Rab8a and Rab8b are essential for several apical transport pathways but insufficient for ciliogenesis.

The small GTP-binding protein Rab8 is known to play an essential role in intracellular transport and cilia formation. We have previously demonstrated that Rab8a is required for localising apical markers in various organisms. Rab8a has a closely related isoform, Rab8b. To determine whether Rab8b can compensate for Rab8a, we generated Rab8b-knockout mice. Although the Rab8b-knockout mice did not display an overt phenotype, Rab8a and Rab8b double-knockout mice exhibited mislocalisation of apical markers and died earlier than Rab8a-knockout mice. The apical markers accumulated in three intracellular patterns in the double-knockout mice. However, the localisation of basolateral and/or dendritic markers of the double-knockout mice seemed normal. The morphology and the length of various primary and/or motile cilia, and the frequency of ciliated cells appeared to be identical in control and double-knockout mice. However, an additional knockdown of Rab10 in double-knockout cells greatly reduced the percentage of ciliated cells. Our results highlight the compensatory effect of Rab8a and Rab8b in apical transport, and the complexity of the apical transport process. In addition, neither Rab8a nor Rab8b are required for basolateral and/or dendritic transport. However, simultaneous loss of Rab8a and Rab8b has little effect on ciliogenesis, whereas additional loss of Rab10 greatly affects ciliogenesis.

The role of PKD in cell polarity, biosynthetic pathways, and organelle/F-actin distribution.

Protein Kinase D (PKD) 1, 2, and 3 are members of the PKD family. PKDs influence many cellular processes, including cell polarity, structure of the Golgi, polarized transport from the Golgi to the basolateral plasma membrane, and actin polymerization. However, the role of the PKD family in cell polarity has not yet been elucidated in vivo. Here, we show that KO mice displayed similar localization of the apical and basolateral proteins, transport of VSV-G and a GPI-anchored protein, and similar localization of actin filaments. As DKO mice were embryonic lethal, we generated MEFs that lacked all PKD isoforms from the PKD1 and PKD2 double floxed mice using Cre recombinase and PKD3 siRNA. We observed a similar localization of various organelles, a similar time course in the transport of VSV-G and a GPI-anchored protein, and a similar distribution of F-actin in the PKD-null MEFs. Collectively, our results demonstrate that the complete deletion of PKDs does not affect the transport of VSV-G or a GPI-anchored protein, and the distribution of F-actin. However, simultaneous deletion of PKD1 and PKD2 affect embryonic development, demonstrating their functional redundancy during 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.

Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector.

The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.

The Rab8 GTPase regulates apical protein localization in intestinal cells.

A number of proteins are known to be involved in apical/basolateral transport of proteins in polarized epithelial cells. The small GTP-binding protein Rab8 was thought to regulate basolateral transport in polarized kidney epithelial cells through the AP1B-complex-mediated pathway. However, the role of Rab8 (Rab8A) in cell polarity in vivo remains unknown. Here we show that Rab8 is responsible for the localization of apical proteins in intestinal epithelial cells. We found that apical peptidases and transporters localized to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes led to a marked reduction in the absorption rate of nutrients in the small intestine, and ultimately to death. Ultrastructurally, a shortening of apical microvilli, an increased number of enlarged lysosomes, and microvillus inclusions in the enterocytes were also observed. One microvillus inclusion disease patient who shows an identical phenotype to Rab8-deficient mice expresses a reduced amount of RAB8 (RAB8A; NM_005370). Our results demonstrate that Rab8 is necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine.

Neuron-specific recombination by Cre recombinase inserted into the murine tau locus.

To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 'floxed' mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo.

Neuron-specific and inducible recombination by Cre recombinase in the mouse.

To investigate the neuronal function of genes in vivo, a neuron-specific and inducible gene targeting system is desirable. In this study, we generated a knockin mouse line that expresses a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR) in neurons. The neuron-specific expression of CrePR was attained by inserting CrePR gene into the tau locus, because tau is expressed strongly in neurons but scarcely in glias and other tissues. By crossing this knockin mouse line (tau(CrePR)) with ROSA26 lacZ reporter mouse line (R26R), we observed that the antiprogesterone RU486 could induce recombinase activity of the CrePR specifically in neurons. Thus, tau (CrePR) knockin line is a useful tool for studying neuronal gene functions.

14-3-3 proteins and protein phosphatases are not reduced in tau-deficient mice.

Tau is an axonal microtubule-associated protein, whose dysfunction causes neurodegenerative diseases such as Alzheimer's disease and other tauopathies. Earlier studies have shown the interactions of tau with glycogen synthase kinase-3beta, 14-3-3zeta, protein phosphatase 1 and protein phosphatase 2A. In this study, we compared the amounts of these tau-interacting proteins in brain microtubule-enriched fractions from wild-type and tau-deficient mice. Contrary to our expectation, we detected no difference in the amount of these proteins between wild-type and tau-deficient mice. Our findings indicate that only a small portion of tau-interacting proteins are bound to tau in vivo, and suggest the existence of other scaffolding proteins. We propose that tau-deficient mice are an ideal system for confirming the function of tau-interacting proteins.

Opposing roles for SNAP23 in secretion in exocrine and endocrine pancreatic cells.

