Extramacrochaetae promotes branch and bouton number via the sequestration of daughterless in the cytoplasm of neurons.

The Class I basic helix-loop-helix (bHLH) proteins are highly conserved transcription factors that are ubiquitously expressed. A wealth of literature on Class I bHLH proteins has shown that these proteins must homodimerize or heterodimerize with tissue-specific HLH proteins in order to bind DNA at E-box consensus sequences to control tissue-specific transcription. Due to its ubiquitous expression, Class I bHLH proteins are also extensively regulated posttranslationally, mostly through dimerization. Previously, we reported that in addition to its role in promoting neurogenesis, the Class I bHLH protein daughterless also functions in mature neurons to restrict axon branching and synapse number. Here, we show that part of the molecular logic that specifies how daughterless functions in neurogenesis is also conserved in neurons. We show that the Type V HLH protein extramacrochaetae (Emc) binds to and represses daughterless function by sequestering daughterless to the cytoplasm. This work provides initial insights into the mechanisms underlying the function of daughterless and Emc in neurons while providing a novel understanding of how Emc functions to restrict daughterless activity within the cell.

Type I bHLH Proteins Daughterless and Tcf4 Restrict Neurite Branching and Synapse Formation by Repressing Neurexin in Postmitotic Neurons.

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, whereas 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 indicate 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.

Amyloid Precursor Protein Translation Is Regulated by a 3'UTR Guanine Quadruplex.

A central event in Alzheimer's disease is the accumulation of amyloid ß (Aß) peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP). APP overexpression leads to increased Aß generation and Alzheimer's disease in humans and altered neuronal migration and increased long term depression in mice. Conversely, reduction of APP expression results in decreased Aß levels in mice as well as impaired learning and memory and decreased numbers of dendritic spines. Together these findings indicate that therapeutic interventions that aim to restore APP and Aß levels must do so within an ideal range. To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation. Such regulation can be mediated by RNA regulatory elements such as guanine quadruplexes (G-quadruplexes), non-canonical structured RNA motifs that affect RNA stability and translation. Via a bioinformatics approach, we identified a candidate G-quadruplex within the APP mRNA in its 3'UTR (untranslated region) at residues 3008-3027 (NM_201414.2). This sequence exhibited characteristics of a parallel G-quadruplex structure as revealed by circular dichroism spectrophotometry. Further, as with other G-quadruplexes, the formation of this structure was dependent on the presence of potassium ions. This G-quadruplex has no apparent role in regulating transcription or mRNA stability as wild type and mutant constructs exhibited equivalent mRNA levels as determined by real time PCR. Instead, we demonstrate that this G-quadruplex negatively regulates APP protein expression using dual luciferase reporter and Western blot analysis. Taken together, our studies reveal post-transcriptional regulation by a 3'UTR G-quadruplex as a novel mechanism regulating APP expression.

Cyclopamine modulates γ-secretase-mediated cleavage of amyloid precursor protein by altering its subcellular trafficking and lysosomal degradation.

Alzheimer disease (AD) is a progressive neurodegenerative disease leading to memory loss. Numerous lines of evidence suggest that amyloid-ß (Aß), a neurotoxic peptide, initiates a cascade that results in synaptic dysfunction, neuronal death, and eventually cognitive deficits. Aß is generated by the proteolytic processing of the amyloid precursor protein (APP), and alterations to this processing can result in Alzheimer disease. Using in vitro and in vivo models, we identified cyclopamine as a novel regulator of γ-secretase-mediated cleavage of APP. We demonstrate that cyclopamine decreases Aß generation by altering APP retrograde trafficking. Specifically, cyclopamine treatment reduced APP-C-terminal fragment (CTF) delivery to the trans-Golgi network where γ-secretase cleavage occurs. Instead, cyclopamine redirects APP-CTFs to the lysosome. These data demonstrate that cyclopamine treatment decreases γ-secretase-mediated cleavage of APP. In addition, cyclopamine treatment decreases the rate of APP-CTF degradation. Together, our data demonstrate that cyclopamine alters APP processing and Aß generation by inducing changes in APP subcellular trafficking and APP-CTF degradation.

Changes in readthrough acetylcholinesterase expression modulate amyloid-beta pathology.

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.

Exploring the binding domain of EmrE, the smallest multidrug transporter.

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 (Glu14) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu14 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, Tyr40 from TM2, and Trp63 in TM3 are involved in substrate binding.