The human serotonin 5-HT4 receptor regulates secretion of non-amyloidogenic precursor protein

The serotonin 5-HT(4) receptor has recently gained a lot of attention for its functional roles in central processes such as memory and cognition. In this study, we show that activation of the human 5-HT(4) (h5-HT(4)) receptor stimulates the secretion of the non-amyloidogenic soluble form of the amyloid precursor protein (sAPPalpha). 5-HT enhanced the level of secreted sAPPalpha in a time- and dose-dependent manner in Chinese hamster ovary cells stably expressing the h5-HT(4(e)) receptor isoform. The increase was inhibited by the selective 5-HT(4) receptor antagonist, GR113808. The 5-HT(4) selective agonists, prucalopride and renzapride, also increased secreted sAPPalpha in IMR32 human neuroblastoma cells. The stimulatory effect of 5-HT was mimicked by forskolin, a direct activator of adenylyl cyclase, and 8-bromo-cAMP, a membrane-permeant cAMP analogue. On the contrary, inhibition of protein kinase A (PKA) by H89 potentiated the 5-HT-induced increase in both secreted and cellular sAPPalpha. This phenomenon involves a novel PKA-independent stimulatory process that overcomes a PKA-dependent inhibitory one. Finally, activation of the h5-HT(4(e)) receptor did not modify extracellular amyloid beta-protein in Chinese hamster ovary cells transfected with the human APP695. Given the neuroprotective and enhancing memory effects of sAPPalpha, our results may open a new avenue for the treatment of Alzheimer's disease.     

Increased processing of APLP2 and APP with concomitant formation of APP intracellular domains in BDNF and retinoic acid-differentiated human neuroblastoma cells

The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPalpha, as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPalpha as well as the levels of AICDs were increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the alpha-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1.     

Sialylation enhances the secretion of neurotoxic amyloid-beta peptides

Alzheimer's disease (AD) is characterized by amyloid-beta peptide (Abeta) deposition in the brain. Abeta is produced by sequential cleavage of amyloid precursor protein (APP) by beta-secretase (BACE1: beta-site APP-cleaving enzyme 1) and gamma-secretase. Previously, we demonstrated that BACE1 also cleaves beta-galactoside alpha2,6-sialyltransferase (ST6Gal-I) and down-regulates its transferase activity. Here, we report that overexpression of ST6Gal-I in Neuro2a cells enhanced alpha2,6-sialylation of endogenous APP and increased the extracellular levels of its metabolites [Abeta by two-fold, soluble APPbeta (sAPPbeta) by three-fold and sAPPalpha by 2.5-fold). Sialylation-deficient mutant (Lec-2) cells secreted half as much Abeta as wild-type Chinese hamster ovary (CHO) cells. Furthermore, wild-type CHO cells showed enhanced secretion of the APP metabolites upon ST6Gal-I overexpression, whereas Lec-2 cells did not, indicating that the secretion enhancement requires sialylation of cellular protein(s). Secretion of metabolites from a mutant APP (APP-Asn467,496Ala) that lacked N-glycosylation sites was not enhanced upon ST6Gal-I overexpression, suggesting that the N-glycans on APP itself are required for the enhanced secretion. In the mouse brain, the amount of alpha2,6-sialylated APP appeared to be correlated with the sAPPbeta level. These results suggest that sialylation of APP promotes its metabolic turnover and could affect the pathology 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.     

