Secreted calmodulin-like skin protein ameliorates scopolamine-induced memory impairment.

Humanin, a short bioactive peptide, inhibits cell death in a variety of cell-based death models through Humanin receptors in vitro. In vivo, Humanin ameliorates both muscarinic receptor antagonist-induced memory impairment in normal mice and memory impairment in Alzheimer's disease (AD)-relevant mouse models including aged transgenic mice expressing a familial AD-linked gene. Recently, calmodulin-like skin protein (CLSP) has been shown to be secreted from skin tissues, contain a region minimally similar to the core region of Humanin, and inhibit AD-related neuronal death through the heterotrimeric Humanin receptor on the cell surface in vitro. As CLSP is much more potent than Humanin and efficiently transported through blood circulation across the blood-brain barrier to the central nervous system, CLSP is considered as a physiological agonist that binds to the heterotrimeric Humanin receptor and triggers the Humanin-induced signals in central nervous system. However, it remains unknown whether CLSP ameliorates memory impairment in mouse dementia models as Humanin does. In this study, we show that recombinant CLSP, administered intracerebroventricularly or intraperitoneally, ameliorates scopolamine-induced dementia in mice.

Functional expression of choline transporter-like protein 1 (CTL1) in human neuroblastoma cells and its link to acetylcholine synthesis.

We examined the molecular and functional characterization of choline uptake into human neuroblastoma cell lines (SH-SY5Y: non-cholinergic and LA-N-2: cholinergic neuroblastoma), and the association between choline transport and acetylcholine (ACh) synthesis in these cells. Choline uptake was saturable and mediated by a single transport system. Removal of Na(+) from the uptake buffer strongly enhanced choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium. The increase in choline uptake under Na(+)-free conditions was inhibited by a Na(+)/H(+) exchanger (NHE) inhibitor. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), NHE1 and NHE5 mRNA are mainly expressed. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. ChAT mRNA was expressed at a much higher level in LA-N-2 cells than in SH-SY5Y cells. The conversion of choline to ACh was confirmed in both cells, and was enhanced in Na(+)-free conditions. These findings suggest that CTL1 is functionally expressed in both SH-SY5Y and LA-N-2 cells and is responsible for choline uptake that relies on a directed H(+) gradient as a driving force, and this transport functions in co-operation with NHE1 and NHE5. Furthermore, choline uptake through CTL1 is associated with ACh synthesis in cholinergic neuroblastoma cells.

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells.

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K(m)) of 16.5 microM and a maximal velocity (V(max)) of 133.9 pmol/mg protein/min. The V(max) value of choline uptake was strongly enhanced in the absence of Na(+) without any change in K(m) values. The increase in choline uptake under Na(+)-free conditions was inhibited by Na(+)/H(+) exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H(+) electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.

Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells.

We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in component of choline uptake under Na(+)-free conditions was inhibited by a Na(+)/H(+) exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H(+) electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention.

Expression and functional characterization of choline transporter in human keratinocytes.

Choline is essential for synthesis of the major membrane phospholipid phosphatidylcholine. Moreover, it serves as a precursor for synthesis of the neurotransmitter acetylcholine (ACh). Keratinocytes of the epidermis synthesize and release ACh. The uptake of choline is the rate-limiting step in both ACh synthesis and choline phospholipid metabolism, and it is a prerequisite for keratinocyte proliferation. However, the nature of the choline transport system in keratinocytes is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake into cultured human keratinocytes. Choline uptake into keratinocytes was independent of extracellular Na(+), saturable, and mediated by a single transport system with an apparent Michaelis-Menten constant of 12.3 muM. Choline uptake was reduced when the keratinocyte membrane potential was depolarized by high K(+). These results provide evidence that the choline transport activity is potential-sensitive. Various organic cations inhibit the choline transport system. RT-PCR demonstrated that keratinocytes expressed mRNA for choline transporter-like protein 1 (CTL1), mainly the CTL1a subtype. The present biochemical and pharmacological data suggest that CTL1a is functionally expressed in human keratinocytes and is responsible for the uptake of choline and organic cations in these cells.

Structure analysis of activity-dependent neurotrophic factor 9 by circular dichroism and sedimentation equilibrium.

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 neuroprotective activity against V642I-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 secondary structure analysis of a potent Ser14Gly analog of antiAlzheimer peptide, Humanin, by circular dichroism.

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.

