Berberine alters the processing of Alzheimer's amyloid precursor protein to decrease Abeta secretion

Berberine is an isoquinoline alkaloid isolated from Coptidis rhizoma, a major herb widely used in Chinese herbal medicine. Berberine's biological activity includes antidiarrheal, antimicrobial, and anti-inflammatory effects. Recent findings show that berberine prevents neuronal damage due to ischemia or oxidative stress and that it might act as a novel cholesterol-lowering compound. The accumulation of amyloid-beta peptide (Abeta) derived from amyloid precursor protein (APP) is a triggering event leading to the pathological cascade of Alzheimer's disease (AD); therefore the inhibition of Abeta production should be a rational therapeutic strategy in the prevention and treatment of AD. Here, we report that berberine reduces Abeta levels by modulating APP processing in human neuroglioma H4 cells stably expressing Swedish-type of APP at the range of berberine concentration without cellular toxicity. Our results indicate that berberine would be a promising candidate for the treatment of AD.     

Comparison of 5-HT4 and 5-HT7 receptor expression and function in the circular muscle of the human colon

Serotonin receptors are potential targets for treating functional bowel disorders. This study investigated the functional roles and expression of the 5-HT4 and the 5-HT7 receptor, which coexist in human colon circular smooth muscle. 5-HT3 receptor expression was also investigated. Part of the relaxant response to 5-HT was due to activation of 5-HT4 receptors as the apparent pKB value of the selective 5-HT4 antagonist, GR 113808, was 9.36. 5-HT4 mRNA levels were low in five tissues and undetectable in four others, but all responded to 5-HT with an EC50 value of 102.54+/-19.32 nM. The contribution of 5-HT7 receptors to the response was not readily demonstrated using the selective 5-HT7 antagonist, SB-269970, as its apparent pKB value of 7.19 (5-HT4 block with 1 microM GR 113808) was lower than the value obtained using the 5-HT7 guinea pig ileum assay (8.62). Nevertheless, the 5-HT7 receptor was expressed more consistently than the 5-HT4, but at similar levels. The 5-HT(3Ashort) and 5-HT(3B) subunits were co-expressed at similar levels, but the 5-HT(3Along) subunit was detected in only five of the nine samples tested. The findings show that 5-HT4-induced relaxation occurs at low to undetectable levels of tissue mRNA, as measured by qPCR. Although 5-HT7 receptor mRNA is detected at low, but consistent levels, the functional activity of this receptor is not readily identified given the currently available drugs.     

The crystal structure of death receptor 6 (DR6): a potential receptor of the amyloid precursor protein (APP)

Death receptors belong to the tumor necrosis factor receptor (TNFR) super family and are intimately involved in the signal transduction during apoptosis, stress response and cellular survival. Here we present the crystal structure of recombinantly expressed death receptor six (DR6), one family member that was recently shown to bind to the amyloid precursor protein (APP) and hence to be probably involved in the development of Alzheimer's disease. The extracellular cysteine rich region of DR6, the typical ligand binding region of all TNFRs, was refined to 2.2 Å resolution and shows that its four constituting cysteine rich domains (CRDs) are arranged in a rod-like overall structure, which presents DR6-specific surface patches responsible for the exclusive recognition of its ligand(s). Based on the structural data, the general ligand binding modes of TNFRs and molecular modeling experiments we were able to elucidate structural features of the potential DR6-APP signaling complex.     

GGA1 overexpression attenuates amyloidogenic processing of the amyloid precursor protein in Niemann-Pick type C cells

Alzheimer’s disease (AD) and a rare inherited disorder of cholesterol transport, Niemann-Pick type C (NPC) share several similarities including aberrant APP processing and increased Aβ production. Previously, we have shown that the AD-like phenotype in NPC model cells involves cholesterol-dependent enhanced APP cleavage by β-secretase and accumulation of both APP and BACE1 within endocytic compartments. Since retrograde transport of BACE1 from endocytic compartments to the trans-Golgi network (TGN) is regulated by the Golgi-localized γ-ear containing ADP ribosylation factor-binding protein 1 (GGA1), we analyzed in this work a potential role of GGA1 in the AD-like phenotype of NPC1-null cells. Overexpression of GGA1 caused a shift in APP processing towards the non-amyloidogenic pathway by increasing the localization of APP at the cell surface. However, the observed effect appear to be independent on the subcellular localization and phosphorylation state of BACE1. These findings show that the AD-like phenotype of NPC model cells can be partly reverted by promoting a non-amyloidogenic processing of APP through the upregulation of GGA1 supporting its preventive role against AD.     

