Notch and EGFR regulate apoptosis in progenitor cells to ensure gut homeostasis in Drosophila.

The regenerative activity of adult stem cells carries a risk of cancer, particularly in highly renewable tissues. Members of the family of inhibitor of apoptosis proteins (IAPs) inhibit caspases and cell death, and are often deregulated in adult cancers; however, their roles in normal adult tissue homeostasis are unclear. Here, we show that regulation of the number of enterocyte-committed progenitor (enteroblast) cells in the adult Drosophila involves a caspase-mediated physiological apoptosis, which adaptively eliminates excess enteroblast cells produced by intestinal stem cells (ISCs) and, when blocked, can also lead to tumorigenesis. Importantly, we found that Diap1 is expressed by enteroblast cells and that loss and gain of Diap1 led to changes in enteroblast numbers. We also found that antagonistic interplay between Notch and EGFR signalling governs enteroblast life/death decisions via the Klumpfuss/WT1 and Lozenge/RUNX transcription regulators, which also regulate enteroblast differentiation and cell fate plasticity. These data provide new insights into how caspases drive adult tissue renewal and protect against the formation of tumours.

1-(2',5'-Dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (RGM079): A Positive Allosteric Modulator of α7 Nicotinic Receptors with Analgesic and Neuroprotective Activity.

Acetylcholine α7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain as well as in neuropsychiatric, neurodegenerative, and inflammatory processes. Positive allosteric modulators (PAMs) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here, we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the α7 receptors and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in Alzheimer's disease (AD)-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aß1-42, with cell death almost completely prevented at 10 and 30 µM, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the complete Freund's adjuvant (CFA)-induced paw inflammation model after intraperitoneal administration.

Amino acid and peptide prodrugs of diphenylpropanones positive allosteric modulators of α7 nicotinic receptors with analgesic activity.

α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels implicated in a number of CNS pathological processes, including pain and psychiatric, cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive allosteric modulation of these channels constitutes an interesting approach to achieve selectivity versus other nicotinic receptors. We have recently described new chalcones and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs, which proved to have good analgesic activities but poor pharmacokinetic properties. Here we report the preparation of amino acid and peptide derivatives as prodrugs of these modulators with the aim of improving their in vivo biological activity. While the valine derivative showed very short half life in aqueous solutions to be considered a prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-diphenylpropanones, via chemical and enzymatic transformation, respectively. Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2',5',4-trihydroxy-1,3-diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays. The best compound, 21, displayed a better profile in the analgesia test than its parent compound 3, exhibiting about the same potency but long-lasting effects.

N-Benzylpiperidine Derivatives as α7 Nicotinic Receptor Antagonists.

A series of multitarget directed propargylamines, as well as other differently susbstituted piperidines have been screened as potential modulators of neuronal nicotinic acetylcholine receptors (nAChRs). Most of them showed antagonist actions on α7 nAChRs. Especially, compounds 13, 26, and 38 displayed submicromolar IC50 values on homomeric α7 nAChRs, whereas they were less effective on heteromeric α3ß4 and α4ß2 nAChRs (up to 20-fold higher IC50 values in the case of 13). Antagonism was concentration dependent and noncompetitive, suggesting that these compounds behave as negative allosteric modulators of nAChRs. Upon the study of a series of less complex derivatives, the N-benzylpiperidine motif, common to these compounds, was found to be the main pharmacophoric group. Thus, 2-(1-benzylpiperidin-4-yl)-ethylamine (48) showed an inhibitory potency comparable to the one of the previous compounds and also a clear preference for α7 nAChRs. In a neuroblastoma cell line, representative compounds 13 and 48 also inhibited, in a concentration-dependent manner, cytosolic Ca(2+) signals mediated by nAChRs. Finally, compounds 38 and 13 inhibited 5-HT3A serotonin receptors whereas they had no effect on α1 glycine receptors. Given the multifactorial nature of many pathologies in which nAChRs are involved, these piperidine antagonists could have a therapeutic potential in cases where cholinergic activity has to be negatively modulated.

Single-channel study of the binding-gating coupling in the slowly desensitizing chimeric alpha7-5HT3A receptor.

We have studied the role of the highly conserved residue alphaLysine145 in the early steps of activation by acetylcholine of the nicotinic acetylcholine receptor (nAChR). Both macroscopic and single-channel currents were recorded in the slowly desensitizing chimeric mutant receptor alpha7V201-5HT3A/R432Q/R436D/R440A, made of alpha7 nAChRs and serotonin receptors of subtype 3A (ch1), and its corresponding mutant K145A (ch1/K145A) expressed in Xenopus oocytes. Mutant ch1/K145A receptors had a reduced gating function similar to that produced by the same mutation in the wild type receptor alpha7. The mutated receptor has reduced opening rate constants, beta, and increased closing rate constants, alpha.

