Eurycomanone from Eurycoma longifolia Jack upregulates neurotrophin-3 gene expression in retinal Müller cells in vitro.

Photoreceptor degeneration decreases light sensitivity and leads to vision loss and various retinal diseases. Neurotrophin-3, originating from Müller glial cells in the retina, plays a key role in protecting photoreceptors from damage induced by light or hypoxia. This neuroprotective approach is important because there are no established methods to regenerate lost photoreceptors. Dietary supplements are one of the useful methods for improving eye health. Eurycoma longifolia (E. longifolia) Jack, which is native to the tropical forest of Malaysia and other Southeast Asian countries, exhibits several medicinal properties. In the present study, we demonstrated that the water extract of E. longifolia roots enhanced neurotrophin-3 gene expression in primary rat Müller cells. Using a stepwise bioassay-guided fractionation and purification of E. longifolia root extracts, we isolated the active compound underlying neurotrophin-3 gene-enhancing activities. Mass spectrometry and nuclear magnetic resonance spectral data identified the compound as eurycomanone. This study provides evidence for the efficacy of E. longifolia and eurycomanone in enhancing neurotrophin-3 expression in Müller cells in vitro. Although the biological significance of this effect and its underlying mechanism remain to be elucidated, this study suggests that E. longifolia may be promising for improving eye health and must be further investigated.

Discovery of novel 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine derivatives as γ-secretase modulators (Part 2).

γ-Secretase modulators (GSMs), which lower pathogenic amyloid beta (Aß) without affecting the production of total Aß or Notch signal, have emerged as a potential therapeutic agent for Alzheimer's disease (AD). A novel series of 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine derivatives was discovered and characterized as GSMs. Optimization of substituents at the 8-position of the core scaffold using ligand-lipophilicity efficiency (LLE) as a drug-likeness guideline led to identification of various types of high-LLE GSMs. Phenoxy compound (R)-17 exhibited especially high LLE as well as potent in vivo Aß42-lowering effect by single administration. Furthermore, multiple oral administration of (R)-17 significantly reduced soluble and insoluble brain Aß42, and ameliorated cognitive deficit in novel object recognition test (NORT) using Tg2576 mice as an AD model.

Pharmacological properties of a novel and potent γ-secretase modulator as a therapeutic option for the treatment of Alzheimer's disease.

Previous studies of γ-secretase inhibitors (GSIs) and Notch-sparing GSIs have shown reduced amyloid-ß (Aß) peptide levels but increased Notch-related and -unrelated adverse effects. In this study, we examined the effects of compound-1 on Aß processing and cognitive function and assessed Notch-related and -unrelated adverse effects. Compound-1 reduced Aß40 and Aß42 levels but inversely increased Aß37 in Neuro2a cells, leading to no net changes in total Aß levels, indicating that compound-1 is a γ-secretase modulator (GSM). In time-course experiments, compound-1 reduced Aß40 and Aß42 levels in tris-soluble fractions, with peak reduction at approximately 3h after oral administration in C57BL mice. Moreover, at >1mg/kg, compound-1 dose dependently reduced Aß40 and Aß42 levels in Tg2576 mice. Chronic treatment with compound-1 in Tg2576 mice for 4 months significantly reduced both soluble and insoluble Aß42 levels and ameliorated cognitive impairments, even after drug withdrawal for 10 days following oral administration for 2 months. In contrast with compound-1, at 100-fold higher doses (100mg/kg), the GSI LY450139 decreased HES1 mRNA expression in thymus tissues and increased the intensity of periodic acid-Schiff (PAS)-positive areas in the intestine. Moreover, the Notch-sparing GSI BMS708163 led to amyloid precursor protein (APP)-ß-C-terminal fragment accumulation in mouse primary neurons. BMS708163 significantly hampered cognitive function in normal mice 1 month after administration, whereas compound-1 significantly improved cognitive function. Taken together, the present novel and orally active GSM is a promising molecule for the treatment of pathologies associated with Aß42 and Aß40.

Single administration of a novel γ-secretase modulator ameliorates cognitive dysfunction in aged C57BL/6J mice.

