Pathological amino-acid substitutions in the amyloid precursor protein (APP) and chemical g-secretase modulators affect the processing of APP by the g-secretase complex and the production of the amyloid-beta peptide Ab42, the accumulation of which is considered causative of Alzheimer's disease. Here we demonstrate that mutations in the transmembrane domain of APP causing aggressive early-onset familial Alzheimer's disease affect both g-and e-cleavage sites, by raising the Ab42/40 ratio and inhibiting the production of AICD50-99, one of the two physiological APP intracellular domains (ICDs). This is in sharp contrast to g-secretase modulators, which shift Ab42 production towards the shorter Ab38, but unequivocally spare the e-site and APP-and Notch-ICDs production. Molecular simulations suggest that familial Alzheimer's disease mutations modulate the flexibility of the APP transmembrane domain and the presentation of its g-site, modifying at the same time, the solvation of the e-site.

Renal tubulo-interstitial inflammation is frequently associated with polyuria and urine concentration defects. This led us to investigate the effects of the major pro-inflammatory nuclear factor B (NF-B) pathway on aquaporin 2 (AQP2) expression by the collecting duct. Using immortalized collecting duct principal cells (mpkCCD cl4 ), we found that, acting independently of vasopressin, activation of NF-B by lipopolysaccharide (LPS) decreased AQP2 mRNA and protein levels in a time-and dose-dependent manner but did not decrease AQP2 mRNA stability. Consistently, constitutively active IB kinase ␤ decreased AQP2 expression. The LPSinduced decrease in AQP2 mRNA levels was confirmed in rat kidney slices and was reproduced both under conditions of elevated cAMP concentration and V 2 receptor antagonism. Computer analysis of the AQP2 promoter revealed two putative B elements. Mutation of either B element abolished the LPS-induced decrease of luciferase activity in cells expressing AQP2 promoter-luciferase plasmid constructs. Chromatin immunoprecipitation revealed that LPS challenge decreased p65, increased p50 and p52, and had no effect on RelB and c-Rel binding to B elements of the AQP2 promoter. RNA-mediated interference silencing of p65, p50, and p52 confirmed controlled AQP2 transcription by these NF-B subunits. We additionally found that hypertonicity activated NF-B in mpkCCD cl4 cells, an effect that may counteract the Tonicity-responsive enhancer binding protein (TonEBP)-dependent increase in AQP2 gene transcription. Taken together, these findings indicate that NF-B is an important physiological regulator of AQP2 transcription.Selective transcellular water permeability across the renal epithelium is facilitated by the aquaporin 1 (AQP1) 3 water channel, expressed in the proximal tubule and thin descending limb of Henle's loop (1, 2) and AQP2, -3, and -4, expressed in collecting duct (CD) principal cells (3). AQP2 inserted in the apical plasma membrane enhances CD apical water permeability. Water exits these cells via basolateral AQP3 and AQP4. By controlling both short term (plasma membrane insertion) and long term (transcriptional and post-transcriptional) AQP2 expression, the antidiuretic hormone [8-arginine]vasopressin (AVP) plays a major role in regulating water reabsorption (3-7). In addition to AVP, AQP2 expression is influenced by numerous factors that act independently of AVP including other hormones, i.e. aldosterone and insulin (8, 9), extracellular calcium (10), and environmental tonicity (11-13).The NF-B family of transcription factors consists of five members (p65, p50/p105, p52/p100, RelB, and c-Rel) that contain a Rel-homology domain required for the formation of various combinations of homo-and heterodimers and for DNA binding. Generally, dimers that contain p65 or c-Rel are transcriptional activators, whereas p50 and p52 homodimers act as repressors. The differential dimerization that occurs between NF-B family members allows for cellspecific transcriptional modulation of target genes in response t...

Recent evidence suggests that arginine vasopressin (AVP)-dependent aquaporin-2 expression is modulated by the extracellular calcium-sensing receptor (CaSR) in principal cells of the collecting duct, but the signaling pathways mediating this effect are unknown. Using a mouse cortical collecting duct cell line (mpkCCD cl4 ), we found that increasing the concentration of apical extracellular calcium or treating with the CaSR agonists neomycin or Gd 3ϩ attenuated AVP-dependent accumulation of aquaporin-2 mRNA and protein; CaSR gene-silencing prevented this effect. Calcium reduced the AVP-induced accumulation of cAMP, but this did not occur by increased degradation of cAMP by phosphodiesterases or by direct inhibition of adenylate cyclase. Notably, the effect of extracellular calcium on AVP-dependent aquaporin-2 expression was prevented by inhibition of calmodulin. In summary, our results show that high concentrations of extracellular calcium attenuate AVP-induced aquaporin-2 expression by activating the CaSR and reducing coupling efficiency between V 2 receptor and adenylate cyclase via a calmodulindependent mechanism in cultured cortical collecting duct cells.

Background: Amyloid-β neurotoxicity depends on the specificity of the proteolytic cleavage of the amyloid precursor protein (APP) transmembrane domain.Results: The APP transmembrane α-helix is straight in a biological membrane bilayer.Conclusion: The flexibility of APP is key for adapting to the lipid environment and modulating proteolytic processing by γ-secretase.Significance: The dynamic characterization of APP is expected to rationalize the design of γ-secretase modulators.