Role of the N-terminal alpha-helix in biogenesis of alpha7 nicotinic receptors.

We studied the role of the alpha-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in alpha7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the alpha-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of alpha7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (alpha3beta4 and alpha4beta2) and 5-HT(3A) receptors also abolished their expression at the membrane. We conclude that the N-terminal alpha-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.

Molecular characterization and localization of the RIC-3 protein, an effector of nicotinic acetylcholine receptor expression.

The RIC-3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC-3 (hRIC-3) protein enhances expression of alpha7 receptors and abolishes expression of alpha4beta2 receptors. In vitro translation of hRIC-3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC-3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for alpha7 expression enhancement but not for alpha4beta2 inhibition. A combination of increased alpha7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in alpha7 membrane expression induced by hRIC-3. Antibodies against hRIC-3 showed its expression in SH-SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC-3 in rat brain localized, in general, in places where alpha7 nAChRs were found.

The cysteine-rich with EGF-like domains 2 (CRELD2) protein interacts with the large cytoplasmic domain of human neuronal nicotinic acetylcholine receptor alpha4 and beta2 subunits.

Using a yeast two-hybrid screening we report the isolation of a novel human protein, hCRELD2beta, that interacts specifically with the large cytoplasmic regions of human nicotinic acetylcholine receptor (nAChR) alpha4 and beta2 subunits, both in yeast cells and in vitro. This interaction is not detected with nAChR alpha7 and alpha3 subunits. The hCRELD2 gene encodes for multiple transcripts, likely to produce multiple protein isoforms. A previously reported one has been renamed as CRELD2alpha. Isoforms alpha and beta are expressed in all tissues examined and have the same N-terminal and central regions but alternative C-terminal regions. Both isoforms interact with the alpha4 subunit. Within this subunit the interaction was localized to the N-terminal region of the large cytoplasmic loop. The CRELD2beta protein is present at the endoplasmic reticulum where colocalized with alpha4beta2 nAChRs upon cell transfection. Immunohistochemistry experiments demonstrated the presence of CRELD2 in the rat brain at sites where alpha4beta2 receptors have been previously detected. Labeling was restricted to neuronal perikarya. Finally, CRELD2 decreases the functional expression and impairs membrane transport of alpha4beta2 nAChRs in Xenopus leavis oocytes, without affecting alpha3beta4 and alpha7 nAChR expression. These results suggest that CRELD2 can act as a specific regulator of alpha4beta2 nAChR expression.

Effects of benzothiazepines on human neuronal nicotinic receptors expressed in Xenopus oocytes.

1. We have investigated the effect of diltiazem and its newly synthesized derivative (+,-)-trans-3-acetoxy-8-chloro-2,3-dihydro-5[2-diisopropylamine)ethyl]-2-(4-methoxyphenyl)-1,5-benzothiazepin-4-(5H)-ona hydrochloride (JAC-65) on several recombinant human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. 2. At 3 microM, both drugs have little effect on the maximal currents evoked by brief pulses of acetylcholine (ACh) in five subtypes of nAChRs (alpha7, alpha3beta2, alpha4beta2, alpha3beta4, and alpha4beta4), showing little selectivity among subtypes. 3. However, both drugs accelerate the decay of the ionic currents evoked upon continuous stimulation of ACh, being this effect larger with JAC-65, and in beta4*-nAChRs. Such an effect was dependent on the concentrations of both the drug and of the agonist used, and showed the characteristics of a non-competitive antagonism. 4. We have further investigated the effect of both drugs when combined with submicromolar concentrations of nicotine, such as those present in plasma of cigarette smokers, and found that JAC-65, but not diltiazem, is able to greatly enhance the desensitizing effect of these low concentrations of nicotine, specially in beta4*-nAChRs. 5. Experiments in alpha4beta4-nAChRs failed to show voltage dependence of the action of JAC-65. Moreover, recovery from desensitization followed the same time course regardless of the presence of the drug, suggesting that the main mechanism of action of JAC-65 does not involve open channel block. 6. In summary, both drugs, diltiazem and JAC-65, seem to act through a non-competitive mechanism, accelerating the decay of the ionic currents, being JAC-65 more effective than diltiazem at the concentrations used in beta4*-nAChRs. Thus, the differences between both benzothiazepines when measuring various parameters suggest that their mechanisms of action could be slightly different. This would require further investigation.