A Farnesyltransferase Inhibitor Restores Cognitive Deficits in Tsc2+/- Mice through Inhibition of Rheb1.

Tuberous sclerosis complex (TSC) is caused by mutations in Tsc1 or Tsc2, whose gene products inhibit the small G-protein Rheb1. Rheb1 activates mTORC1, which may cause refractory epilepsy, intellectual disability, and autism. The mTORC1 inhibitors have been used for TSC patients with intractable epilepsy. However, its effectiveness for cognitive symptoms remains unclear. We found a new signaling pathway for synapse formation through Rheb1 activation, but not mTORC1. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib increased unfarnesylated (inactive) Rheb1 levels and restored synaptic abnormalities in cultured Tsc2+/- neurons, whereas rapamycin did not enhance spine synapse formation. Lonafarnib treatment also restored the plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in cultured Tsc2+/- neurons. Lonafarnib action was partly dependent on the Rheb1 reduction with syntenin. Oral administration of lonafarnib increased unfarnesylated protein levels without affecting mTORC1 and MAP (mitogen-activated protein (MAP)) kinase signaling, and restored dendritic spine morphology in the hippocampi of male Tsc2+/- mice. In addition, lonafarnib treatment ameliorated contextual memory impairments and restored memory-related Arc expression in male Tsc2+/- mice in vivo Heterozygous Rheb1 knockout in male Tsc2+/- mice reproduced the results observed with pharmacological treatment. These results suggest that the Rheb1 activation may be responsible for synaptic abnormalities and memory impairments in Tsc2+/- mice, and its inhibition by lonafarnib could provide insight into potential treatment options for TSC-associated neuropsychiatric disorders.SIGNIFICANCE STATEMENT Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that causes neuropsychiatric symptoms, including intractable epilepsy, intellectual disability (ID) and autism. No pharmacological treatment for ID has been reported so far. To develop a pharmacological treatment for ID, we investigated the mechanism of TSC and found that Rheb1 activation is responsible for synaptic abnormalities in TSC neurons. To inhibit Rheb1 function, we used the farnesyltransferase inhibitor lonafarnib, because farnesylation of Rheb1 is required for its activation. Lonafarnib treatment increased inactive Rheb1 and recovered proper synapse formation and plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in TSC neurons. Furthermore, in vivo lonafarnib treatment restored contextual memory and Arc induction in TSC mice. Together, Rheb1 inhibition by lonafarnib could provide insight into potential treatments for TSC-associated ID.

Mast Cell-Specific Deletion of Group III Secreted Phospholipase A2 Impairs Mast Cell Maturation and Functions.

Tissue-resident mast cells (MCs) have important roles in IgE-associated and -independent allergic reactions. Although microenvironmental alterations in MC phenotypes affect the susceptibility to allergy, understanding of the regulation of MC maturation is still incomplete. We previously reported that group III secreted phospholipase A2 (sPLA2-III) released from immature MCs is functionally coupled with lipocalin-type prostaglandin D2 (PGD2) synthase in neighboring fibroblasts to supply a microenvironmental pool of PGD2, which in turn acts on the PGD2 receptor DP1 on MCs to promote their proper maturation. In the present study, we reevaluated the role of sPLA2-III in MCs using a newly generated MC-specific Pla2g3-deficient mouse strain. Mice lacking sPLA2-III specifically in MCs, like those lacking the enzyme in all tissues, had immature MCs and displayed reduced local and systemic anaphylactic responses. Furthermore, MC-specific Pla2g3-deficient mice, as well as MC-deficient KitW-sh mice reconstituted with MCs prepared from global Pla2g3-null mice, displayed a significant reduction in irritant contact dermatitis (ICD) and an aggravation of contact hypersensitivity (CHS). The increased CHS response by Pla2g3 deficiency depended at least partly on the reduced expression of hematopoietic PGD2 synthase and thereby reduced production of PGD2 due to immaturity of MCs. Overall, our present study has confirmed that MC-secreted sPLA2-III promotes MC maturation, thereby facilitating acute anaphylactic and ICD reactions and limiting delayed CHS response.

OHC-TRECK: A Novel System Using a Mouse Model for Investigation of the Molecular Mechanisms Associated with Outer Hair Cell Death in the Inner Ear.