Altered processing of amyloid precursor protein in the human neuroblastoma SH-SY5Y by chronic hypoxia

Alzheimer's disease (AD) is more prevalent following an ischemic or hypoxic episode, such as stroke. Indeed, brain levels of amyloid precursor protein (APP) and the cytotoxic amyloid beta peptide (Abeta) fragment are enhanced in these patients and in animal models following experimental ischaemia. We have investigated the effect of chronic hypoxia (CH; 2.5% O2, 24 h) on processing of APP in the human neuroblastoma, SH-SY5Y. We demonstrate that constitutive and muscarinic-receptor-enhanced secretion of the alpha-secretase cleaved fragment of APP, sAPPalpha, was reduced by approximately 60% in CH cells. The caspase inhibitor BOC-D(Ome)FMK did not reverse this effect of CH, and CH cells were as viable as controls, based on MTT assays. Thus, loss of sAPPalpha is not related to cell death or caspase processing of APP. Pre-incubation with antioxidants did not reverse the effect of CH, and the effect could not be mimicked by H2O2, discounting the involvement of reactive oxygen species in hypoxic loss of sAPPalpha. CH did not affect muscarinic activation of extracellular-signal regulated kinase. However, expression of ADAM 10 (widely believed to be alpha-secretase) was decreased approximately 50% following CH. Thus, CH selectively decreases processing of APP by the alpha-secretase pathway, most likely by decreasing levels of ADAM 10.     

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.     

The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT(2A) serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTT proliferation assay. We have demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT(2A) receptor present in this cell line is identical to the 5-HT(2A) receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT(2A) receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT(2A) receptor subtype, which is fully expressed in this cell line.     

A 5-HT7 Heteroreceptor-Mediated Inhibition of [3H]Serotonin Release in Raphe Nuclei Slices of the Rat: Evidence for a Serotonergic–Glutamatergic Interaction

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with [3H]serotonin ([3H]5-HT), superfused, and the electrically induced efflux of radioactivity was determined. The nonselective 5-HT receptor agonist 5-carboxamido-tryptamine (5-CT; 0.001 to 1 microM) inhibited the electrically stimulated [3H]5-HT overflow from raphe nuclei slices (IC50 of 3.34 +/- 0.37 nM). This effect of 5-CT on [3H]5-HT overflow was antagonized by the 5-HT7 receptor antagonist SB-258719 (10 microM) and the 5-HT(1B/1D) antagonist SB-216641 (1 microM), the IC50 values for 5-CT in the presence of SB-258719 and SB-216641 were 94.23 +/- 4.84 and 47.81 +/- 4.66 nM. The apparent pA2 values for SB-258719 and SB-216641 against 5-CT were 6.43 and 7.12, respectively. The inhibitory effect of 5-CT on [3H]5-HT overflow was weakly antagonized by 10 microM of WAY-100635, a 5-HT1A receptor antagonist (IC50 6.65 +/- 0.56 nM, apparent pA2 4.99). The antagonist effect of SB-258719 (10 microM) on 5-CT-evoked [3H]5-HT overflow inhibition was also determined in the presence of 1 microM SB-216641 or 1 microM SB-216641 and 10 microM WAY-100635, and additive interactions were found between the antagonists of 5-HT7 and 5-HT1 receptor subtypes. Addition of the Na+ channel blocker tetrodotoxin (1 microM) in the presence of SB-216641 (1 microM) and WAY-100635 (10 microM) attenuated the inhibitory effect of 5-CT on KCl-induced [3H]5-HT overflow. These findings indicate that 5-CT inhibits [3H]5-HT overflow from raphe nuclei slices of the rat by stimulation of 5-HT7 and 5-HT(1B/1D receptors, whereas the role of 5-HT1A receptors in this inhibition is less pronounced. They also suggest that 5-HT7 receptors are probably not located on serotonergic neurons and thus may serve as heteroreceptors in regulation of 5-HT release in the raphe nuclei. 5-CT (0.1 microM) also inhibited [3H]glutamate release, and SB-258719 (10 microLM) suspended this effect. We therefore speculated that the axon terminals of the glutamatergic cortico-raphe neurons may possess 5-HT7 receptors that inhibit glutamate release, which consequently leads to decreased activity of serotonergic neurons. The postulated glutamatergic-serotonergic interaction in the raphe nuclei was further evidenced by the finding that N-methyl-D-aspartate and AMPA enhanced [3H]5-HT release.     