Mutations in presenilin 1 (PS1) and presenilin 2 (PS2) are known to cause early onset of Alzheimer's disease (AD). These proteins comprise the catalytic domain of γ-secretase, which catalyzes the cleavage of ß-amyloid (Aß) from amyloid precursor protein (APP). In recent reports, PS1 and PS2 were linked to the modulation of intracellular calcium ion (Ca(2+)) dynamics, a key regulator of synaptic function. Ca(2+) dysregulation and synaptic dysfunction are leading hypothesis of cognitive dysfunctions during aging and AD progression. Accordingly, manipulations of presenilins by small molecules may have therapeutic potential for the treatment of cognitive dysfunction. In an accompanying report, we showed that chronic treatment with compound-1, a novel γ-secretase modulator (GSM), reduced Aß production and ameliorated cognitive dysfunction in Tg2576 APP transgenic mice. Accordingly, in the present study we showed that single oral administration of compound-1 at 1 and 3mg/kg ameliorated cognitive dysfunction in aged non-transgenic mice. Moreover, compound-1 enhanced synaptic plasticity in hippocampal slices from aged C57BL/6J mice and increased messenger RNA (mRNA) expression of the immediate early gene c-fos, which has been shown to be related to synaptic plasticity in vivo. Finally, compound-1 modulated Ca(2+) signals through PS1 in mouse embryonic fibroblast cells. Taken together, compound-1 ameliorates both Aß pathology and age-related cognitive dysfunctions. Hence, compound-1 may have potential as an early intervention for the cognitive declines that are commonly diagnosed in aged subjects, such as mild cognitive impairment (MCI) and prodromal AD.

Discovery of novel 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine derivatives as γ-secretase modulators.

Novel 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine derivatives were designed, synthesized, and evaluated as γ-secretase modulators (GSMs). An optimization study of this series resulted in the identification of (R)-11j, which showed a potent Aß42-lowering effect, high bioavailability and good blood-brain barrier permeability in mice. Oral administration of (R)-11j significantly reduced brain Aß42 in mice at a dose of 10 mg/kg.

Design and synthesis of piperazine derivatives as a novel class of γ-secretase modulators that selectively lower Aß42 production.

Novel piperazine derivatives as γ-secretase modulators (GSMs) were prepared and tested for their ability to selectively lower Aß42 production. Lead compound 3, with selective Aß42-lowering activity, was modified by replacing its imidazolylphenyl moiety with an oxazolylphenyl moiety. Optimization of the urea group significantly improved mouse microsomal stability, while retaining both activity and selectivity. These efforts led to the successful identification of an orally available and brain-penetrant GSM, 6j, which selectively reduced brain Aß42 in mice.

Neuronal expression of F-box and leucine-rich-repeat protein 2 decreases over Braak stages in the brains of Alzheimer's disease patients.

BACKGROUND: The dysfunction of protein degradation through the ubiquitin-proteasome system is now widely accepted as one of the causes of Alzheimer's disease (AD), the pathological hallmarks of which are abnormal protein accumulation such as senile plaques and neurofibrillary tangles in the brain. OBJECTIVE: To examine the expression of F-box and leucine-rich-repeat protein 2 (FBL2), a member of the ubiquitin-protein ligase complex expected to be involved in the ubiquitin-proteasome system. METHODS AND RESULTS: We investigated the expression profile of FBL2 in the brains of AD patients by quantitative PCR and immunohistochemical analysis. In healthy subjects, the FBL2 mRNA level was very high in the brain when compared to other tissues. FBL2 immunoreactivities were detected in somata and dendrites in the neurons, but not detected in astrocytes or microglia. The FBL2 mRNA level decreased progressively in the brains of AD patients over Braak stages; this was more prominent in the temporal cortex (known to be a vulnerable region) than in the frontal cortex. Interestingly, the decrease was more severe in AD patients carrying the apolipoprotein E4 allele. The FBL2 IR also decreased over Braak stages, and was hardly detected at Braak stage 5 in both NeuN-positive and EAAC1-positive glutamatergic neurons. CONCLUSION: These results suggest that the involvement of the reduction of FBL2 level is related to AD progression.

FBL2 regulates amyloid precursor protein (APP) metabolism by promoting ubiquitination-dependent APP degradation and inhibition of APP endocytosis.

