Intracellular domains of amyloid precursor-like protein 2 interact with CP2 transcription factor in the nucleus and induce glycogen synthase kinase-3beta expression.

Amyloid precursor protein (APP) is a member of a gene family that includes two APP-like proteins, APLP1 and 2. Recently, it has been reported that APLP1 and 2 undergo presenilin-dependent gamma-secretase cleavage, as does APP, resulting in the release of an approximately 6 kDa intracellular C-terminal domain (ICD), which can translocate into the nucleus. In this study, we demonstrate that the APLP2-ICDs interact with CP2/LSF/LBP1 (CP2) transcription factor in the nucleus and induce the expression of glycogen synthase kinase 3beta (GSK-3beta), which has broad-ranged substrates such as tau- and beta-catenin. The significance of this finding is substantiated by the in vivo evidence of the increase in the immunoreactivities for the nuclear C-terminal fragments of APLP2, and for GSK-3beta in the AD patients' brain. Taken together, these results suggest that APLP2-ICDs contribute to the AD pathogenesis, by inducing GSK-3beta expression through the interaction with CP2 transcription factor in the nucleus.

Ionic effects of the Alzheimer's disease beta-amyloid precursor protein and its metabolic fragments.

Alzheimer's disease is a progressive dementia characterized in part by deposition of proteinaceous plaques in various areas of the brain. The main plaque protein component is beta-amyloid, a metabolic product of the beta-amyloid precursor protein. Substantial evidence has implicated beta-amyloid (and other amyloidogenic fragments of the precursor protein) with the neurodegeneration observed in Alzheimer's disease. Recently, beta-amyloid precursor protein and its amyloidogenic metabolic fragments have been shown to alter cellular ionic activity, either through interaction with existing channels or by de novo channel formation. Such alteration in ionic homeostasis has also been linked with cellular toxicity and might provide a molecular mechanism underlying the neurodegeneration seen in Alzheimer's disease.

Use-dependent effects of amyloidogenic fragments of (beta)-amyloid precursor protein on synaptic plasticity in rat hippocampus in vivo.

The Alzheimer's disease-related beta-amyloid precursor protein (beta-APP) is metabolized to a number of potentially amyloidogenic peptides that are believed to be pathogenic. Application of relatively low concentrations of the soluble forms of these peptides has previously been shown to block high-frequency stimulation-induced long-term potentiation (LTP) of glutamatergic transmission in the hippocampus. The present experiments examined how these peptides affect low-frequency stimulation-induced long-term depression (LTD) and the reversal of LTP (depotentiation). We discovered that beta-amyloid peptide (Abeta1-42) and the Abeta-containing C -terminus of beta-APP (CT) facilitate the induction of LTD in the CA1 area of the intact rat hippocampus. The LTD was frequency- and NMDA receptor-dependent. Thus, although low-frequency stimulation alone was ineffective, after intracerebroventricular injection of Abeta1-42, it induced an LTD that was blocked by d-(-)-2-amino-5-phosphonopentanoic acid. Furthermore, an NMDA receptor-dependent depotentiation was induced in a time-dependent manner, being evoked by injection of CT 10 min, but not 1 hr, after LTP induction. These use- and time-dependent effects of the amyloidogenic peptides on synaptic plasticity promote long-lasting reductions in synaptic strength and oppose activity-dependent strengthening of transmission in the hippocampus. This will result in a profound disruption of information processing dependent on hippocampal synaptic plasticity.

Neural stem cell transplantation at critical period improves learning and memory through restoring synaptic impairment in Alzheimer's disease mouse model.

Alzheimer's disease (AD) is characterized by neuronal loss in several regions of the brain. Recent studies have suggested that stem cell transplantation could serve as a potential therapeutic strategy to halt or ameliorate the inexorable disease progression. However, the optimal stage of the disease for stem cell transplantation to have a therapeutic effect has yet to be determined. Here, we demonstrated that transplantation of neural stem cells into 12-month-old Tg2576 brains markedly improved both cognitive impairments and neuropathological features by reducing ß-amyloid processing and upregulating clearance of ß-amyloid, secretion of anti-inflammatory cytokines, endogenous neurogenesis, as well as synapse formation. In contrast, the stem cell transplantation did not recover cognitive dysfunction and ß-amyloid neuropathology in Tg2576 mice aged 15 months when the memory loss is manifest. Overall, this study underscores that stem cell therapy at optimal time frame is crucial to obtain maximal therapeutic effects that can restore functional deficits or stop the progression of AD.

Overexpression of short heterodimer partner recovers impaired glucose-stimulated insulin secretion of pancreatic beta-cells overexpressing UCP2.

The short heterodimer partner (SHP) (NR0B2) is an orphan nuclear receptor whose function in pancreatic beta-cells is unclear. Mitochondrial uncoupling protein (UCP2) in beta-cells is upregulated in obesity-related diabetes, causing impaired glucose-stimulated insulin secretion (GSIS). We investigated whether SHP plays a role in UCP2-induced GSIS impairment. We overexpressed SHP in normal islet cells and in islet cells overexpressing UCP2 by an adenovirus-mediated infection technique. We found that SHP overexpression enhanced GSIS in normal islets, and restored GSIS in UCP2-overexpressing islets. SHP overexpression increased the glucose sensitivity of ATP-sensitive K+ (KATP) channels and enhanced the ATP/ADP ratio. A peroxisome proliferator-activated receptor gamma (PPARgamma) antagonist, GW9662, did not block the SHP effect on GSIS. SHP overexpression also corrected the impaired sensitivity of UCP2-overexpressing beta-cells to methylpyruvate, another energy fuel that bypasses glycolysis and directly enters the Krebs cycle. KATP channel inhibition mediated by dihydroxyacetone, which gives reducing equivalents directly to complex II of the electron transport system, was similar in Ad-Null-, Ad-UCP2- and Ad-UCP2+Ad-SHP-infected cells. The mitochondrial metabolic inhibitor sodium azide totally blocked the effect of SHP overexpression on GSIS. These results suggest that SHP positively regulates GSIS in beta-cells and restores glucose sensitivity in UCP2-overexpressing beta-cells by enhancing mitochondrial glucose metabolism, independent of PPARgamma activation.

Lack of interactions between amyloid precursor protein and hydrophilic domains of presenilin 1 and 2 using the yeast two hybrid system.

Mutations in the two related genes, presenilin 1 (PS1) and presenilin 2 (PS2), which are predicted multispanning membrane proteins, are responsible for the majority of early-onset familial Alzheimer's disease (FAD). To demonstrate direct interactions between presenilins (PS) and amyloid precursor protein (APP), the authors utilized a yeast two-hybrid system. Various hydrophilic domains derived from PS and those of APP were coexpressed in yeast and tested for the interaction. No detectable interactions were found in any PS/APP set examined. The authors' studies suggest that PS and APP do not interact through their hydrophilic domains in yeast, raising the possibility that interaction may occur indirectly or require proper conformation or subunit formation.

Progesterone inhibits the estrogen-induced prolactin gene expression in the rat pituitary.

The present study examines the inhibitory action of progesterone (P) on prolactin (PRL) gene expression in the anterior pituitary of ovariectomized, estradiol (OVX+E) treated adult rats. A single injection of P (1 mg) was administered s.c. to OVX+E treated rats, and animals were killed at 3, 6, 9, and 36 h following P administration. Northern blot analysis showed that P suppressed the E-enhanced PRL mRNA level at 3, 6, and 9 h, but not at 36 h following P administration. When the second injection of P (1 mg) was given at 30 h after the first P, it again suppressed the E-induced PRL mRNA level, indicating that the inhibitory effect of P may not last until 36 h. The inhibitory action of P was dose-dependent, and the pretreatment of RU486 (100 micrograms/rat), a P receptor antagonist at 1 h before P injection partially restored PRL mRNA level which was inhibited by P. These data indicate that P plays a crucial role in the regulation of PRL gene expression in the rat pituitary.

Inhibition of the NGF and IL-1beta-induced expression of Alzheimer's amyloid precursor protein by antisense oligonucleotides.

Alzheimer's disease (AD) is a neurodegenerative disorder of the brain characterized by the extracellular deposition of amyloid in senile plaques and along the walls of the cerebral vasculature. The principal constituent of amyloid deposit is amyloid beta peptide (Abeta) derived from its larger precursor protein, amyloid precursor protein (APP). The overexpression of APP is known to be a risk factor for Abeta deposit in AD and in Down syndrome (DS). The inhibition of APP expression has been thought to be beneficial to patients with AD and DS. In this study, we investigated the effects of antisense oligonucleotide (AO) on the overexpression of APP induced by IL-1beta and NGF. Using phosphorothioate-oligonucleotides against initiation codon significantly reduced the protein levels of APP induced by NGF and IL-1beta to basal level in PC12 cell culture systems. These results showed that these antisense oligonucleotides may have a potential to be a therapeutic agent for some patients with AD and DS.

Negative regulation of gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor gene expression by a gonadotrophin-releasing hormone agonist in the rat hypothalamus.

There exists evidence for the presence of ultrashort loop feedback circuits of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. It is, however, uncertain whether a similar mechanism is involved in the regulation of GnRH gene expression in vivo. Furthermore, little is known about the regulation of GnRH receptor (GnRHR) expression in the brain. In the present study, we examined the regulation of GnRH and its receptor gene expression by GnRH in vivo. A GnRH agonist, [D-Ala6, des-Gly10]GnRH-ethylamide (des-Gly GnRH), was administered by intracerebroventricular (i.c.v.) injection via the lateral ventricle of ovariectomized and estradiol (OVX + E)-treated rats. The amounts of GnRH and GnRHR mRNA were measured in the preoptic area (POA) and posterior mediobasal hypothalamus (pMBH) micropunch samples from individual rat brain slices by respective competitive reverse transcription-polymerase chain reactions. The i.c.v. administration of des-Gly GnRH significantly decreased GnRH and GnRHR mRNA expression in a dose-and time-related manner: des-Gly GnRH (6 ng) suppressed GnRH and GnRHR mRNA expression within 2 h, and the suppression was maintained without significant variation until 8 h after treatment. Treatment with Antide, [N-Ac-D-Nal(2)1, pCl-D-Phe2, D-Pal(3)3, Lys(Nic)5, D-Lys(Nic)6, Lys(iPR)8, D-Ala10]GnRH (10 ng), a potent GnRH antagonist, did not alter GnRH mRNA expression, but prevented des-Gly GnRH-induced suppression of GnRH mRNA expression. Antide alone decreased GnRHR mRNA expression, but failed to alter agonist-induced suppression of GnRHR mRNA expression. These results demonstrate the existence of an ultrashort loop feedback mechanism for GnRH gene expression in the POA, along with homologous down-regulation of GnRHR mRNA expression in the pMBH.

Molecular physiology, biochemistry, and pharmacology of Alzheimer's amyloid precursor protein (APP).

The function of APP is not yet known in detail but growing evidence exists that APP may mediate cell interactions with the cell surface or soluble glycoproteins and defense mechanisms in the CNS involving the immune system. We describe here the finding that almost all CD4+ lymphocytes and the majority of CD8+ lymphocytes were positive for A beta and the antibodies against A beta or APP did not inhibit the [3H]-thymidine uptake of mitogen-treated lymphocytes significantly. There were no differences in the A beta immunoreactivity on the cell surface of lymphocytes between Alzheimer's disease (AD) and control samples. Excessive amyloidogenic pathway of APP processing may be the final common pathway involved in the pathogenesis of AD. Thus, the identification of proteases or factors leading to aberrant proteolysis which process APP to yield a variety of potentially amyloidogenic fragments would promise pharmacological targets to develop anti-AD drugs. In attempts to define the proteases or factors which alter the balance between nonamyloidogenic and amyloidogenic processing pathways, our study indicates that thrombin or acetylcholinesterase(AChE)-associated protease may be involved in the amyloidogenic processing pathway of APP in vivo to generate amyloidogenic intermediates linked to amyloid deposition. Highly specific and dose-dependent direct modulation of APP processing by biologically available metal ions including Ca2+, Zn2+, Fe2+/Fe3+ and Al3+ suggest the disrupted metal homeostasis as factors leading to overaccumulation of APP and subsequent aberrant proteolysis utilizing excessive amyloidogenic processing pathway. There is mounting evidence that at least some of the neurotoxicity associated with AD is due to fragments from APP. Most research has focused on the toxic effect and the ion channel activity of A beta in causation of the disease. The possible role of other cleaved products of APP is less clear. We investigated the channel-forming ability of various products of APP when applied to Xenopus oocytes and their neurotoxicity in vitro. CT105 peptide was found to be exceedingly potent at 500 nM concentration in forming nonselective ion channels during application from either outside or inside the oocyte and more toxic than either of the A beta fragments, A beta 25-35, or A beta 1-40. Taken together, these results suggest the possible involvement of CT peptide in inducing the neurotoxicity characteristic of AD through the direct damage on the cell membrane. Therefore, we hypothesize that amyloidogenic CT may make nonselective ion channels or pores in the membrane and may cause neuronal death in the early stage of AD and then further metabolized to more stable and less toxic A beta which may be finally deposited in the brain where it could inflict further toxicity to neurons. Here we report successful inhibition of APP gene expression by antisense oligodeoxynucleotides at the mRNA or the protein level in in vitro and cell culture systems.

Age-related changes in the expression of Alzheimer's beta APP in the brain of senescence accelerated mouse (SAM)-P/10.

Patients with Alzheimer's disease (AD) show loss of memory and cognitive deficits the molecular mechanisms of which are not completely known. We examined age-related changes in the expression levels of beta-amyloid precursor protein (beta APP) in the brain of the senescence accelerated mouse (SAM) P/10, which shows age-dependent brain atrophy and impairment in learning and memory, and in the senescence resistant mouse (SAM)-R/1 using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Levels of both beta APP mRNA and protein increased with age, reaching a peak at 8 months of age in the hippocampus of SAM-P/10. In contrast, beta APP protein level decreased with age in the hippocampus of SAM-R/1 while beta APP mRNA level did not change significantly. Levels of beta APP mRNA and protein showed no change with ageing in other brain regions, including cerebral cortex, thalamus/midbrain and cerebellum brain stem. These results suggest that the beta APP over-expression in the hippocampus might be related to the characteristic memory loss in SAM-P/10.

Block of LTP in rat hippocampus in vivo by beta-amyloid precursor protein fragments.

The effects of beta-amyloid precursor protein (beta-APP) fragments on plasticity of glutamtatergic synaptic transmission were examined in the hippocampus of urethane anaesthetized rats. I.c.v. injection of beta-amyloid (A beta) 1-40 and 1-42 and the C-terminal fragment CT105 greatly shortened the duration of high frequency stimulation-induced long-term potentiation (LTP) of field excitatory postsynaptic potentials in the CA1 area. Whereas in vehicle injected animals LTP was stable over a 5 h recording period, doses of these peptides (A beta 1-40, 0.4 and 3.5 nmol; A beta1-42, 0.01 nmol; CT105, 0.05 nmol) which did not affect baseline synaptic transmission abolished LTP within 3-5 h. The reduced duration of this form of synaptic plasticity may contribute to the cognitive deficits in Alzheimer's disease.

C-terminal fragment of amyloid precursor protein inhibits calcium uptake into rat brain microsomes by Mg2+-Ca2+ ATPase.

Numerous lines of evidence suggest that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid beta peptide (A beta). However, the possible role of other cleaved products of APP is less clear. In this study, the effects of a recombinant carboxy terminal 105 amino acid (CT105) fragment of APP on the calcium uptake by endoplasmic reticulum Mg2+-Ca2+ ATPase, the major mechanism for sequestering calcium in this organelle, were investigated. We found that CT 105 is a potent inhibitor of Mg2+-Ca2+ ATPase of endoplasmic reticulum, whereas A beta shows no effect. These results demonstrate that CT 105 inhibits the ability of brain microsomes to sequester calcium and suggest that this inhibitory effect of CT 105 may contribute to disruption of intracellular calcium concentration, possibly being involved in inducing the neural toxicity characteristic of AD.

C-terminal fragment of Alzheimer's amyloid precursor protein inhibits sodium/calcium exchanger activity in SK-N-SH cell.

Numerous lines of evidence suggest that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid beta peptide (Abeta). However, the possible role of other cleaved products of APP is less clear. We have previously shown that a recombinant carboxy-terminal 105 amino acid fragment (CT 105) of APP induced strong non-selective inward currents and also showed neurotoxicity in PC 12 cells and primary cortical neurons and blocked later phase of long term potentiation (LTP) in rat hippocampus in vivo. In this study, we investigated the effects of CT 105 on Na+-Ca2+ exchanger activity in SK-N-SH human neuroblastoma, in the presence of ouabain and monensin, which are considered to drive Na+-Ca2+ exchanger in the reverse mode. CT 105 inhibited the activity of this exchanger in SK-N-SH cells by approximately 50%.

Toxic effect of the beta-amyloid precursor protein C-terminus fragment and Na+/Ca2+ gradients.

There is evidence to suggest that certain metabolic fragments of the beta-amyloid precursor protein (betaAPP) containing the whole of the beta-amyloid (Abeta) sequence are toxic to cells. We showed previously that the 105-amino acid C-terminal peptide (CT105) fragment, incorporating Abeta, is particularly toxic to Xenopus oocytes as well as to mammalian neurons. Here, we investigated the contributions of Na+ and Ca2+ gradients to intracellular CT105-induced toxicity in oocytes, monitored by measuring the membrane resting potential. The concentration gradients of Na+ and Ca2+ were manipulated to determine the involvement of the trans-membrane concentration gradients of these ions in the mode of action of CT105. The results suggested that Na+ influx and intracellular events are mainly responsible for the observed CT105-induced toxicity.

Cation selective channels formed by a C-terminal fragment of beta-amyloid precursor protein.

The C-terminal 105 amino acid fragment of beta-amyloid precursor protein (CT105) is highly neurotoxic. To obtain insights into its cytotoxic effect, we examined the ionophoric effects of CT105 (10-1000 nM) on artificial lipid membranes. Macroscopic membrane conductance increased with CT105 concentration and its ionophoric effect was comparable to that of amyloid beta protein. The mean unitary conductance of CT105-induced channels was 120 pS and open-state probability was close to 1 at voltages from -80 to +80 mV. CT105induced channels were selective to cations (PK/ P(Cl) = 10.2), being most selective to Ca2+. These findings suggest that CT105 can cause direct neurotoxic effects by forming Ca2+ permeable cation channels on neuronal membranes.

The cDNA cloning and ontogeny of mouse alpha-synuclein.

Alpha-synuclein has been implicated in the pathogenesis of Parkinson's disease. To investigate the role of alpha-synuclein in the brain, the cDNA clone encoding the mouse cognate of the human alpha-synuclein was isolated from a mouse brain cDNA library. The open reading frame coded for 140 amino acids that share 95% identity with human alpha-synuclein. Northern blot analysis showed that alpha-synuclein mRNA was primarily expressed in brain and spleen of adult mouse. In situ hybridization histochemistry revealed the highest expression of alpha-synuclein mRNA in the hippocampal formation and neocortex of the adult mouse. alpha-Synuclein mRNA expression in the brain was first observed in the hippocampus and neocortex on postnatal day 1. Levels of alpha-synuclein mRNA in these forebrain areas were nearly maximal at postnatal day 7 and remained relatively high until the adult stage. alpha-Synuclein mRNA was expressed in the liver transiently during embryogenesis.

Post-ischemic changes in the expression of Alzheimer's APP isoforms in rat cerebral cortex.

A significant porportion (25%) of patients with Alzheimer's disease (AD) also shows vascular pathology. Recent ultrastructural studies demonstrated characteristic and extensive angio-architectural distortions of cerebral capillaries in AD brains. We examined the expression of APP mRNA isoforms of cerebral cortex after transient ischemia by middle cerebral artery occlusion, using RT-PCR. Neuronal damage and glial fibrillary acidic protein immunohistochemistry were also examined histologically. After transient ischemia, the Kunitz protease inhibitor-bearing isoforms (KPI-APP) were increased whereas APP 695, which lacks KPI domain, was decreased. Neuronal damage and GFAP-immunoreactive astrocytes were also observed. These results show that focal, transient ischemia alters KPI-APP/APP 695 ratio in cerebral cortex and this shift in APP isoforms could be related to neurodegeneration and/or activation of astrocytes during the ischemic process.

Cerebral cortical phospholipase A2 activity of senescence-accelerated mouse is increased in an age-dependent manner.

The PLA2 activities of both the cytosolic and the membrane fractions from the cerebral cortex of 17-month old senescence-accelerated mouse-prone/10 (SAM-P/10) were significantly increased by 1.3 times compared with those of 2-month old mice. The PLA2 activities were independent of Ca2+ ion and not affected either by dithiothreitol (DTT) or by trifluoromethyl ketone analogs of arachidonic acid (AACOCF3). The PLA2 activities were eluted in a single peak of molecular mass of 170 kDa, using a gel filtration column. These findings suggest that the enhanced PLA2 activity may be related to neuronal degeneration and accelerated senescence of SAM-P/10, and cerebral cortical PLA2 activity categorized as a Ca2+-independent PLA2.

Aggregation of amyloid precursor proteins by aluminum in vitro.

The effect of various metal ions on aggregation of human recombinant amyloid precursor protein (APP) in vitro was investigated based on characterizations of altered migration on SDS-PAGE or immunoblots. Most biological metal ions tested had no significant effect on aggregation of APP. In contrast, AlCl3 in particular promoted aggregation of APP or APP-CT105 in a dose dependent manner. This effect of AlCl3 on APP mobility shift was prevented or reversed by the metal chelator, EDTA. Amorphous aggregates were observed in AlCl3 treated APP when examined by EM. These results suggest that aluminum may play a role in the pathogenesis of AD by directly promoting aggregation of APP.