Phosphatidylinositol 4-phosphate 5-kinase Iγ_v6, a new splice variant found in rodents and humans

It has been shown that anti-cancer drug induces secretion of serotonin (5-HT) from small intestine which activates serotonin type 3 (5-HT₃) receptor to cause nausea and vomiting. In general, antagonist for 5-HT₃ receptor is used as anti-emetics during chemotherapy. However, we found that anti-cancer drug irinotecan itself inhibits 5-HT-gated current through the homomeric 5-HT_3A and heteromeric 5-HT_3AB receptor in a concentration-dependent manner. The inhibitory effect of irinotecan on 5-HT_3A receptor was more potent than that on 5-HT_3AB receptor. On the other hand, SN-38, a metabolite of irinotecan, had no effect on the responsiveness. Our findings suggest that irinotecan itself could have anti-emetic activities through inhibition of the 5-HT_3A and 5-HT_3AB receptor.     

5-HT<Subscript>6/7</Subscript> Receptor Antagonists Facilitate Dopamine Release in the Cochlea via a GABAergic Disinhibitory Mechanism

In humans, serotonin (5-HT) has been implicated in numerous physiological and pathological processes in the peripheral auditory system. Dopamine (DA), another transmitter of the lateral olivocochlear (LOC) efferents making synapses on cochlear nerve dendrites, controls auditory nerve activation and protects the sensory nerve against overactivation. Using in vitro microvolume superfusion techniques we tested 5-HT₆ and 5-HT₇ receptor antagonists whether they can influence dopamine (DA) release from the guinea-pig cochlea in control and in ischemic conditions using currently available and new 5-HT₆ and 5-HT₇ antagonists and mixed antagonists, which were synthesized and characterized for the current study. While the 5-HT₇ antagonist SB-258719 was ineffective, SB-271046, which blocks the 5-HT₆ receptor, caused a significant increase in cochlear DA release what is contradictory with the excitatory nature of this type of receptor. Moreover, the mixed 5-HT_6/7 antagonist EGIS-12233 induced an even more pronounced increase in the resting DA release. To understand why the block of an excitatory receptor results in an increase instead of a decrease in function, we investigated the possible involvement of an indirect neural mechanism through an inhibitory system. In the presence of the GABA_A receptor blocker bicuculline, EGIS-12233 failed to increase the release of DA, suggesting that the serotonin receptor modulation of DA release from the lateral olivocochlear efferents in the cochlea was produced indirectly by decreasing the GABAergic inhibitory tone on dopaminergic nerve endings. The mixed 5-HT₇/D₄ receptor antagonist EGIS-11983 significantly increased both the stimulation-evoked and the resting DA release, while the selective D4 blocker L-741,741 alone had no significant effect. Ischemia, simulated by oxygen and glucose deprivation from the perfusion solution had no action on the effect of the drugs. Drugs that can increase the release of DA from LOC terminals in the cochlea may have a role in the treatment of sensorineural hearing loss.     

Nicotinic component of galantamine in the regulation of amyloid precursor protein processing

Current therapies for Alzheimer's disease treatment rely mainly on acetylcholinesterase inhibitors, improving central cholinergic neurotransmission. Among these molecules, galantamine (GAL) has an interesting pharmacological profile as it is both a reversible acetylcholinesterase inhibitor and an allosteric potentiator of nicotinic cholinergic receptors. We investigated the effect of GAL on the metabolism of the amyloid precursor protein (APP) in differentiated SH-SY5Y neuroblastoma cells. The rationale was based on the suggestion that cholinergic activity may also be involved in the regulation of APP metabolism. We studied the acute effect on APP metabolism measuring the secretion of sAPPalpha in the conditioned medium of cells. Following 2h treatment, GAL 10microM promoted a strong increase in the release of sAPPalpha, the maximal effect approaching on average three-fold baseline value. The compound appeared to increase the release of sAPPalpha, with a mechanism dependent upon an indirect cholinergic stimulation. The effect of GAL was prevented by pre-treatment with alpha-bungarotoxin (40nM) but not low (nanomolar) atropine concentrations, suggesting the specific involvement of nicotinic cholinergic receptors.     

