Rer1-mediated quality control system is required for neural stem cell maintenance during cerebral cortex development.

Rer1 is a retrieval receptor for endoplasmic reticulum (ER) retention of various ER membrane proteins and unassembled or immature components of membrane protein complexes. However, its physiological functions during mammalian development remain unclear. This study aimed to investigate the role of Rer1-mediated quality control system in mammalian development. We show that Rer1 is required for the sufficient cell surface expression and activity of γ-secretase complex, which modulates Notch signaling during mouse cerebral cortex development. When Rer1 was depleted in the mouse cerebral cortex, the number of neural stem cells decreased significantly, and malformation of the cerebral cortex was observed. Rer1 loss reduced γ-secretase activity and downregulated Notch signaling in the developing cerebral cortex. In Rer1-deficient cells, a subpopulation of γ-secretase complexes and components was transported to and degraded in lysosomes, thereby significantly reducing the amount of γ-secretase complex on the cell surface. These results suggest that Rer1 maintains Notch signaling by maintaining sufficient expression of the γ-secretase complex on the cell surface and regulating neural stem cell maintenance during cerebral cortex development.

The role of VAMP7/TI-VAMP in cell polarity and lysosomal exocytosis in vivo.

VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.

Structure of liposome encapsulating proteins characterized by X-ray scattering and shell-modeling.

Lipid liposomes are promising drug delivery systems because they have superior curative effects owing to their high adaptability to a living body. Lipid liposomes encapsulating proteins were constructed and the structures examined using synchrotron radiation small- and wide-angle X-ray scattering (SR-SWAXS). The liposomes were prepared by a sequential combination of natural swelling, ultrasonic dispersion, freeze-throw, extrusion and spin-filtration. The liposomes were composed of acidic glycosphingolipid (ganglioside), cholesterol and phospholipids. By using shell-modeling methods, the asymmetric bilayer structure of the liposome and the encapsulation efficiency of proteins were determined. As well as other analytical techniques, SR-SWAXS and shell-modeling methods are shown to be a powerful tool for characterizing in situ structures of lipid liposomes as an important candidate of drug delivery systems.

RAB35 is required for murine hippocampal development and functions by regulating neuronal cell distribution.

RAB35 is a multifunctional small GTPase that regulates endocytic recycling, cytoskeletal rearrangement, and cytokinesis. However, its physiological functions in mammalian development remain unclear. Here, we generated Rab35-knockout mice and found that RAB35 is essential for early embryogenesis. Interestingly, brain-specific Rab35-knockout mice displayed severe defects in hippocampal lamination owing to impaired distribution of pyramidal neurons, although defects in cerebral cortex formation were not evident. In addition, Rab35-knockout mice exhibited defects in spatial memory and anxiety-related behaviors. Quantitative proteomics indicated that the loss of RAB35 significantly affected the levels of other RAB proteins associated with endocytic trafficking, as well as some neural cell adhesion molecules, such as contactin-2. Collectively, our findings revealed that RAB35 is required for precise neuronal distribution in the developing hippocampus by regulating the expression of cell adhesion molecules, thereby influencing spatial memory.

Structure of Ultrafine Bubbles and Their Effects on Protein and Lipid Membrane Structures Studied by Small- and Wide-Angle X-ray Scattering.

Ultrafine bubbles (UFBs) are defined as small gas-filled bubbles with a diameter smaller than 1 µm. UFBs are stable for several weeks in aqueous solutions due to their small size. Although the mechanism of the stability of UFBs remains under intensive investigation, industrial applications of UFBs have recently arisen in various fields such as agricultural and fishery industries and medical therapy. The relevance of ions (protons and hydroxide anions) in UFB solutions has been discussed; however, the mechanism underlying the behavior of UFBs is still ambiguous and there is little direct evidence of the effect of UFBs on biological materials. This study deals with gaseous UFBs in aqueous solutions. Using small- and wide-angle X-ray scattering, we have investigated the structures of UFBs (air-UFBs, O2-UFBs, and N2-UFBs) and their effect on protein and lipid membrane structures. X-ray scattering and modeling data suggest that UFBs present a dynamic diffusive boundary (interface) due to the continuous release and absorption of gas. UFBs were found to not affect the structures of proteins at all hierarchal structure levels (from quaternary to tertiary, to internal, to secondary), whereas they did influence the packing and fluctuation of the hydrocarbon chains in the liposomes but not their shapes.