Neuronal cell death in Alzheimer's disease and a neuroprotective factor, humanin.

Brain atrophy caused by neuronal loss is a prominent pathological feature of Alzheimer's disease (AD). Amyloid beta (Abeta), the major component of senile plaques, is considered to play a central role in neuronal cell death. In addition to removal of the toxic Abeta, direct suppression of neuronal loss is an essential part of AD treatment; however, no such neuroprotective therapies have been developed. Excess amount of Abeta evokes multiple cytotoxic mechanisms, involving increase of the intracellular Ca(2+) level, oxidative stress, and receptor-mediated activation of cell-death cascades. Such diversity in cytotoxic mechanisms induced by Abeta clearly indicates a complex nature of the AD-related neuronal cell death. We have identified a 24-residue peptide, Humanin (HN), which suppresses in vitro neuronal cell death caused by all AD-related insults, including Abeta, so far tested. The anti-AD effect of HN has been further confirmed in vivo using mice with Abeta-induced amnesia. Altogether, such potent neuroprotective activity of HN against AD-relevant cytotoxicity both in vitro and in vivo suggests the potential clinical applications of HN in novel AD therapies aimed at controlling neuronal death.

Implanted cannula-mediated repetitive administration of Abeta25-35 into the mouse cerebral ventricle effectively impairs spatial working memory.

Amyloid beta (Abeta) is closely related to the onset of Alzheimer's disease (AD). To construct AD animal models, a bolus administration of a large dose of toxic Abeta into the cerebral ventricles of rodents has been performed in earlier studies. In parallel, a continuous infusion system via an osmotic pump into the cerebral ventricle has been developed to make a rat AD model. In this study, we developed a mouse AD model by repetitive administration of Abeta25-35 via a cannula implanted into the cerebral ventricle. Using this administration system, we reproducibly constructed a mouse with impaired spatial working memory. In accordance with the occurrence of the abnormal mouse behavior, we found that the number of choline acetyltransferase (ChAT)-positive neurons was reduced in paraventricular regions of brains of Abeta25-35-administered mice in a dose-dependent manner. Considering that the repetitive administration of a small dose of toxic Abeta via an implanted cannula leads to a brain status more resembling that of the AD patients than a bolus injection of a large dose of Abeta, and therapeutic as well as toxic agents are able to be repeatedly and reliably administered via an implanted cannula, we concluded that the implanted cannula-bearing AD mouse model is useful for development of new AD therapy.

A humanin derivative, S14G-HN, prevents amyloid-beta-induced memory impairment in mice.

Humanin (HN) is a 24-amino acid peptide that protects neuronal cells from death caused by Alzheimer's disease (AD)-related genes and amyloid-beta (Abeta). Multiple studies have revealed its biochemical and neuroprotective characteristics in vitro; however, little has been known regarding whether HN is effective in vivo in AD model systems. We examined the effect of S14G-HN, a 1,000-fold more potent derivative of HN in vitro, on amnesia induced by Abeta25-35 in mice. The Y-maze test revealed that at least 50 pmol of S14G-HN by intracerebroventricular injection prevented Abeta-induced impairment of short-term/spatial working memory; however, 5 nmol of S14A-HN, a neuroprotection-defective mutant in vitro, did not prevent Abeta-induced amnesia. These results are in agreement with the structure-function correlation shown previously in vitro. In the water-finding task, S14G-HN prevented prolongation of finding latency (the time to find water) observed in Abeta-amnesic mice, indicating that S14G-HN also blocked Abeta-induced impairment of latent learning. In accordance with these observations, immunohistochemical analysis showed that S14G-HN sustained the number of cholinergic neurons in the basal forebrain and the striata nearly to the normal level. Furthermore, genistein, a specific inhibitor of tyrosine kinases, blocked recovery from scopolamine-induced amnesia by S14G-HN, suggesting that certain tyrosine kinase(s) are involved in the inhibitory function of S14G-HN in vivo. Taking these findings together, we conclude that S14G-HN has rescue activity against memory impairment caused by AD-related insults in vivo by activating the same intracellular neuroprotective machinery as elucidated previously in vitro.

Neuroprotective effect of activity-dependent neurotrophic factor against toxicity from familial amyotrophic lateral sclerosis-linked mutant SOD1 in vitro and in vivo.

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.

Cytotoxic mechanisms by M239V presenilin 2, a little-analyzed Alzheimer's disease-causative mutant.

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 N141I-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.

