Presenilin (PS) is essential for the ␥-cleavage required for the generation of the C terminus of amyloid ␤-protein (A␤). However, the mechanism underlying PS-mediated ␥-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of ␥-cleavage. Herp was originally identified as a homocysteine-responsive protein, and its expression is up-regulated by endoplasmic reticulum stress. Herp is an endoplasmic reticulum-localized membrane protein that has a ubiquitin-like domain. Here, we report that a high expression of Herp in cells increases the level of A␤ generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated A␤ generation, possibly through its binding to PS. Immunohistochemical analysis of a normal human brain section with an anti-Herp antibody revealed the exclusive staining of neurons and vascular smooth muscle cells. Moreover, the antibody strongly stained activated microglia in senile plaques in the brain of patients with Alzheimer disease. Taken together, Herp could be involved in A␤ accumulation, including the formation of senile plaques and vascular A␤ deposits.

Changes in the cytoskeletal architecture underpin the dynamic changes in tissue shape that occur during development. It is clear that such changes must be coordinated so that individual cell behaviors are synchronized; however, the mechanisms by which morphogenesis is instructed and coordinated are unknown. After its induction in non-neural ectoderm, the inner ear undergoes morphogenesis, being transformed from a flat ectodermal disk on the surface of the embryo to a hollowed sphere embedded in the head. We provide evidence that this shape change relies on extrinsic signals subsequent to genetic specification. By using specific inhibitors, we find that local fibroblast growth factor (FGF) signaling triggers a phosphorylation cascade that activates basal myosin II through the activation of phospholipase Cgamma. Myosin II exhibits a noncanonical activity that results in the local depletion of actin filaments. Significantly, the resulting apical actin enrichment drives morphogenesis of the inner ear. Thus, FGF signaling directly exerts profound cytoskeletal effects on otic cells, coordinating the morphogenesis of the inner ear. The iteration of this morphogenetic signaling system suggests that it is a more generally applicable mechanism in other epithelial tissues undergoing shape change.

Immotile cilia at the ventral node of mouse embryos are required for sensing leftward fluid flow that breaks left-right symmetry of the body. However, the flow-sensing mechanism has long remained elusive. In this work, we show that immotile cilia at the node undergo asymmetric deformation along the dorsoventral axis in response to the flow. Application of mechanical stimuli to immotile cilia by optical tweezers induced calcium ion transients and degradation of Dand5 messenger RNA (mRNA) in the targeted cells. The Pkd2 channel protein was preferentially localized to the dorsal side of immotile cilia, and calcium ion transients were preferentially induced by mechanical stimuli directed toward the ventral side. Our results uncover the biophysical mechanism by which immotile cilia at the node sense the direction of fluid flow.

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.

Herp is an endoplasmic reticulum (ER)-stress-inducible membrane protein, which has a ubiquitin-like domain (ULD). However, its biological function is as yet unknown. Previously, we reported that a high expression level of Herp in cells increases the generation of amyloid L L-protein (AL L) and that Herp interacts with presenilin (PS). Here, we addressed the role of the ULD of Herp in AL L generation and intracellular Herp stability. We found that the ULD is not essential for the enhancement of AL L generation by Herp expression and the interaction of Herp with PS, but is involved in the rapid degradation of Herp, most likely via the ubiquitin/proteasome pathway. Thus, the ULD of Herp most likely plays a role in the regulation of the intracellular level of Herp under ER stress.

After induction, the inner ear is transformed from a superficially located otic placode into an epithelial vesicle embedded in the mesenchyme of the head. Invagination of this epithelium is biphasic: phase 1 involves the expansion of the basal aspect of the otic cells, and phase 2, the constriction of their apices. Apical constriction is important not only for otic invagination, but also the invagination of many other epithelia; however, its molecular basis is still poorly understood. Here we show that phase 2 otic morphogenesis, like phase 1 morphogenesis, results from the activation of myosin-II. However unlike the actin depolymerising activity observed basally, active myosin-II results in actomyosin contractility. Myosin-II activation is triggered by the accumulation of the planar cell polarity (PCP) core protein, Celsr1 in apical junctions (AJ). Apically polarized Celsr1 orients and recruits the Rho Guanine exchange factor (GEF) ArhGEF11 to apical junctions, thus restricting RhoA activity to the junctional membrane where it activates the Rho kinase ROCK. We suggest that myosin-II and RhoA activation results in actomyosin dependent constriction in an apically polarised manner driving otic epithelium invagination.

