Secreted Amyloid Precursor Protein β and Secreted Amyloid Precursor Protein α Induce Axon Outgrowth In Vitro through Egr1 Signaling Pathway

BACKGROUND: sAPPα released after α secretase cleavage of Amyloid Precursor Protein (APP) has several functions including the stimulation of neurite outgrowth although detailed morphometric analysis has not been done. Two domains involved in this function have been described and are present in sAPPβ released at the first step of amyloid peptide cleavage, raising the possibility that sAPPβ could also stimulate neurite outgrowth. We investigated the morphological effects of sAPPα and sAPPβ on primary neurons and identified a key signaling event required for the changes observed. METHODOLOGY/PRINCIPAL FINDINGS: Final concentrations of 50 to 150 nM bacterial recombinant sAPPα or sAPPβ added to primary neuronal cultures after 1 day in vitro decreased cell adhesion 24 hours later and primary dendrite length 96 hours later. 150 nM sAPPα and sAPPβ induced a similar increase of axon outgrowth, although this increase was already significant at 100 nM sAPPα. These morphological changes induced by sAPPs were also observed when added to differentiated neurons at 5 days in vitro. Real time PCR and immunocytochemistry showed that sAPPα and sAPPβ stimulated Egr1 expression downstream of MAPK/ERK activation. Furthermore, in primary neurons from Egr1 -/- mice, sAPPs affected dendritic length but did not induce any increase of axon length. CONCLUSION/SIGNIFICANCE: sAPPα and sAPPβ decrease cell adhesion and increase axon elongation. These morphological changes are similar to what has been observed in response to heparan sulfate. The sAPPα/sAPPβ stimulated increase in axon growth requires Egr1 signaling. These data suggest that sAPPβ is not deleterious per se. Since sAPPβ and sAPPα are present in the embryonic brain, these two APP metabolites might play a role in axon outgrowth during development and in response to brain damage

Compared pharmacology of human histamine H(3) and H(4) receptors: structure–activity relationships of histamine derivatives

1. Various histamine derivatives were investigated at the human H(3) receptor (H(3)R) and H(4) receptor (H(4)R) stably expressed in human embryonic kidney (HEK)-293 cells using [(125)I]iodoproxyfan and [(3)H]histamine binding, respectively. 2. In Tris buffer, [(3)H]histamine binding to membranes of HEK(hH(4)R) cells was monophasic (K(D) of 3.8±0.8 nM). In phosphate buffer, the Hill coefficient was decreased (n(H)=0.5±0.1) and a large fraction of the binding was converted into a low-affinity component (K(D)=67±27 nM). 3. The inhibition of [(3)H]histamine binding by two agonists, a protean agonist and five antagonists/inverse agonists confirms that the potency of many H(3)R ligands is retained or only slightly reduced at the H(4)R. 4. Histamine derivatives substituted with methyl groups in α, β or N(α) position of the side chain retained a nanomolar potency at the H(3)R, but their affinity was dramatically decreased at the H(4)R. With relative potencies to histamine of 282 and 0.13% at the H(3)R and H(4)R, respectively, (±)-α,β-dimethylhistamine is a potent and selective H(3)R agonist. 5. Chiral α-branched analogues exhibited a marked stereoselectivity at the H(3)R and H(4)R, the enantiomers with a configuration equivalent to L-histidine being preferred at both receptors. 6. The methylsubstitution of the imidazole ring was also studied. The relative potency to histamine of 4-methylhistamine (4-MeHA) at the H(4)R (67%) was similar to that reported at H(2) receptors but, owing to its high affinity at the H(4)R (K(i)=7.0±1.2 nM) and very low potency at H(1)- and H(3)-receptors, it can be considered as a potent and selective H(4)R agonist. 7. On inhibition of forskolin-induced cAMP formation, all the compounds tested, including 4-MeHA, behaved as full agonists at both receptors. However, the maximal inhibition achieved at the H(4)R (∼−30%) was much lower than at the H(3)R (∼−80%). Thioperamide behaved as an inverse agonist at both receptors and increased cAMP formation with the same maximal effect (∼+25%). 8. In conclusion, although the pharmacological profiles of the human H(3)R and H(4)R overlap, the structure–activity relationships of histamine derivatives at both receptors strongly differ and lead to the identification of selective compounds