Outer hair cells (OHCs) are responsible for the amplification of sound, and the death of these cells leads to hearing loss. Although the mechanisms for sound amplification and OHC death have been well investigated, the effects on the cochlea after OHC death are poorly understood. To study the consequences of OHC death, we established an OHC knockout system using a novel mouse model, Prestin-hDTR, which uses the prestin promoter to express the human diphtheria toxin (DT) receptor gene (hDTR). Administration of DT to adult Prestin-hDTR mice results in the depletion of almost all OHCs without significant damage to other cochlear and vestibular cells, suggesting that this system is an effective tool for the analysis of how other cells in the cochlea and vestibula are affected after OHC death. To evaluate the changes in the cochlea after OHC death, we performed differential gene expression analysis between the untreated and DT-treated groups of wild-type and Prestin-hDTR mice. This analysis revealed that genes associated with inflammatory/immune responses were significantly upregulated. Moreover, we found that several genes linked to hearing loss were strongly downregulated by OHC death. Together, these results suggest that this OHC knockout system is a useful tool to identify biomarkers associated with OHC death.

Skin CD4+ Memory T Cells Play an Essential Role in Acquired Anti-Tick Immunity through Interleukin-3-Mediated Basophil Recruitment to Tick-Feeding Sites.

Ticks, blood-sucking arthropods, serve as vectors for transmission of infectious diseases including Lyme borreliosis. After tick infestation, several animal species can develop resistance to subsequent infestations, reducing the risk of transmission. In a mouse model, basophils reportedly infiltrate tick-feeding sites during the second but not first infestation and play a crucial role in the expression of acquired tick resistance. However, the mechanism underlying basophil recruitment to the second tick-feeding site remains ill-defined. Here, we investigated cells and their products responsible for the basophil recruitment. Little or no basophil infiltration was detected in T-cell-deficient mice, and adoptive transfer of CD4+ but not CD8+ T cells reconstituted it. Il3 gene expression was highly upregulated at the second tick-feeding site, and adoptive transfer of interleukin-3 (IL-3)-sufficient but not IL-3-deficient CD4+ T cells conferred the basophil infiltration on T-cell-deficient mice, indicating that the CD4+ T-cell-derived IL-3 is essential for the basophil recruitment. Notably, IL-3+ resident CD4+ memory T cells were detected even before the second infestation in previously uninfested skin distant from the first tick-feeding site. Taken together, IL-3 produced locally by skin CD4+ memory T cells appears to play a crucial role in basophil recruitment to the second tick-feeding site.

The parathyroid hormone regulates skin tumour susceptibility in mice.

Using a forward genetics approach to map loci in a mouse skin cancer model, we previously identified a genetic locus, Skin tumour modifier of MSM 1 (Stmm1) on chromosome 7, conferring strong tumour resistance. Sub-congenic mapping localized Parathyroid hormone (Pth) in Stmm1b. Here, we report that serum intact-PTH (iPTH) and a genetic polymorphism in Pth are important for skin tumour resistance. We identified higher iPTH levels in sera from cancer-resistant MSM/Ms mice compared with susceptible FVB/NJ mice. Therefore, we performed skin carcinogenesis experiments with MSM-BAC transgenic mice (Pth MSM-Tg) and Pth knockout heterozygous mice (Pth +/-). As a result, the higher amounts of iPTH in sera conferred stronger resistance to skin tumours. Furthermore, we found that the coding SNP (rs51104087, Val28Met) localizes in the mouse Pro-PTH encoding region, which is linked to processing efficacy and increased PTH secretion. Finally, we report that PTH increases intracellular calcium in keratinocytes and promotes their terminal differentiation. Taken together, our data suggest that Pth is one of the genes responsible for Stmm1, and serum iPTH could serve as a prevention marker of skin cancer and a target for new therapies.

PNPLA1 has a crucial role in skin barrier function by directing acylceramide biosynthesis.