Aequorin luminescence-based assay for 5-hydroxytryptamine (serotonin) type 3 receptor characterization

The classical electrophysiological method to measure the function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT(3)) receptor, a cation-permeable ligand-gated ion channel, is time-consuming and not suitable for high-throughput screening. Therefore, we have optimized the conditions for a sensitive assay suitable to measure 5-HT(3) receptor responses in cell suspension based on aequorin bioluminescence caused by Ca(2+) influx. The assay, carried out in 96-well plates, was applied for the pharmacological characterization of 5-HT(3) receptors on human embryonic kidney (HEK) 293 cells transiently coexpressing apoaequorin and either the human homopentameric 5-HT(3A) receptor or the human heteromeric 5-HT(3A/B) receptor in the same subset of cells. Thus, the luminescence signal originates exclusively from transfected cells, leading to a high signal/noise ratio, a major advantage compared with fluorescence techniques using Ca(2+)-sensitive dyes. The potencies of two 5-HT(3A) receptor agonists and two antagonists as well as the potency and efficacy of serotonin at the heteromeric 5-HT(3A/B) receptor were comparable to those reported using other functional methods. In conclusion, the aequorin assay described here provides a convenient and highly sensitive method for functional characterization of 5-HT(3) receptors that is well suited for high-throughput screening.     

Keratinocytes from APP/APLP2-deficient mice are impaired in proliferation, adhesion and migration in vitro

Growing evidence shows that the soluble N-terminal form (sAPPalpha) of the amyloid precursor protein (APP) represents an epidermal growth factor fostering keratinocyte proliferation, migration and adhesion. APP is a member of a protein family including the two mammalian amyloid precursor-like proteins APLP1 and APLP2. In the mammalian epidermis, only APP and APLP2 are expressed. APP and APLP2-deficient mice die shortly after birth but do not display a specific epidermal phenotype. In this report, we investigated the epidermis of APP and/or APLP2 knockout mice. Basal keratinocytes showed reduced proliferation in vivo by about 40%. Likewise, isolated keratinocytes exhibited reduced proliferation rates in vitro, which could be completely rescued by either exogenously added recombinant sAPPalpha, or by co-culture with dermal fibroblasts derived from APP knockout mice. Moreover, APP-knockout keratinocytes revealed reduced migration velocity resulting from severely compromised cell substrate adhesion. Keratinocytes from double knockout mice died within the first week of culture, indicating essential functions of APP-family members for survival in vitro. Our data indicate that sAPPalpha has to be considered as an essential epidermal growth factor which, however, in vivo can be functionally compensated to a certain extent by other growth factors, e.g., factors released from dermal fibroblasts.     

Hypothalamic Ahi1 mediates feeding behavior through interaction with 5-HT2C receptor

It is indicated that there are important molecules interacting with brain nervous systems to regulate feeding and energy balance by influencing the signaling pathways of these systems, but relatively few of the critical players have been identified. In the present study, we provide the evidence for the role of Abelson helper integration site 1 (Ahi1) protein as a mediator of feeding behavior through interaction with serotonin receptor 2C (5-HT(2C)R), known for its critical role in feeding and appetite control. First, we demonstrated the co-localization and interaction between hypothalamic Ahi1 and 5-HT(2C)R. Ahi1 promoted the degradation of 5-HT(2C)R through the lysosomal pathway. Then, we investigated the effects of fasting on the expression of hypothalamic Ahi1 and 5-HT(2C)R. Fasting resulted in an increased Ahi1 expression and a concomitant decreased expression of 5-HT(2C)R. Knockdown of hypothalamic Ahi1 led to a concomitant increased expression of 5-HT(2C)R and a decrease of food intake and body weight. Last, we found that Ahi1 could regulate the expression of neuropeptide Y and proopiomelanocortin. Taken together, our results indicate that Ahi1 mediates feeding behavior by interacting with 5-HT(2C)R to modulate the serotonin signaling pathway.     