The ubiquitin-proteasome pathway is a major protein degradation pathway whose dysfunction is now widely accepted as a cause of neurodegenerative diseases, including Alzheimer's disease. Here we demonstrate that the F-box and leucine rich repeat protein2 (FBL2), a component of the E3 ubiquitin ligase complex, regulates amyloid precursor protein (APP) metabolism through APP ubiquitination. FBL2 overexpression decreased the amount of secreted amyloid ß (Aß) peptides and sAPPß, whereas FBL2 mRNA knockdown by siRNA increased these levels. FBL2 overexpression also decreased the amount of intracellular Aß in Neuro2a cells stably expressing APP with Swedish mutation. FBL2 bound with APP specifically at its C-terminal fragment (CTF), which promoted APP/CTF ubiquitination. FBL2 overexpression also accelerated APP proteasome-dependent degradation and decreased APP protein localization in lipid rafts by inhibiting endocytosis. These effects were not observed in an F-box-deleted FBL2 mutant that does not participate in the E3 ubiquitin ligase complex. Furthermore, a reduced insoluble Aß and Aß plaque burden was observed in the hippocampus of 7-month-old FBL2 transgenic mice crossed with double-transgenic mice harboring APPswe and PS1(M146V) transgenes. These findings indicate that FBL2 is a novel and dual regulator of APP metabolism through FBL2-dependent ubiquitination of APP.

Central forkhead domain of human TFIIE beta plays a primary role in binding double-stranded DNA at transcription initiation.

The human general transcription factor, TFIIE, consists of two subunits, alpha and beta. Structural analyses indicated the presence of a forkhead motif within the central region of TFIIEbeta. This motif was essential for transcription and possessed a double-stranded DNA-binding activity. Protein-DNA photo-cross-linking studies indicated that TFIIEbeta binds within the promoter region, adjacent to the transcription initiation site where promoter melting begins at transcription initiation. Furthermore, neither TFIIE nor the other general transcription factor TFIIH, were required for basal transcription of adenovirus major late promoter artificially pre-melted at the initiation site. These data suggest a model in which TFIIE binds to a position adjacent to the initiation site via the forkhead domain, enabling TFIIH to begin opening the promoter. Here, we used systematic point mutations to further investigate the functional roles of this domain. The mutant proteins were expressed in bacteria, purified and used to examine transcription of two different forms of template, phosphorylation of the C-terminal domain of RNA polymerase II, as well as dsDNA-binding. Taken together, our results strongly demonstrated that the primary function of the forkhead region is dsDNA-binding in transcription. In addition, we identified three positively charged lysine residues which play a key role in this function.

Studies of Schizosaccharomyces pombe TFIIE indicate conformational and functional changes in RNA polymerase II at transcription initiation.

The general transcription factor TFIIE plays essential roles in transcription by RNA polymerase II (PolII). Despite recent progress, the elucidation of its precise mechanisms including biological functions awaits further characterization. We report the isolation and characterization of Schizosaccharomyces pombe TFIIE (spTFIIE). Like human and other eukaryotic TFIIE proteins, spTFIIE consists of alpha and beta subunits and the genes encoding both subunits are essential for viability. Chromatin immunoprecipitation assays demonstrated that spTFIIE localizes to promoters in vivo. Mutational analysis of the C-terminal basic helix-loop region of TFIIEbeta, which is involved in the transition from transcription initiation to elongation, revealed that transcription-defective mutants affected in this region are also cold sensitive. The spTFIIEbeta subunit binds both spTFIIEbeta and spTFIIEalpha but spTFIIEalpha binds only spTFIIEbeta. These results indicate that TFIIE forms an alpha2beta2 heterotetramer in which two alphabeta heterodimers are connected via beta subunits. Further analysis of binding specificities showed that spTFIIEbeta binds the Rpb2 and Rpb12 subunits of PolII, whereas spTFIIEalpha predominantly binds Rpb5, which is located at the clamp region and changes conformation upon transcription initiation.

The carboxy terminus of the small subunit of TFIIE regulates the transition from transcription initiation to elongation by RNA polymerase II.

The general transcription factor TFIIE plays essential roles in both transcription initiation and the transition from initiation to elongation. Previously, we systematically deleted the structural motifs and characteristic sequences of the small subunit of human TFIIE (hTFIIE beta) to map its functional regions. Here we introduced point mutations into two regions located near the carboxy terminus of hTFIIE beta and identified the functionally essential amino acid residues that bind to RNA polymerase II (Pol II), the general transcription factors, and single-stranded DNA. Although most residues identified were essential for transcription initiation, use of an in vitro transcription assay with a linearized template revealed that several residues in the carboxy-terminal helix-loop region are crucially involved in the transition stage. Mutations in these residues also affected the ability of hTFIIE beta to stimulate TFIIH-mediated phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of Pol II. Furthermore, these mutations conspicuously augmented the binding of hTFIIE beta to the p44 subunit of TFIIH. The antibody study indicated that they thus altered the conformation of one side of TFIIH, consisting of p44, XPD, and Cdk-activating kinase subunits, that is essential for the transition stage. This is an important clue for elucidating the molecular mechanisms involved in the transition stage.