Expression of tau reduces secretion of Abeta without altering the amyloid precursor protein content in CHOsw cells

Insoluble deposits of tau and amyloid precursor protein (APP) peptides Abeta characterize Alzheimer's disease. We studied the role of tau in the metabolism of APP in cells stably expressing APP Swedish mutation (CHOsw). Transient expression of tau in CHOsw cells caused morphological changes, bundling of microtubules and perinuclear aggregation of Golgi-derived vesicles. It also reduced the secretion of Abeta(1-40) and Abeta(1-42) without altering the APP steady state levels. This was accompanied by a reduction in the gamma-secretase and an increase in the insulin degrading enzyme activities. Our results suggest that tau may play an inhibitory role in the amyloidogenic activity of APP.     

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells

Serotonin 5-HT4 receptor isoforms are G protein-coupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT4R ligands did not influence the constitutive energy transfer of the h5-HT4(d)R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT4(d)R and h5-HT4(g)R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT4R and beta2-adrenergic receptor (beta2AR) led to their heterodimerization. Given the large number of h5-HT4R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.     

Epac signaling pathway involves STEF, a guanine nucleotide exchange factor for Rac, to regulate APP processing

The amyloid precursor protein (APP) is a key protein involved in the development of Alzheimer's disease. We previously identified a signal transduction secretory pathway in which the small G protein Rac sets downstream of the cAMP/Epac/Rap1 signalling cascade regulating the alpha cleavage of APP [Maillet, M. et al. (2003) Crosstalk between Rap and Rac regulates secretion of sAPP alpha. Nat. Cell Biol. 5, 633-639]. We now report that Rap1 can physically and specifically associate with the guanine nucleotide exchange factor (GEF) STEF through its TSS region. A deleted TSS domain of STEF cells fails to activate Rac1 and dramatically decreases secretion of the non-amyloidogenic soluble form of APP (sAPP alpha) induced by the cAMP-binding protein Epac. Altogether, our data show that upon Epac activation, Rap1 recruits STEF through its TSS region and activates Rac1, which mediates APP processing.     

ADAM19 autolysis is activated by LPS and promotes non-classical secretion of cysteine-rich protein 2

ADAM family proteins are type I transmembrane, zinc-dependent metalloproteases. This family has multiple conserved domains, including a signal peptide, a pro-domain, a metalloprotease domain, a disintegrin (DI) domain, a cysteine-rich (Cys) domain, an EGF-like domain, a transmembrane domain, and a cytoplasmic domain. The Cys and DI domains may play active roles in regulating proteolytic activity or substrate specificity. ADAM19 has an autolytic processing activity within its Cys domain, and the processing is necessary for its proteolytic activity. To identify a new physiological function of ADAM19, we screened for associating proteins by using the extracellular domain of ADAM19 in a yeast two-hybrid system. Cysteine-rich protein 2 (CRIP2) showed an association with ADAM19 through its DI and Cys domains. Sequence analysis revealed that CRIP2 is a secretable protein without a classical signal. CRIP2 secretion was increased by overexpression of ADAM19 and decreased by suppression of ADAM19 expression. Moreover, CRIP2 secretion increased in parallel with the autolytic processing of ADAM19 stimulated by lipopolysaccharide. These findings suggest that ADAM19 autolysis is activated by lipopolysaccharide and that ADAM19 promotes the secretion of CRIP2.     

Amyloid precursor protein traffics from the Golgi directly to early endosomes in an Arl5b‐ and AP4‐dependent pathway

The intracellular trafficking and proteolytic processing of the membrane‐bound amyloid precursor protein (APP) are coordinated events leading to the generation of pathogenic amyloid‐beta (Aβ) peptides. The membrane transport of newly synthesized APP from the Golgi to the endolysosomal system is not well defined, yet it is likely to be critical for regulating its processing by β‐secretase (BACE1) and γ‐secretase. Here, we show that the majority of newly synthesized APP is transported from the trans‐Golgi network (TGN) directly to early endosomes and then subsequently to the late endosomes/lysosomes with very little transported to the cell surface. We show that Arl5b, a small G protein localized to the TGN, and AP4 are essential for the post‐Golgi transport of APP to early endosomes. Arl5b is physically associated with AP4 and is required for the recruitment of AP4, but not AP1, to the TGN. Depletion of either Arl5b or AP4 results in the accumulation of APP, but not BACE1, in the Golgi, and an increase in APP processing and Aβ secretion. These findings demonstrate that APP is diverted from BACE1 at the TGN for direct transport to early endosomes and that the TGN represents a site for APP processing with the subsequent secretion of Aβ.      