Effect of protein-encapsulation on thermal structural stability of liposome composed of glycosphingolipid/cholesterol/phospholipid.

We have studied the thermal structural stability of liposomes encapsulating proteins by using synchrotron radiation small- and wide-angle X-ray scattering (SR-SWAXS). Liposomes are known to be effective drug-delivery systems (DDSs) because they can reduce drug toxicity due to biodegradability and biocompatibility and can offer promising carriers of various types of drugs. However, in spite of numerous studies of liposomes, physicochemical characteristics of liposomes entrapping proteins are rarely known. The liposome studied is characterized by the lipid composition (mixture of acidic glycosphingolipid (ganglioside)/cholesterol/phospholipid). Gangliosides are one of the major constituents of so-called lipid rafts playing the role of a platform of cell-signaling. We have found that the encapsulation of proteins elevates the thermal transition temperature of the liposome membrane and suppresses the deformation of its shape. The present results suggest that not only membrane proteins, but also water-soluble proteins affect liposome stability through the revalence between osmotic pressure and membrane elasticity. In addition, we have found the presence of the size-effect depending on the molar content of gangliosides in the liposome, indicating the ability of ganglioside molecule controlling both the size and effective surface charge of the liposome. The present results would have significance from two different points of view. One is the confinement effect of proteins within a limited space like cell, and the other is a stability of a new type of DDS using gangliosides. Due to the intrinsic properties, gangliosides are expected to be promising agents for targeting and long-circulation properties of liposomal DDSs.

Rer1 and calnexin regulate endoplasmic reticulum retention of a peripheral myelin protein 22 mutant that causes type 1A Charcot-Marie-Tooth disease.

Peripheral myelin protein 22 (PMP22) resides in the plasma membrane and is required for myelin formation in the peripheral nervous system. Many PMP22 mutants accumulate in excess in the endoplasmic reticulum (ER) and lead to the inherited neuropathies of Charcot-Marie-Tooth (CMT) disease. However, the mechanism through which PMP22 mutants accumulate in the ER is unknown. Here, we studied the quality control mechanisms for the PMP22 mutants L16P and G150D, which were originally identified in mice and patients with CMT. We found that the ER-localised ubiquitin ligase Hrd1/SYVN1 mediates ER-associated degradation (ERAD) of PMP22(L16P) and PMP22(G150D), and another ubiquitin ligase, gp78/AMFR, mediates ERAD of PMP22(G150D) as well. We also found that PMP22(L16P), but not PMP22(G150D), is partly released from the ER by loss of Rer1, which is a Golgi-localised sorting receptor for ER retrieval. Rer1 interacts with the wild-type and mutant forms of PMP22. Interestingly, release of PMP22(L16P) from the ER was more prominent with simultaneous knockdown of Rer1 and the ER-localised chaperone calnexin than with the knockdown of each gene. These results suggest that CMT disease-related PMP22(L16P) is trapped in the ER by calnexin-dependent ER retention and Rer1-mediated early Golgi retrieval systems and partly degraded by the Hrd1-mediated ERAD system.

p31 deficiency influences endoplasmic reticulum tubular morphology and cell survival.

p31, the mammalian orthologue of yeast Use1p, is an endoplasmic reticulum (ER)-localized soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) that forms a complex with other SNAREs, particularly syntaxin 18. However, the role of p31 in ER function remains unknown. To determine the role of p31 in vivo, we generated p31 conditional knockout mice. We found that homozygous deletion of the p31 gene led to early embryonic lethality before embryonic day 8.5. Conditional knockout of p31 in brains and mouse embryonic fibroblasts (MEFs) caused massive apoptosis accompanied by upregulation of ER stress-associated genes. Microscopic analysis showed vesiculation and subsequent enlargement of the ER membrane in p31-deficient cells. This type of drastic disorganization in the ER tubules has not been demonstrated to date. This marked change in ER structure preceded nuclear translocation of the ER stress-related transcription factor C/EBP homologous protein (CHOP), suggesting that ER stress-induced apoptosis resulted from disruption of the ER membrane structure. Taken together, these results suggest that p31 is an essential molecule involved in the maintenance of ER morphology and that its deficiency leads to ER stress-induced apoptosis.