The membrane fusion of secretory granules with plasma membranes is crucial for the exocytosis of hormones and enzymes. Secretion disorders can cause various diseases such as diabetes or pancreatitis. Synaptosomal-associated protein 23 (SNAP23), a soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) molecule, is essential for secretory granule fusion in several cell lines. However, the in vivo functions of SNAP23 in endocrine and exocrine tissues remain unclear. In this study, we show opposing roles for SNAP23 in secretion in pancreatic exocrine and endocrine cells. The loss of SNAP23 in the exocrine and endocrine pancreas resulted in decreased and increased fusion of granules to the plasma membrane after stimulation, respectively. Furthermore, we identified a low molecular weight compound, MF286, that binds specifically to SNAP23 and promotes insulin secretion in mice. Our results demonstrate opposing roles for SNAP23 in the secretion mechanisms of the endocrine and exocrine pancreas and reveal that the SNAP23-binding compound MF286 may be a promising drug for diabetes treatment.

In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris.

Patients with pemphigus vulgaris (PV) have circulating anti-desmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic strength of anti-Dsg3 IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantly by Dsg3, we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting, and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PV sera (n=10, 77.4+/-21.4) than controls (n=11, 16.0+/-9.6; p=0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n=6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies.

Functional redundancy of protein kinase D1 and protein kinase D2 in neuronal polarity.

Mammalian protein kinase D (PKD) isoforms have been proposed to regulate diverse biological processes, including the establishment and maintenance of neuronal polarity. To investigate the function of PKD in neuronal polarization in vivo, we generated PKD knockout (KO) mice. Here, we show that the brain, particularly the hippocampus, of both PKD1 KO and PKD2 KO mice was similar to that of control animals. Neurite length in cultured PKD1 KO and PKD2 KO hippocampal neurons was similar to that of wild-type neurons. However, hippocampal neurons deficient in both PKD1 and PKD2 genes showed a reduction in axonal elongation and an increase in the percentage of neurons with multiple axons relative to control neurons. These results reveal that whereas PKD1 and PKD2 are essential for neuronal polarity, there exists a functional redundancy between the two proteins.

Suppression of the immune response against exogenous desmoglein 3 in desmoglein 3 knockout mice: an implication for gene therapy.

Gene therapies for recessive genetic diseases may provoke unwanted immune responses against the introduced gene product because patients, especially those with null mutation of a certain protein, have no tolerance for the protein of interest. This study used desmoglein 3 knockout (Dsg3-/-) mice as a disease model for a genetic defect in DSG3, to investigate whether nonviral gene therapy induces an immune response against Dsg3 and whether the reaction against Dsg3 can be prevented. When mouse Dsg3 cDNA was injected in the skin of Dsg3-/- mice, 50% of treated Dsg3-/- mice developed anti-Dsg3 IgG, which can bind native Dsg3 in vivo. To prevent this response, we used an anti-CD40L monoclonal antibody, MR1, which blocks the costimulatory interaction between CD40 and CD40L. To evaluate the effect of MR1, we grafted Dsg3+/+skin on Dsg3-/- mice, to mimic stable gene transfer of Dsg3. After skin grafting, all the recipient Dsg3-/- mice were treated with either MR1 (n=8) or control hamster IgG (n=8). All of the control IgG-treated mice developed circulating anti-Dsg3 IgG about 2 wk after grafting, and IgG deposition was observed on the surfaces of keratinocytes in the grafted Dsg3+/+skin. Such anti-Dsg3 IgG production was significantly prevented, however, when the recipient mice were treated with MR1. These findings suggested that gene therapies for recessive diseases may provoke an immune response against the transgene product, and that the CD40-CD40L interaction might be a reasonable target for effective prevention of such undesirable immune responses, leading, in turn, to a successful gene therapy.

Localization of cAMP-dependent protein kinase in the actin and microtubule cytoskeletons in mouse hippocampal neurons.

Cyclic adenosine monophosphate-dependent protein kinase (PKA) is involved in various biological functions in neurons. To investigate the subcellular localization of PKA, we stained cultured hippocampal neurons with anti-PKA catalytic subunit antisera. PKA catalytic subunit colocalized with microtubules (MTs) in dendrites as well as with the actin filaments (F-actin) in growth cones. After treatment with cytochalasin B, the colocalization of PKA catalytic subunits with MTs was enhanced, whereas the colocalization with F-actin was suppressed. This result indicates that PKA is anchored to the actin and MT cytoskeletons, and disruption of F-actin releases PKA to the cytoplasm, which then leads to an increase in the amount of PKA in MT domains in the neuron.

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.

Reduced expression of kinase-associated phosphatase in cortical dendrites of MAP2-deficient mice.

We previously demonstrated that cAMP-dependent protein kinase was reduced in the dendrites of MAP2-deficient mice. In this study, we compared the expression of various protein phosphatases (PPs) between wild-type and map2(-/-) dendrites. Kinase-associated phosphatase (KAP) was the only PP which showed difference between the two phenotypes: (1) the expression of KAP was reduced in map2(-/-) cortical dendrites, and (2) the amount of KAP bound to microtubules was reduced in map2(-/-) brains. We also demonstrated in cultured neuroblastoma cells that KAP is not only expressed in dividing cells, but also in the neurites of differentiated cells. Our findings propose that KAP, which has been reported to function in cell-cycle control, has an as yet uncovered role in regulating dendritic functions. We also propose MAP2-deficient mice as an ideal system for identifying protein phosphatases essential for dendritic functions.