The effect of the selective 5-HT(3) receptor agonist on ferret gut motility

The effect of the selective 5-hydroxytryptamine (5-HT)(3) receptor agonist YM-31636 (2-(1H-imidazol-4-ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate) on gut motility of fed ferrets was investigated. YM-31636 (0.1 mg/kg p.o.) induced a giant migrating contraction (GMC)-like, high-amplitude, ungrouped colonic contraction although it did not change the basal colonic motility pattern. This GMC-like contraction was always accompanied by defecation. Both GMC-like contraction and defecation were inhibited with the selective 5-HT(3) receptor antagonist ramosetron. YM-31636 affected gastric, duodenal and ileal motility pattern only slightly. These results suggest that 5-HT(3) receptor agonists such as YM-31636 are useful in treating constipation since they facilitate GMC-like contractions and defecation without undesired changes in gut motility pattern.     

Discovery of a novel allosteric modulator of 5-HT3 receptors: inhibition and potentiation of Cys-loop receptor signaling through a conserved transmembrane intersubunit site

The ligand-gated ion channels in the Cys-loop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA, and glycine. Cys-loop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel class of negative allosteric modulators of the 5-HT(3) receptors (5-HT(3)Rs). PU02 (6-[(1-naphthylmethyl)thio]-9H-purine) is a potent and selective antagonist displaying IC(50) values of ~1 μM at 5-HT(3)Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5-HT(3)A receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)-subunit and TM1 and TM2 in the (-)-subunit. The Ser(248), Leu(288), Ile(290), Thr(294), and Gly(306) residues are identified as important molecular determinants of PU02 activity with minor contributions from Ser(292) and Val(310), and we propose that the naphthalene group of PU02 docks into the hydrophobic cavity formed by these. Interestingly, specific mutations of Ser(248), Thr(294), and Gly(306) convert PU02 into a complex modulator, potentiating and inhibiting 5-HT-evoked signaling through these mutants at low and high concentrations, respectively. The PU02 binding site in the 5-HT(3)R corresponds to allosteric sites in anionic Cys-loop receptors, which emphasizes the uniform nature of the molecular events underlying signaling through the receptors. Moreover, the dramatic changes in the functional properties of PU02 induced by subtle changes in its binding site bear witness to the delicate structural discrimination between allosteric inhibition and potentiation of Cys-loop receptors.     

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.     

Nerve growth factor modulates the expression and secretion of beta-amyloid precursor protein through different mechanisms in PC12 cells

The beta-amyloid protein, component of the senile plaques found in Alzheimer brains is proteolytically derived from the beta-amyloid precursor protein (APP), a larger membrane-associated protein that is expressed in both neural and non-neural cells. Overexpression of APP might be one of the mechanisms that more directly contributes to the development of Alzheimer's disease. The APP gene expression is regulated by a number of cellular mediators including nerve growth factor (NGF) and other ligands of tyrosine kinase receptors. We have previously described that NGF increases APP mRNA levels in PC12 cells. However, the molecular mechanisms and the precise signalling pathways that mediate its regulation are not yet well understood. In the present study we present evidence that NGF, and to a lesser extent fibroblast growth factor and epidermal growth factor, stimulate APP promoter activity in PC12 cells. This induction is mediated by DNA sequences located between the nucleotides - 307 and - 15, and involves activation of the Ras-MAP kinase signalling pathway. In contrast, we have also found that NGF-induced secretion of soluble fragments of APP into the culture medium is mediated by a Ras independent mechanism.     

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.