Mutations in patatin-like phospholipase domain-containing 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, but the mechanism involved remains unclear. Here we show that PNPLA1, an enzyme expressed in differentiated keratinocytes, plays a crucial role in the biosynthesis of ω-O-acylceramide, a lipid component essential for skin barrier. Global or keratinocyte-specific Pnpla1-deficient neonates die due to epidermal permeability barrier defects with severe transepidermal water loss, decreased intercellular lipid lamellae in the stratum corneum, and aberrant keratinocyte differentiation. In Pnpla1-/- epidermis, unique linoleate-containing lipids including acylceramides, acylglucosylceramides and (O-acyl)-ω-hydroxy fatty acids are almost absent with reciprocal increases in their putative precursors, indicating that PNPLA1 catalyses the ω-O-esterification with linoleic acid to form acylceramides. Moreover, acylceramide supplementation partially rescues the altered differentiation of Pnpla1-/- keratinocytes. Our findings provide valuable insight into the skin barrier formation and ichthyosis development, and may contribute to novel therapeutic strategies for treatment of epidermal barrier defects.

Establishment of a heteroplasmic mouse strain with interspecific mitochondrial DNA haplotypes and improvement of a PCR-RFLP-based measurement system for estimation of mitochondrial DNA heteroplasmy.

Mitochondrial DNA segregation is one of the characteristic modes of mitochondrial inheritance in which the heteroplasmic state of mitochondrial DNA is transmitted to the next generation in variable proportions. To analyze mitochondrial DNA segregation, we produced a heteroplasmic mouse strain with interspecific mitochondrial DNA haplotypes, which contains two types of mitochondrial DNA derived from C57BL/6J and Mus spretus strains. The strain was produced on a C57BL/6J nuclear genomic background by microinjection of donor cytoplasm into fertilized eggs. The PCR-RFLP semi-quantitative analysis method, which was improved to reduce the effect of heteroduplex formation, was used to measure the proportion of heteroplasmic mitochondrial DNA in tissues. Founder mice contained up to approximately 14% of exogenous Mus spretus mitochondrial DNA molecules in their tails, and the detected proportions differed among tissues of the same individual. Heteroplasmic mitochondrial DNA is transmitted to the next generation in varying proportions under the maternal inheritance mode. This mitochondrial heteroplasmic mouse strain and the improved PCR-RFLP measurement system enable analysis of the transmission of heteroplasmic mitochondrial DNA variants between tissues and generations.

Dual Roles of Group IID Phospholipase A2 in Inflammation and Cancer.

Phospholipase A2 enzymes have long been implicated in the promotion of inflammation by mobilizing pro-inflammatory lipid mediators, yet recent evidence suggests that they also contribute to anti-inflammatory or pro-resolving programs. Group IID-secreted phospholipase A2 (sPLA2-IID) is abundantly expressed in dendritic cells in lymphoid tissues and resolves the Th1 immune response by controlling the steady-state levels of anti-inflammatory lipids such as docosahexaenoic acid and its metabolites. Here, we show that psoriasis and contact dermatitis were exacerbated in Pla2g2d-null mice, whereas they were ameliorated in Pla2g2d-overexpressing transgenic mice, relative to littermate wild-type mice. These phenotypes were associated with concomitant alterations in the tissue levels of ω3 polyunsaturated fatty acid (PUFA) metabolites, which had the capacity to reduce the expression of pro-inflammatory and Th1/Th17-type cytokines in dendritic cells or lymph node cells. In the context of cancer, however, Pla2g2d deficiency resulted in marked attenuation of skin carcinogenesis, likely because of the augmented anti-tumor immunity. Altogether, these results underscore a general role of sPLA2-IID as an immunosuppressive sPLA2 that allows the microenvironmental lipid balance toward an anti-inflammatory state, exerting beneficial or detrimental impact depending upon distinct pathophysiological contexts in inflammation and cancer.

Heterozygous mutation of Ush1g/Sans in mice causes early-onset progressive hearing loss, which is recovered by reconstituting the strain-specific mutation in Cdh23.