Two serine residues distinctly regulate the rescue function of Humanin, an inhibiting factor of Alzheimer's disease-related neurotoxicity: functional potentiation by isomerization and dimerization.

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 A beta. 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 microM A beta1-43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics.

A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insults.

Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer's disease (AD)-related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways.

[Neuronal cell death by Alzheimer's disease-relevant insults and its rescue].

Neuronal cell death accounts for the clinical manifestations in Alzheimer's disease (AD). To establish the curative therapy of AD, neuroprotection is one of the primary therapeutic targets, and the elucidation of the mechanism of neuronal cell death is mandatory. Detailed characterization of neuronal cell death caused by familial AD (FAD)-linked mutant genes revealed that different cell death pathways are evoked by different types of mutants. Humanin (HN), a newly identified neuroprotective peptide, suppresses neuronal cell death caused by all known FAD mutants and A beta, while it has no effect on neuronal cell death caused by AD-irrelevant insults. The functional target of HN is the antagonism to neuronal death, not the modulation of A beta production, suggesting that HN-based medication can be combined with other remedies targeting A beta. HN is a promising seed for a novel therapy aiming at complete cure of AD through the suppression of neuronal loss.

Death and survival of neuronal cells exposed to Alzheimer's insults.

Neuronal cell death is the central abnormality occurring in brains suffering from Alzheimer's disease (AD). The notion that AD is a disease caused by loss of neurons points toward suppression of neuronal death as the most important therapeutic target. Nevertheless, the mechanisms for neuronal death in AD are still relatively unclear. Three known mutant genes cause familial AD (FAD): amyloid precursor protein, presenilin 1, and presenilin 2. Detailed analysis of cytotoxic mechanisms of the FAD-linked mutant genes reveals that they cause neuronal cell death at physiologically low expression levels. Unexpectedly, cytotoxic mechanisms vary depending on the type of mutations and genes, suggesting that various mechanisms for neuronal cell death are involved in AD patients. In support of this, activity-dependent neurotrophic factor, basic fibroblast growth factor, and insulin-like growth factor-I can completely protect neurons from beta-amyloid (A beta) cytotoxicity but exhibit incomplete or little effect on cytotoxicity by FAD mutant genes. By contrast, Humanin, a newly identified 24-residue peptide, suppresses neuronal cell death by various FAD mutants and A beta, whereas this factor has no effect on cytotoxicity from AD-irrelevant insults. Studies investigating death and survival of neuronal cells exposed to AD insults will open a new horizon in developing therapy aimed at neuroprotection.

Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer's disease-related insults.

An unbiased functional screening with brain cDNA library from an Alzheimer's disease (AD) brain identified a novel 24-residue peptide Humanin (HN), which suppresses AD-related neurotoxicity. As the 1567-base cDNA containing the open reading frame (ORF) of HN is 99% identical to mitochondrial 16S ribosomal RNA as well as registered human mRNA, it was elusive whether HN is produced in vivo. Here, we raised anti-HN antibody and found that long cDNAs containing the ORF of HN (HN-ORF) produced the HN peptide in mammalian cells, dependent on the presence of full-length HN-ORF. Immunoblot analysis detected a 3-kDa protein with HN immunoreactivity in the testis and the colon in 3-week-old mice and in the testis in 12-week-old mice. HN immunoreactivity was also detected in an AD brain, but little in normal brains. This study suggests that HN peptide could be produced in vivo, and would provide a novel insight into the pathophysiology of AD.

Molecular mechanisms for neuronal cell death by Alzheimer's amyloid precursor protein-relevant insults.

To develop a therapeutic intervention for Alzheimer's disease (AD), it is necessary to clarify the mechanisms underlying the pathogenesis of AD, in which senile plaques, neurofibrillary tangles and neuronal loss in the cerebrum are the central abnormalities. A number of studies have focused on the major component of the senile plaques, which is amyloid-beta (Abeta) and its precursor protein APP, and have investigated the roles of these molecules in the onset, progression and inhibition of AD. For multiple reasons, however, their roles in AD, especially in neuronal death, remain elusive and a unified concept for their roles has not yet been established. Recently, it has been found that APP functions normally as a neuronal surface transmembrane protein. In this article, we review the molecular mechanisms of neuronal cell death by these APP-relevant insults and discuss the functions of APP in regard to intracellular signal transducers, including c-Jun N-terminal kinase. We also revise the roles of Abeta in neuronal death and survival.