Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.

The Caenorhabditis elegans sel-10 protein is structurally similar to E3 ubiquitin ligases and is a negative regulator of Notch (lin-12) and presenilin signaling. In this report, we characterize the mammalian Sel-10 homolog (mSel-10) and analyze its effects on Notch signaling. We find that mSel-10 localizes to the cell nucleus, and that it physically interacts with the Notch 1 intracellular domain (IC) and reduces Notch 1 IC-mediated activation of the HES 1 promoter. Notch 1 IC is ubiquitinated by mSel-10, and ubiquitination requires the presence of the most carboxyl-terminal region of the Notch IC, including the PEST domain. In the presence of the proteasome inhibitor MG132, the amount of Notch 1 IC and its level of ubiquitination are increased. Interestingly, this accumulation of Notch 1 IC in the presence of MG132 is accompanied by decreased activation of the HES 1 promoter, suggesting that ubiquitinated Notch 1 IC is a less potent transactivator. Finally, we show that mSel-10 itself is ubiquitinated and degraded by the proteasome. In conclusion, these data reveal the importance of ubiquitination and proteasome-mediated degradation for the activity and turnover of Notch ICs, and demonstrate that mSel-10 plays a key role in this process.

Notch receptors and their ligands play important roles in both normal animal development and pathogenesis. We show here that the F-box/WD40 repeat protein SEL-10 negatively regulates Notch receptor activity by targeting the intracellular domain of Notch receptors for ubiquitin-mediated protein degradation. Blocking of endogenous SEL-10 activity was done by expression of a dominant-negative form containing only the WD40

In Alzheimer's disease brains, more than 90% of pyramidal neurons in lamina V and 70% in lamina III displayed 2- to 5-fold elevated levels of cathepsin D (Cat D) mRNA by in situ hybridization compared with neurologically normal controls. Most of these cells appeared histologically normal. The less vulnerable nonpyramidal neuron population in lamina IV had relatively normal message levels. Neuronal populations expressing more Cat D mRNA also displayed quantitatively increased Cat D immunoreactive protein. Cat D mRNA expression was only moderately increased in astrocytes. Degenerating neurons exhibited intense immunoreactivity but lowered Cat D mRNA levels. The upregulation of Cat D synthesis and accumulation of hydrolase-laden lysosomes indicate an early activation of the endosomal-lysosomal system in vulnerable neuronal populations, possibly reflecting early regenerative or repair processes. These abnormalities also represent a basis for altered regulation of amyloid precursor protein processing.

The ␣7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel ␣7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (K i ϭ 10.8 nM) and human (K i ϭ 16.7 nM) ␣7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the ␣7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that ␣7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of ␣7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked ␣7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that ␣7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia.

Mutations in two related genes, presenilin 1 and 2, account for most early-onset familial Alzheimer's disease. Although structural features indicate that the presenilins are membrane proteins, their function(s) is unknown. We have localized the presenilins to the nuclear membrane, its associated interphase kinetochores, and the centrosomes-all subcellular structures involved in cell cycle regulation and mitosis. The colocalization of the presenilins with kinetochores on the nucleoplasmic surface of the inner nuclear membrane, together with other results, suggests that they may play a role in chromosome organization and segregation, perhaps as kinetochore binding proteins/receptors. We discuss a pathogenic pathway for familial Alzheimer's disease in which defective presenilin function causes chromosome missegregation during mitosis, resulting in apoptosis and/or trisomy 21 mosaicism.

The ␣7 nicotinic acetylcholine receptor (nAChR), a homopentameric, rapidly activating and desensitizing ligand-gated ion channel with relatively high degree of calcium permeability, is expressed in the mammalian central nervous system, including regions associated with cognitive processing. Selective agonists targeting the ␣7 nAChR have shown efficacy in animal models of cognitive dysfunction. Use of positive allosteric modulators selective for the ␣7 receptor is another strategy that is envisaged in the design of active compounds aiming at improving attention and cognitive dysfunction. The recent discovery of novel positive allosteric modulators such as 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethylphenyl)urea (NS-1738) and 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea (PNU-120596) that are selective for the ␣7 nAChRs but display significant phenotypic differences in their profile of allosteric modulation, suggests that these molecules may act at different sites on the receptor. Taking advantage of the possibility to obtain functional receptors by the fusion of proteins domains from the ␣7 and the 5-HT 3 receptor, we examined the structural determinants required for positive allosteric modulation. This strategy revealed that the extracellular N-terminal domain of ␣7 plays a critical role in allosteric modulation by NS-1738. In addition, ␣7-5HT 3 chimeras harboring the M2-M3 segment showed that spontaneous activity in response to NS-1738, which confirmed the critical contribution of this small extracellular segment in the receptor gating. In contrast to NS-1738, positive allosteric modulation by PNU-120596 could not be restored in the ␣7-5HT 3 chimeras but was selectively observed in the reverse 5HT 3 -␣7 chimera. All together, these data illustrate the existence of distinct allosteric binding sites with specificity of different profiles of allosteric modulators and open new possibilities to investigate the ␣7 receptor function.The ␣7 nicotinic acetylcholine receptor (nAChR) belongs to the family of ionotropic receptors that share four transmembrane domains as a common structural feature (for review, see Hogg et al., 2005). By virtue of its high expression levels in brain regions involved in learning and memory, such as hippocampus and cerebral cortex, and its unique physiological properties, partly attributed to a high permeability to Ca 2ϩ , the ␣7 nAChR has received considerable attention as drug target for development of drugs intended to treat cognitive/attention disorders underlying neuropsychiatric and neurodegenerative diseases (for review, see Dani and Bertrand, 2007). Furthermore, despite its low sensitivity to acetylcholine, the ␣7 nAChR has been shown to modulate the release of other neurotransmitters and, in some cases, to contribute directly to signal transmission (for review, see Wonnacott et al., 2006).Gene knock-out and antisense studies together with pharmacological studies using small-molecule selective agonists and positive allosteric modulators have demon...

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