Various cellular replacement therapies using in vitro generated cells to replace damaged tissue have been proposed as strategies to alleviate hearing loss. All such therapies must involve a complete understanding of the earliest steps in inner ear development; its induction as a thickened plate of cells in the non-neural, surface ectoderm of the embryo, to its internalization as an otocyst embedded in the head mesenchyme of the embryo. Such knowledge informs researchers addressing the feasibility of the proposed strategy and present alternatives if needed. In this review we describe the mechanisms of inner ear induction, concentrating on the factors that steer the fate of ectoderm into precursors of the inner ear. Induction then leads to inner ear morphogenesis and we describe the cellular changes that occur as the inner ear is converted from a superficial placode to an internalized otocyst, and how they are coordinated with a particular emphasis on how the signaling environment surrounding the inner ear influences these processes.

Presenilin (PS) is essential for ␥-cleavage, which is required for the generation of amyloid ␤-protein (A␤) from the ␤-amyloid precursor protein. However, it remains to be clarified how ␥-cleavage is regulated. To elucidate the regulation of PS-mediated ␥-cleavage, we developed a new functional screening method for identifying cDNA that enhances ␥-cleavage. This screening system utilizes our own developed cell line, where the expression of cDNA that enhances ␥-cleavage confers puromycin resistance. The cDNA library is retrovirally delivered to the above-mentioned cell line, allowing the identification of our target cDNAs by a combination of puromycin resistance selection and A␤ assay screening. With this screening method, we isolated several cDNAs enhancing ␥-cleavage, including the previously reported Herp. Here we also demonstrate that Rab1A, identified with this screening, can be a regulator of A␤ generation. Thus, our established screening method is a powerful tool for identifying multiple regulators involved in ␥-cleavage in the A␤ generation pathway, including modulators of ␥-secretase activity or the intracellular trafficking of factors necessary for ␥-cleavage. A␤,1 which is the major component of senile plaques in the brain of patients with Alzheimer's disease (AD), is generated from APP through its sequential proteolytic cleavage catalyzed by ␤-and ␥-secretase (reviewed in Ref. 1). Although ␤-secretase was identified as a membrane-tethered aspartyl protease (2), the molecule responsible for ␥-secretase activity remains to be clarified. Mutations in the presenilin (PS) genes, PS1 and PS2, cause early-onset familial AD (reviewed in Ref. 1). Accumulating evidence showed that PS is required for the proteolytic cleavage catalyzed by ␥-secretase, which occurs in the transmembrane domain of APP (␥-cleavage) (Refs. 3-5; reviewed in Ref. 6). Interestingly, although the ␥-cleavage is a critical step toward 〈␤ production, the major intramembranous cleavage site of APP is distinct from the ␥-cleavage (named as ⑀-cleavage site) (7,8). In addition, recent studies revealed that PS mediates several intramembranous cleavages including those of APP, Notch (3-5), ErbB4 (9, 10), and E-cadherin (11), indicating that the PS-mediated intramembranous cleavage plays a critical role in biological functions. The PS complex appears to be responsible for inducing ␥-secretase activity (12, 13); however, it is still controversial whether PS itself is ␥-secretase (Refs. 14 -16, reviewed in Ref. 17). The discrepancy between the intracellular major distribution of the PS complex and the intracellular site of ␥-cleavage also remains to be clarified (reviewed in Ref. 17). Therefore, the understanding of the mechanism underlying ␥-cleavage will require clarification of multiple factors involved in ␥-cleavage, including the components of the ␥-secretase complex and modulators of the ␥-secretase activity or the trafficking of factors necessary for ␥-cleavage. To elucidate how PS-mediated ␥-cleavage is regulated, we developed a new functiona...