Rearrangement of microtubule polarity orientation during conversion of dendrites to axons in cultured pyramidal neurons.

Axons and dendrites of neurons differ in the polarity orientation of their microtubules. Whereas the polarity orientation of microtubules in axons is uniform, with all plus ends distal, that in dendrites is nonuniform. The mechanisms responsible for establishment and maintenance of microtubule polarity orientation in neuronal processes remain unclear, however. We previously described a culture system in which dendrites of rat cortical neurons convert to axons. In the present study, we examined changes in microtubule polarity orientation in such dendrites. With the use of the hooking procedure and electron microscopy, we found that microtubule polarity orientation changed from nonuniform to uniform, with a plus end-distal arrangement, in dendrites that gave rise to axons during culture of neurons for 24 h. Microtubule polarity orientation remained nonuniform in dendrites that did not elongate. Axon regeneration at the dendritic tip thus triggered the disappearance of minus end-distal microtubules from dendrites. These minus end-distal microtubules also disappeared from dendrites during axon regeneration in the presence of inhibitors of actin polymerization, suggesting that actin-dependent transport of microtubules is not required for this process and implicating a previously unidentified mechanism in the establishment and maintenance of microtubule polarity orientation in neuronal processes.

Inhibitory neurons from fetal rat cerebral cortex exert delayed axon formation and active migration in vitro.

Inhibitory and excitatory neurons exhibit distinct patterns of development in the mammalian cerebral cortex. The morphological development of inhibitory and excitatory neurons derived from fetal rat cerebral cortex has now been compared in vitro. Inhibitory neurons were identified by immunofluorescence staining with antibodies to gamma-aminobutyric acid, and axon formation was detected by staining with antibodies to phosphorylated neurofilaments. In chemically defined, glia-free and low-density cultures, excitatory neurons formed axons within three days of plating. By contrast, inhibitory neurons required more than six days to form axons. Time-lapse analysis over six days revealed that most inhibitory neurons were bipolar and that their two processes exhibited alternate growth and retraction without giving rise to axons. Movement of the cell body towards the growing process was apparent in about one-half of inhibitory neurons, whereas such movement was never seen in excitatory neurons. The migratory behavior of neurons was further investigated by culture on a glial cell monolayer. Inhibitory neurons migrated over substantially larger distances than did excitatory neurons. The centrosome of inhibitory neurons translocated to the base of the newly emerging leading process, suggesting the existence of a force that pulls intracellular organelles towards the leading process. Centrosome translocation was not detected in excitatory neurons. These observations suggest that the developmental programs of excitatory and inhibitory neurons differ. Inhibitory neurons thus possess a more effective cytoskeletal machinery for migration than excitatory neurons and they form axons later.

A novel, brain-specific mouse drebrin: cDNA cloning, chromosomal mapping, genomic structure, expression, and functional characterization.

Drebrin A, a major neuronal actin-binding protein, regulates the dendritic spine shapes of neurons. Here, we have cloned and characterized a novel mouse cDNA clone encoding a truncated form of drebrin A, named s-drebrin A. Analysis of the genomic organization of the mouse drebrin gene (Dbn1), which mapped to the central portion of chromosome 13, revealed that isoforms including s-drebrin A are generated by alternative splicing from a single drebrin gene. The s-drebrin A mRNA was expressed in the brain, but not in non-neuronal tissues. The s-drebrin A expression was barely detected in the embryonic brain, but was upregulated during postnatal development of the brain. Overexpression of GFP-tagged s-drebrin A in fibroblasts showed it to be associated with actin filaments and with changes in actin cytoskeleton organization. These findings suggest that s-drebrin A has a role in spine morphogenesis, possibly by competing the actin-binding activity with drebrin A.