No abstract

In vitro reconstitution of functions of membrane proteins is often hampered by aggregation, misfolding, or lack of post-translational modifications of the proteins attributable to overexpression. To overcome this technical obstacle, we have developed a method to express multimeric integral membrane proteins in extracellular (budded) baculovirus particles that are released from Sf9 cells co-infected with multiple transmembrane proteins. We applied this method to the reconstitution of ␥-secretase, a membrane protease complex that catalyzes the intramembrane cleavage of ␤-amyloid precursor protein to release A␤ peptides, the major component of amyloid deposits in Alzheimer brains as well as of Notch. When we co-infected Sf9 cells with human presenilin 1 (PS1), nicastrin, APH-1a, and PEN-2, a high-molecular-weight membrane protein complex that contained PS1 exclusively in its fragment form associated with three other cofactor proteins was reconstituted and recovered in a highly ␥-secretase-active state in budded virus particles, whereas nonfunctional PS1 holoproteins massively contaminated the parental Sf9 cell membranes. The relative ␥-secretase activity (per molar PS1 fragments) was concentrated by ϳ2.5 fold in budded virus particles compared with that in Sf9 membranes. The budded baculovirus system will facilitate structural and functional analyses of ␥-secretase, as well as screening of its binding molecules or inhibitors, and will also provide a versatile methodology for the characterization of a variety of membrane protein complexes.

␥-Secretase is an atypical aspartyl protease that cleaves amyloid ␤-precursor protein to generate A␤ peptides that are causative for Alzheimer disease. ␥-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2. Pen-2 directly binds to transmembrane domain 4 of PS and confers proteolytic activity on ␥-secretase, although the mechanism of activation and its role in catalysis remain unknown. Here we show that an addition of amino acid residues to the N terminus of Pen-2 specifically increases the generation of A␤42, the longer and more aggregable species of A␤. The effect of the N-terminal elongation of Pen-2 on A␤42 generation was independent of the amino acid sequences, the expression system and the presenilin species. In vitro ␥-secretase assay revealed that Pen-2 directly affects the A␤42-generating activity of ␥-secretase. The elongation of Pen-2 N terminus caused a reduction in the water accessibility of the luminal side of the catalytic pore of PS1 in a similar manner to that caused by an A␤42-raising ␥-secretase modulator, fenofibrate, as determined by substituted cysteine accessibility method. These data suggest a unique mechanism of A␤42 overproduction associated with structural changes in the catalytic pore of presenilins caused commonly by the N-terminal elongation of Pen-2 and fenofibrate.

␥-Secretase cleaves type I transmembrane proteins, including ␤-amyloid precursor protein and Notch, and requires the formation of a protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2 for its activity. Aph-1 is implicated in the stabilization of this complex, although its precise mechanistic role remains unknown. Substitution of the first glycine within the transmembrane GXXXG motif of Aph-1 causes a loss-of-function phenotype in Caenorhabditis elegans. Here, using an untranslated region-targeted RNA interference/rescue strategy in Drosophila Schneider 2 cells, we show that Aph-1 contributes to the assembly of the ␥-secretase complex by multiple mechanisms involving intermolecular and intramolecular interactions depending on or independent of the conserved glycines. Aph-1 binds to nicastrin forming an early subcomplex independent of the conserved glycines within the endoplasmic reticulum. Certain mutations in the conserved GXXXG motif affect the interaction of the Aph-1⅐nicastrin subcomplex with presenilin that mediates trafficking of the presenilin⅐Aph-1⅐nicas-trin tripartite complex to the Golgi. The same mutations decrease the stability of Aph-1 polypeptides themselves, possibly by affecting intramolecular associations through the transmembrane domains. Our data suggest that the proper assembly of the Aph-1⅐nicastrin subcomplex with presenilin is the prerequisite for the trafficking as well as the enzymatic activity of the ␥-secretase complex and that Aph-1 functions as a stabilizing scaffold in the assembly of this complex. Mutations in presenilin (PS)1 genes account for the majority of early onset familial Alzheimer's disease cases, causing an overproduction of those amyloid ␤ peptides (A␤) ending at position 42 (A␤42) that most readily form amyloid deposits (1). A␤ is derived from the ␤-amyloid precursor protein (␤APP) through sequential cleavages by ␤-and ␥-secretases (2). Genetic and biochemical studies suggest that PS is essential for the ␥-secretase-mediated intramembrane cleavage of ␤APP. Additional type 1 transmembrane proteins (e.g. notch, ErbB4, and CD44) also are cleaved by ␥-secretase to release intracellular domains that harbor biological activities (3). These data suggest that PS-dependent ␥-cleavage is involved in a novel mode of intramembrane proteolysis-dependent signal transduction.PS is a highly conserved, polytopic integral membrane protein that spans the membrane eight times and undergoes endoproteolysis to generate amino-and carboxyl-terminal fragments (reviewed in Ref. 4). The endoproteolytic fragments of PS form a heterodimer and are then incorporated into a highly stabilized, high molecular weight (HMW) protein complex, whereas the PS holoprotein forms a low molecular weight complex and is rapidly degraded (5, 6). We have shown by a systematic mutational analysis that the stabilization and HMW complex formation of PS are required for ␥-secretase activity, which is mediated by the carboxyl terminus of PS (7-9). PS carries two highly conserved intramembrane aspartates ...

Direct connections between corticospinal (CS) axons and motoneurons (MNs) appear to be present only in higher primates, where they are essential for discrete movement of the digits. Their presence in adult rodents was once claimed but is now questioned. We report that MNs innervating forearm muscles in infant rats receive monosynaptic input from CS axons, but MNs innervating proximal muscles do not, which is a pattern similar to that in primates. Our experiments were carefully designed to show monosynaptic connections. This entailed selective electrical and optogenetic stimulation of CS axons and recording from MNs identified by retrograde labelling from innervated muscles. Morphological evidence was also obtained for rigorous identification of CS axons and MNs. These connections would be transient and would regress later during development. These results shed light on the development and evolution of direct CS-MN connections, which serve as the basis for dexterity in humans. Recent evidence suggests there is no direct connection between corticospinal (CS) axons and spinal motoneurons (MNs) in adult rodents. We previously showed that CS synapses are present throughout the spinal cord for a time, but are eliminated from the ventral horn during development in rodents. This raises the possibility that CS axons transiently make direct connections with MNs located in the ventral horn of the spinal cord. This was tested in the present study. Using cervical cord slices prepared from rats on postnatal days (P) 7-9, CS axons were stimulated and whole cell recordings were made from MNs retrogradely labelled with fluorescent cholera toxin B subunit (CTB) injected into selected groups of muscles. To selectively activate CS axons, electrical stimulation was carefully limited to the CS tract. In addition we employed optogenetic stimulation after injecting an adeno-associated virus vector encoding channelrhodopsin-2 (ChR2) into the sensorimotor cortex on P0. We were then able to record monosynaptic excitatory postsynaptic currents from MNs innervating forearm muscles, but not from those innervating proximal muscles. We also showed close contacts between CTB-labelled MNs and CS axons labelled through introduction of fluorescent protein-conjugated synaptophysin or the ChR2 expression system. We confirmed that some of these contacts colocalized with postsynaptic density protein 95 in their partner dendrites. It is intriguing from both phylogenetic and ontogenetic viewpoints that direct and putatively transient CS-MN connections were found only on MNs innervating the forearm muscles in infant rats, as this is analogous to the connection pattern seen in adult primates.