Regulation of beta-amyloid precursor protein expression by brain-derived neurotrophic factor involves activation of both the Ras and phosphatidylinositide 3-kinase signalling pathways

Brain-derived neurotrophic factor (BDNF) stimulates beta-amyloid precursor protein (APP) promoter activity by a Ras-dependent mechanism in TrkB-expressing SH-SY5Y cells. To determine the signalling pathways involved in the BDNF-induced response, we have analysed the ability of TrkB mutated forms to mediate promoter stimulation. Brain-derived neurotrophic factor causes a significant induction of promoter activity and mutation K540R in the active site of TrkB completely abolishes the neurotrophin-induced response. A substitution of the Y484 residue by phenylalanine, which blocks binding of Shc, reduces the activation of APP promoter by BDNF by approximately 50% whereas mutation Y785P, which blocks binding of phospholipase C gamma, does not affect the response. In addition, the phosphatidylinositide 3-kinase (PI3K)-specific inhibitors wortmannin and LY294002 reduced BDNF-induced activation. In agreement with a participation of both Ras/MAPK- and PI3K/Akt-mediated mechanisms, transient expression of constitutive active forms of Ras, PI3K and other components of both signalling pathways led to a significant increase of APP promoter activity. Furthermore, the stimulation of the APP promoter by BDNF was completely precluded by expression of dominant-negative forms of Ras and PI3K effectors. Taken together, our results suggest that simultaneous activation of at least two signalling pathways, Ras/MAPK and PI3K/Akt, is necessary to mediate a full activation of the APP promoter by BDNF.     

Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E,and the mRNA 5′ cap

Initiation is the rate-limiting step during mRNA 5′ cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.     

Determination of the investigational anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid and its acyl glucuronide in Caco-2 monolayers by liquid chromatography with fluorescence detection: application to transport studies

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a potent cytokine inducer, with a bioavailability of >70% in the mouse. The aim of this study was to develop and validate HPLC methods for the determination of DMXAA and DMXAA acyl glucuronide (DMXAA-G) in the human intestinal cell line Caco-2 monolayers. The developed HPLC methods were sensitive and reliable, with acceptable accuracy (85-115% of true values) and precision (intra- and inter-assay CV < 15%). The total running time was within 6.8 min, with acceptable separation of the compounds of interest. The limit of quantitation (LOQ) values for DMXAA and DMXAA-G were 14.2 and 24 ng/ml, respectively. The validated HPLC methods were applied to examine the epithelial transport of DMXAA and DMXAA-G by Caco-2 monolayers. The permeability coefficient (Papp) values (overall mean +/- S.D., n = 3-9) of DMXAA over 10-500 microM were independent of concentration for both apical (AP) to basolateral (BL) (4.0 +/- 0.4 x 10(-5)cm/s) and BL-AP (4.3 +/- 0.5 x 10(-5)cm/s) transport, and of similar magnitude in either direction, with net efflux ratio (Rnet) values of 1-1.3. However, the Papp values for the BL to AP transport of DMXAA-G were significantly greater than those for the AP to BL transport, with Rnet values of 17.6, 6.7 and 4.5 at 50, 100 and 200 microM, respectively. Further studies showed that the transport of DMXAA-G was Na+- and energy-dependent, and inhibited by MK-571 [a multidrug resistance associated protein (MRP) 1/2 inhibitor], but not by verapamil and probenecid. These data indicate that the HPLC methods for the determination of DMXAA and DMXAA-G in the transport buffer were simple and reliable, and the methods have been applied to the transport study of both compounds by Caco-2 monolayers. DMXAA across Caco-2 monolayers was through a passive transcellular process, whereas the transport of DMXAA-G was mediated by MRP1/2.     

Role of sphingosine 1-phosphate and lysophosphatidic acid in fibrosis

This review highlights an emerging role for sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) in many different types of fibrosis. Indeed, both LPA and S1P are involved in the multi-process pathogenesis of fibrosis, being implicated in promoting the well-established process of differentiation of fibroblasts to myofibroblasts and the more controversial epithelial–mesenchymal transition and homing of fibrocytes to fibrotic lesions. Therefore, targeting the production of these bioactive lysolipids or blocking their sites/mechanisms of action has therapeutic potential. Indeed, LPA receptor 1 (LPA₁) selective antagonists are currently being developed for the treatment of fibrosis of the lung as well as a neutralising anti-S1P antibody that is currently in Phase 1 clinical trials for treatment of age related macular degeneration. Thus, LPA- and S1P-directed therapeutics may not be too far from the clinic. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.