Iron dysregulates APP processing accompanying with sAPPα cellular retention and ß-secretase inhibition in rat cortical neurons.

Amyloid precursor protein (APP) and iron both play pivotal roles in the central nervous system, but whether and how iron influences the processing of endogenous APP in neurons remain unclear. Here, we investigated the regulatory effects and underlying mechanisms of iron on non-amyloidogenic and amyloidogenic processing of APP in rat primary cortical neurons. Treatment of the neurons with ferric ammonium citrate (FAC, 100 µmol/L) markedly facilitated the non-amyloidogenic processing of APP, as evidenced by a robust increase in α-secretase-derived carboxy-terminal fragment α (CTFα). Furthermore, the distribution of sAPPα was altered after iron treatment, and sAPPα remained in the cellular lysates instead of being secreted into the extracellular milieu. Moreover, the levels of APP amyloidogenic products, including sAPPß and Aß were both decreased. We further revealed that FAC did not alter the expression of ß-secretase, but significantly suppressed its enzymatic activity in iron-treated neurons. In a cell-free ß-secretase activity assay, FAC dose-dependently inhibited the activity of purified ß-secretase with an IC50 value of 21.67 µmol/L. Our data provide the first evidence that iron overload alters the neuronal sAPPα distribution and directly inhibits ß-secretase activity. These findings shed light on the regulatory mechanism of bio-metals on APP processing.

Microsecond molecular dynamics simulation of Aß42 and identification of a novel dual inhibitor of Aß42 aggregation and BACE1 activity.

AIM: To study the conformational changes of Aß42 and discover novel inhibitors of both Aß42 aggregation and ß-secretase (BACE1). METHODS: A molecular dynamics (MD) simulation at a microsecond level was performed to explore stable conformations of Aß42 monomer in aqueous solution. Subsequently, structure-based virtual screening was used to search for inhibitors of both Aß42 aggregation and BACE1. Protein purification and in vitro activity assays were performed to validate the inhibition of the compounds identified via virtual screening. RESULTS: The initial α-helical conformation of Aß42, which was unstable in aqueous solution, turned into a ß-sheet mixed with a coil structure through a transient and fully random coil. The conformation of Aß42 mainly comprising ß-sheets and coils structure was used for further virtual screening. Five compounds were identified as inhibitors for Aß42 aggregation, and one of them, AE-848, was discovered to be a dual inhibitor of both Aß42 aggregation and BACE1, with IC50 values of 36.95 µmol/L and 22.70 µmol/L, respectively. CONCLUSION: A helical to ß-sheet conformational change in Aß42 occurred in a 1.8 microsecond MD simulation. The resulting ß-sheet structure of the peptide is an appropriate conformation for the virtual screening of inhibitors against Aß42 aggregation. Five compounds were identified as inhibitors of Aß42 aggregation by in vitro activity assays. It was particularly interesting to discover a dual inhibitor that targets both Aß42 aggregation and BACE1, the two crucial players in the pathogenesis of Alzheimer's disease.

L655,240, acting as a competitive BACE1 inhibitor, efficiently decreases ß-amyloid peptide production in HEK293-APPswe cells.

AIM: To identify a small molecule L655,240 as a novel ß-secretase (BACE1) inhibitor and to investigate its effects on ß-amyloid (Aß) generation in vitro. METHODS: Fluorescence resonance energy transfer (FRET) was used to characterize the inhibitory effect of L655,240 on BACE1. Surface plasmon resonance (SPR) technology-based assay was performed to study the binding affinity of L655,240 for BACE1. The selectivity of L655,240 toward BACE1 over other aspartic proteases was determined with enzymatic assay. The effects of L655,240 on Aß40, Aß42, and sAPPß production were studied in HEK293 cells stably expressing APP695 Swedish mutant(K595N/M596L) (HEK293-APPswe cells). The activities of BACE1, γ-secretase and α-secretase were assayed, and both the mRNA and protein levels of APP and BACE1 were evaluated using real-time PCR (RT-PCR) and Western blot analysis. RESULTS: L655,240 was determined to be a competitive, selective BACE1 inhibitor (IC(50)=4.47±1.37 µmol/L), which bound to BACE1 directly (K(D)=17.9±0.72 µmol/L). L655,240 effectively reduced Aß40, Aß42, and sAPPß production by inhibiting BACE1 without affecting the activities of γ-secretase and α-secretase in HEK293-APPswe cells. L655,240 has no effect on APP and BACE1 mRNA or protein levels in HEK293-APPswe cells. CONCLUSION: The small molecule L655,240 is a novel BACE1 inhibitor that can effectively decreases Aß production in vitro, thereby highlighting its therapeutic potential for the treatment of Alzheimer's disease.

[Advances in molecular genetics of schizophrenia].

Schizophrenia (MIM 181500) is a complex disorder affecting approximately 1% of the population worldwide. Epidemiologic evidences, together with recent linkage and association studies, have clearly demonstrated the high heritability of schizophrenia (up to 80%). Uncovering the genetic mechanism of schizophrenia has became one of the greatest challenges for both psychiatry and genetics. In recent years, remarkable advances in the genetics of this disorder has been achieved with the rapid growth of human genome information and experiment technologies. Several candidate genes within some of the best-supported linkage regions have been reported and, more importantly, replicated. Moreover, these genes present a significant connection in the signaling pathways implicated in the development of schizophrenia, especially NMDA receptor-mediated glutamate transmission. In this review, we summarize the recent advances in the genetics of schizophrenia, focusing particularly on linkage disequilibrium analysis and the latest understanding of the neurobiology of the disorder.

Case-control study and transmission disequilibrium test provide consistent evidence for association between schizophrenia and genetic variation in the 22q11 gene ZDHHC8.

Genetic variants in the 22q11 gene ZDHHC8, which encodes a putative transmembrane palmitoyltransferase, has been associated to schizophrenia in family-based linkage disequilibrium (LD) studies. The single nucleotide polymorphism (SNP) rs175174 (A/G), which had the strongest association, has been shown recently to regulate the level of the fully functional transcript by modulating the retention of intron 4 of ZDHHC8. In this work, we genotyped three genetic variants within the ZDHHC8 locus and conducted association studies in both population- and family-based samples of the Han Chinese population. The three polymorphisms spanning approximately 5.5 Kb were detected to be in significant LD. Our results provided compelling supportive evidence for association of the variants within the ZDHHC8 locus with schizophrenia but revealed different risk allele at SNP rs175174. The G allele was significantly more common in cases than in controls (69.47 : 59.96%; P=0.000018) and excess transmission of the same allele was confirmed in the family-based transmission disequilibrium test (transmitted/non-transmitted=87 : 54; P=0.0055). Both sample sets even shared the same risk haplotype with similar frequency. Our current data presents consistent association results obtained from both case-control and family-based samples in a same laboratory under the same experimental condition. Despite the potential genetic heterogeneity, our independent findings further support that the 22q11 region is likely to harbor candidate schizophrenia susceptibility genes.