Alzheimer's disease has long been known to involve cholinergic deficits, but the linkage between cholinergic gene expression and the Alzheimer's disease amyloid pathology has remained incompletely understood. One known link involves synaptic acetylcholinesterase (AChE-S), shown to accelerate amyloid fibrils formation. Here, we report that the 'Readthrough' AChE-R splice variant, which differs from AChE-S in its 26 C-terminal residues, inversely exerts neuroprotective effects from amyloid beta (Abeta) induced toxicity. In vitro, highly purified AChE-R dose-dependently suppressed the formation of insoluble Abeta oligomers and fibrils and abolished Abeta toxicity to cultured cells, competing with the prevalent AChE-S protein which facilitates these processes. In vivo, double transgenic APPsw/AChE-R mice showed lower plaque burden, fewer reactive astrocytes and less dendritic damage than single APPsw mice, inverse to reported acceleration of these features in double APPsw/AChE-S mice. In hippocampi from Alzheimer's disease patients (n = 10), dentate gyrus neurons showed significantly elevated AChE-R mRNA and reduced AChE-S mRNA. However, immunoblot analyses revealed drastic reductions in the levels of intact AChE-R protein, suggesting that its selective loss in the Alzheimer's disease brain exacerbates the Abeta-induced damages and revealing a previously unforeseen linkage between cholinergic and amyloidogenic events.

Proneural proteins of the class I/II basic Helix Loop Helix (bHLH) family are highly conserved transcription factors. Class I bHLH proteins are expressed in a broad number of tissues during development, while class II bHLH protein expression is more tissue restricted. Our understanding of the function of class I/II bHLH transcription factors in both invertebrate and vertebrate neurobiology is largely focused on their function as regulators of neurogenesis. Here, we show that the class I bHLH proteins Daughterless and Tcf4 are expressed in postmitotic neurons in Drosophila melanogaster and mice, respectively, where they function to restrict neurite branching and synapse formation. Our data indicates that Daughterless performs this function in part by restricting the expression of the cell adhesion molecule Neurexin. This suggests a role for these proteins outside of their established roles in neurogenesis.

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu 14 ) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu 14 are part of the binding domain. Alkylation of Cys replacements in the same transmembrane domain inhibits the activity of the protein and this inhibition is fully prevented by substrates of EmrE. To monitor directly the reaction we tested also the extent of modification using fluorescein-5-maleimide. While most residues are not accessible or only partially accessible, four, Y4C, I5C, L7C, and A10C, were modified at least 80%. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of two of these residues by up to 80%. To study other essential residues we generated functional hetero-oligomers and challenged them with various methane thiosulfonates. Taken together the findings imply the existence of a binding cavity accessible to alkylating reagents where at least three residues from TM1, Tyr 40 from TM2, and Trp 63 in TM3 are involved in substrate binding.EmrE, a protein from Escherichia coli, provides a unique model for the study of polytopic membrane proteins. It is a small (110 residues) multidrug transporter that extrudes various positively charged drugs in exchange for protons, thus rendering bacteria resistant to these drugs (1-3). The protein has been characterized, purified, and reconstituted in a functional form (1, 4 -6). High affinity substrate binding has been established as a reliable and sensitive assay for activity of the detergentsolubilized transporter (4). Structural and biochemical evidence suggest that the basic EmrE oligomer is a dimer (7-10). EmrE has only one membrane-embedded charged residue, Glu 14 , which is conserved in more than 100 homologous proteins (5). Acidic side chains embedded in the membrane have been shown to be important for activity in various ion-coupled transporters (for a review, see Ref. 11). EmrE is unique in that the same acidic side chain (the carboxyl group of Glu 14 ) is involved in recognition of both substrate and the coupling ion.Within the small multidrug resistance (SMR) 2 family of transporters, a comparative analysis reveals that the face of transmembrane domain 1 (TM1) containing Glu 14 is conserved, displaying a helical periodicity (Fig. 1A and Ref. 5). Previously, using site-directed mutagenesis of this TM1 face we identified a cluster of five amino acids that play a role in substrate and H ϩ recognition and/or translocation with substitutions at most positions yielding either inactive mutants or mutants with modified affinity to substrates (12, 13). We now use Cys replacements in TM1 to study accessibility of the residues in T...