, and L648P-AbetaPP. By using an unbiased functional screening with transfection and expression of a human brain cDNA library, we searched for genes that protect neuronal cells from toxicity by V642I-AbetaPP. One protective clone was identical to the human GTX, a neuronal homeobox gene. Human Gtx (hGtx) inhibited caspase inhibitor-sensitive neuronal cell death not only by V642I-AbetaPP but also by L648P-, NL-, A617G-AbetaPP, apolipoprotein E4, and Abeta. The region of hGtx responsible for this rescue function was specified to be its homeodomain (Lys(148)-His(207)). The rescue function was shared by DLX4, a distal-less family gene with a homeodomain only 38.3% homologous to that of hGtx, suggesting that this function would be generally shared by homeodomains. The neuroprotective function of hGtx was attributable to hGtx-stimulated production and secretion of insulin-like growth factor-I. This study provides molecular clues to understand how neuronal cells developmentally regulate themselves against cell death as well as to develop reagents effective in curative therapeutics of Alzheimer's disease.

Presenilin (PS)1 and its mutants, which consist of the N-terminal and C-terminal fragments, cause certain familial forms of Alzheimer's disease (FAD). Our earlier studies found that FAD-linked M146L-PS1 causes neuronal cell death through nitrogen oxide synthase (NOS) and that FAD-linked N141I-PS2, another member of the PS family, causes neuronal cell death through NADPH oxidase. In this study, we examined 27 different FAD-linked mutants of PS1, and found that PS1 mutants with mutations in the N-terminal fragment caused NOS inhibitor (NOSI)-sensitive neuronal cell death; in contrast, the PS1 mutants with mutations in the C-terminal fragment caused NOSI-resistant neuronal cell death. The former toxicity was resistant to the specific NADPH oxidase inhibitor apocynin and was inhibited by Humanin (HN), a newly identified neuroprotective factor against Alzheimer's disease (AD)-relevant insults, but not by insulin-like growth factor-I (IGF-1). In contrast, the latter toxicity was sensitive to apocynin and inhibited by both IGF-I and HN. This study indicates for the first time that N- and C-terminal fragment PS1 mutants can generate distinct neurotoxic signals, which will provide an important clue to the understanding of the entire array of neurotoxic signals generated by FAD-causative mutations of PS1. (C) 2003 Wiley-Liss, Inc.

Insulin-like growth factor-binding protein-3 (IGFBP-3) regulates IGF bioactivity and also independently modulates cell growth and survival. By using a yeast two-hybrid screen to identify IGFBP-3-interacting proteins, we cloned humanin (HN) as an IGFBP-3-binding partner. HN is a 24-aa peptide that has been shown to specifically inhibit neuronal cell death induced by familial Alzheimer's disease mutant genes and amyloid-beta (Abeta). The physical interaction of HN with IGFBP-3 was determined to be of high affinity and specificity and was confirmed by yeast mating, displaceable pull-down experiments with (His)-6-tagged HN, and ligand blot experiments. Coimmunoprecipitation of IGFBP-3 and HN from mouse testes confirmed the interaction in vivo. In crosslinking experiments, HN bound IGFBP-3 but did not compete with IGF-I-IGFBP-3 binding; competitive ligand dot blot experiments revealed the 18-aa heparin-binding domain of IGFBP-3 as the binding site for HN. Alanine scanning determined that F6A-HN mutant does not bind IGFBP-3. HN but not F6A-HN inhibited IGFBP-3-induced apoptosis in human glioblastoma-A172. In contrast, HN did not suppress IGFBP-3 response in SH-SY5Y neuroblastoma and mouse cortical primary neurons. In primary neurons, IGFBP-3 markedly potentiated HN rescue ability from Abeta(1-43) toxicity. In summary, we have identified an interaction between the survival peptide HN and IGFBP-3 that is pleiotrophic in nature and is capable of both synergistic and antagonistic interaction. This interaction may prove to be important in neurological disease processes and could provide important targets for drug development.

The 24-residue peptide Humanin (HN), containing two Ser residues at positions 7 and 14, protects neuronal cells from insults of various Alzheimer's disease (AD) genes and Abeta. It was not known why the rescue function of (S14G)HN is more potent than HN by two to three orders of magnitude. Investigating the possibility that the post-translational modification of Ser14 might play a role, we found that HN with d-Ser at position 14 exerts neuroprotection more potently than HN by two to three orders of magnitude, whereas d-Ser7 substitution does not affect the rescue function of HN. On the other hand, S7A substitution nullified the HN function. Multiple series of experiments indicated that Ser7 is necessary for self-dimerization of HN, which is essential for neuroprotection by this factor. These findings indicate that the rescue function of HN is quantitatively modulated by d-isomerization of Ser14 and Ser7-relevant dimerization, allowing for the construction of a very potent HN derivative that was fully neuroprotective at 10 pm against 25 mum Abeta1-43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics.