Transcriptional downregulation of FAM3C/ILEI in the Alzheimer's brain.

Amyloid-ß (Aß) accumulation in the brain triggers the pathogenic cascade for Alzheimer's disease (AD) development. The secretory protein FAM3C (also named ILEI) is a candidate for an endogenous suppressor of Aß production. In this study, we found that FAM3C expression was transcriptionally downregulated in the AD brain. To determine the transcriptional mechanism of the human FAM3C gene, we delineated the minimal 5'-flanking sequence required for basal promoter activity. From a database search for DNA-binding motifs, expression analysis using cultured cells, and promoter DNA-binding assays, we identified SP1 and EBF1 as candidate basal transcription factors for FAM3C, and found that SMAD1 was a putative inducible transcription factor and KLF6 was a transcription repressor for FAM3C. Genomic deletion of the basal promoter sequence from HEK293 and Neuro-2a cells markedly reduced endogenous expression of FAM3C and abrogated SP1- or EBF1-mediated induction of FAM3C. Nuclear protein extracts from AD brains contained lower levels of SP1 and EBF1 than did those from control brains, although the relative mRNA levels of these factors did not differ significantly between the groups. Additionally, the ability of nuclear SP1 and EBF1 in AD brains to bind with the basal promoter sequence-containing DNA probe was reduced compared with the binding ability of these factors in control brains. Thus, the transcriptional downregulation of FAM3C in the AD brain is attributable to the reduced nuclear levels and genomic DNA binding of SP1 and EBF1. An expressional decline in FAM3C may be a risk factor for Aß accumulation and eventually AD development.

Protective efficacy of passive immunization with monoclonal antibodies in animal models of H5N1 highly pathogenic avian influenza virus infection.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.

Nicotinic acetylcholine receptor activation induces BACE1 transcription via the phosphorylation and stabilization of nuclear SP1.

Epidemiological studies have shown that cigarette smoking increases the risk of Alzheimer disease. However, inconsistent results have been reported regarding the effects of smoking or nicotine on brain amyloid ß (Aß) deposition. In this study, we found that stimulation of the nicotinic acetylcholine receptor (nAChR) increased Aß production in mouse brains and cultured neuronal cells. nAChR activation triggered the MEK/ERK pathway, which then phosphorylated and stabilized nuclear SP1. Upregulated SP1 acted on two recognition motifs in the BACE1 gene to induce its transcription, resulting in enhanced Aß production. Mouse brain microdialysis revealed that nAChR agonists increased Aß levels in the interstitial fluid of the cerebral cortex but caused no delay of Aß clearance. In vitro assays indicated that nicotine inhibited Aß aggregation. We also found that nicotine modified the immunoreactivity of anti-Aß antibodies, possibly through competitive inhibition and Aß conformation changes. Using anti-Aß antibody that was carefully selected to avoid these effects, we found that chronic nicotine treatment in Aß precursor protein knockin mice increased the Aß content but did not visibly change the aggregated Aß deposition in the brain. Thus, nicotine influences brain Aß deposition in the opposite direction, thereby increasing Aß production and inhibiting Aß aggregation.

Reduction of Amyloid-ß Production without Inhibiting Secretase Activity by MS-275.

Brain amyloid-ß (Aß) governs the pathogenic process of Alzheimer's disease. Clinical trials to assess the disease-modifying effects of inhibitors or modulators of ß- and γ-secretases have not shown clinical benefit and can cause serious adverse events. Previously, we found that the interleukin-like epithelial-to-mesenchymal transition inducer (ILEI, also known as FAM3C) negatively regulates the Aß production through a decrease in Aß immediate precursor, without the inhibition of ß- and γ-secretase activity. Herein, we found that MS-275, a benzamide derivative that is known to inhibit histone deacetylases (HDACs), exhibits ILEI-like activity to reduce Aß production independent of HDAC inhibition. Chronic MS-275 treatment decreased Aß deposition in the cerebral cortex and hippocampus in an Alzheimer's disease mouse model. Overall, our results indicate that MS-275 is a potential therapeutic candidate for efficiently reducing brain Aß accumulation.

Human CRB2 inhibits gamma-secretase cleavage of amyloid precursor protein by binding to the presenilin complex.

Drosophila Crumbs has been reported to attenuate Notch signaling by inhibition of gamma-secretase cleavage at the wing margins. gamma-Secretase is an intramembrane protease that is responsible for the generation of amyloid-beta (Abeta) peptides from the beta-amyloid precursor protein (APP). Here, we re-examined gamma-secretase inhibition by human CRB2, which is the most abundant Crumbs ortholog in the brain. Transfected CRB2 inhibited proteolytic production of Abeta and APP intracellular domains from APP C-terminal fragments in HEK293 and SH-SY5Y cells. Conversely, knockdown of endogenous CRB2 increased gamma-secretase cleavage products in SH-SY5Y cells. CRB2 inhibition of gamma-cleavage was also detected in cell-free assays. CRB2 interacted with the gamma-secretase complex, but was not a competitive substrate for gamma-cleavage. The transmembrane domain of CRB2 was indispensable for inhibition of Abeta generation and mediated CRB2 binding with the gamma-secretase complex. In addition, the cytoplasmic domain appeared to play a supportive role in gamma-secretase inhibition, whereas mutational disruption of the two protein-binding motifs involved in the formation of cell adhesion complexes did not affect gamma-secretase inhibition. Co-overexpression of presenilin-1 or APH-1 abrogated gamma-secretase inhibition probably through prevention of the incorporation of CRB2 into the gamma-secretase complex. Our results suggest that CRB2 functions as an inhibitory binding protein that is involved in the formation of a mature but inactive pool of the gamma-secretase complex.

Extracellular Release of ILEI/FAM3C and Amyloid-ß Is Associated with the Activation of Distinct Synapse Subpopulations.

BACKGROUND: Brain amyloid-ß (Aß) peptide is released into the interstitial fluid (ISF) in a neuronal activity-dependent manner, and Aß deposition in Alzheimer's disease (AD) is linked to baseline neuronal activity. Although the intrinsic mechanism for Aß generation remains to be elucidated, interleukin-like epithelial-mesenchymal transition inducer (ILEI) is a candidate for an endogenous Aß suppressor. OBJECTIVE: This study aimed to access the mechanism underlying ILEI secretion and its effect on Aß production in the brain. METHODS: ILEI and Aß levels in the cerebral cortex were monitored using a newly developed ILEI-specific ELISA and in vivo microdialysis in mutant human Aß precursor protein-knockin mice. ILEI levels in autopsied brains and cerebrospinal fluid (CSF) were measured using ELISA. RESULTS: Extracellular release of ILEI and Aß was dependent on neuronal activation and specifically on tetanus toxin-sensitive exocytosis of synaptic vesicles. However, simultaneous monitoring of extracellular ILEI and Aß revealed that a spontaneous fluctuation of ILEI levels appeared to inversely mirror that of Aß levels. Selective activation and inhibition of synaptic receptors differentially altered these levels. The evoked activation of AMPA-type receptors resulted in opposing changes to ILEI and Aß levels. Brain ILEI levels were selectively decreased in AD. CSF ILEI concentration correlated with that of Aß and were reduced in AD and mild cognitive impairment. CONCLUSION: ILEI and Aß are released from distinct subpopulations of synaptic terminals in an activity-dependent manner, and ILEI negatively regulates Aß production in specific synapse types. CSF ILEI might represent a surrogate marker for the accumulation of brain Aß.

Lipid class composition of membrane and raft fractions from brains of individuals with Alzheimer's disease.

Perturbation of the homeostasis of brain membrane lipids has been implicated in the pathomechanism of Alzheimer's disease (AD). The ε4 allele of the apolipoprotein E gene (APOE) confers an increased risk, in a dosage-dependent manner, for brain amyloid-ß accumulation and the development of sporadic AD. An effect of the APOE genotype on brain lipid homeostasis may underlie the AD risk associated with the ε4 allele. In this research, we examined an effect of APOE ε4 on the lipid class composition of crude membranes and raft-enriched fractions of brains. We applied enzymatic reaction-based methods for the quantification of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and sphingomyelin. Our results indicate that brain lipid class composition was neither significantly altered in AD subjects nor affected by the presence of the APOE ε4 allele.