Most clinical reports have suggested that patients with congenital profound hearing loss have recessive mutations in deafness genes, whereas dominant alleles are associated with progressive hearing loss (PHL). Jackson shaker (Ush1gjs) is a mouse model of recessive deafness that exhibits congenital profound deafness caused by the homozygous mutation of Ush1g/Sans on chromosome 11. We found that C57BL/6J-Ush1gjs/+ heterozygous mice exhibited early-onset PHL (ePHL) accompanied by progressive degeneration of stereocilia in the cochlear outer hair cells. Interestingly, ePHL did not develop in mutant mice with the C3H/HeN background, thus suggesting that other genetic factors are required for ePHL development. Therefore, we performed classical genetic analyses and found that the occurrence of ePHL in Ush1gjs/+ mice was associated with an interval in chromosome 10 that contains the cadherin 23 gene (Cdh23), which is also responsible for human deafness. To confirm this mutation effect, we generated C57BL/6J-Ush1gjs/+, Cdh23c.753A/G double-heterozygous mice by using the CRISPR/Cas9-mediated Cdh23c.753A>G knock-in method. The Cdh23c.753A/G mice harbored a one-base substitution (A for G), and the homozygous A allele caused moderate hearing loss with aging. Analyses revealed the complete recovery of ePHL and stereocilia degeneration in C57BL/6J-Ush1gjs/+ mice. These results clearly show that the development of ePHL requires at least two mutant alleles of the Ush1g and Cdh23 genes. Our results also suggest that because the SANS and CDH23 proteins form a complex in the stereocilia, the interaction between these proteins may play key roles in the maintenance of stereocilia and the prevention of ePHL.

The role of group IIF-secreted phospholipase A2 in epidermal homeostasis and hyperplasia.

Epidermal lipids are important for skin homeostasis. However, the entire picture of the roles of lipids, particularly nonceramide lipid species, in epidermal biology still remains obscure. Here, we report that PLA2G2F, a functionally orphan-secreted phospholipase A2 expressed in the suprabasal epidermis, regulates skin homeostasis and hyperplasic disorders. Pla2g2f(-/-) mice had a fragile stratum corneum and were strikingly protected from psoriasis, contact dermatitis, and skin cancer. Conversely, Pla2g2f-overexpressing transgenic mice displayed psoriasis-like epidermal hyperplasia. Primary keratinocytes from Pla2g2f(-) (/-) mice showed defective differentiation and activation. PLA2G2F was induced by calcium or IL-22 in keratinocytes and preferentially hydrolyzed ethanolamine plasmalogen-bearing docosahexaenoic acid secreted from keratinocytes to give rise to unique bioactive lipids (i.e., protectin D1 and 9S-hydroxyoctadecadienoic acid) that were distinct from canonical arachidonate metabolites (prostaglandins and leukotrienes). Ethanolamine lysoplasmalogen, a PLA2G2F-derived marker product, rescued defective activation of Pla2g2f(-/-) keratinocytes both in vitro and in vivo. Our results highlight PLA2G2F as a previously unrecognized regulator of skin pathophysiology and point to this enzyme as a novel drug target for epidermal-hyperplasic diseases.

BTB-ZF Protein Znf131 Regulates Cell Growth of Developing and Mature T Cells.

Many members of the BTB-ZF family have been shown to play important roles in lymphocyte development and function. The role of zinc finger Znf131 (also known as Zbtb35) in T cell lineage was elucidated through the production of mice with floxed allele to disrupt at different stages of development. In this article, we present that Znf131 is critical for T cell development during double-negative to double-positive stage, with which significant cell expansion triggered by the pre-TCR signal is coupled. In mature T cells, Znf131 is required for the activation of effector genes, as well as robust proliferation induced upon TCR signal. One of the cyclin-dependent kinase inhibitors, p21(Cip1) encoded by cdkn1a gene, is one of the targets of Znf131. The regulation of T cell proliferation by Znf131 is in part attributed to its suppression on the expression of p21(Cip1).

Quantitative trait loci on chromosome 5 for susceptibility to frequency-specific effects on hearing in DBA/2J mice.

The DBA/2J strain is a model for early-onset, progressive hearing loss in humans, as confirmed in the present study. DBA/2J mice showed progression of hearing loss to low-frequency sounds from ultrasonic-frequency sounds and profound hearing loss at all frequencies before 7 months of age. It is known that the early-onset hearing loss of DBA/2J mice is caused by affects in the ahl (Cdh23(ahl)) and ahl8 (Fscn2(ahl8)) alleles of the cadherin 23 and fascin 2 genes, respectively. Although the strong contributions of the Fscn2(ahl8) allele were detected in hearing loss at 8- and 16-kHz stimuli with LOD scores of 5.02 at 8 kHz and 8.84 at 16 kHz, hearing loss effects were also demonstrated for three new quantitative trait loci (QTLs) for the intervals of 50.3-54.5, 64.6-119.9, and 119.9-137.0 Mb, respectively, on chromosome 5, with significant LOD scores of 2.80-3.91 for specific high-frequency hearing loss at 16 kHz by quantitative trait loci linkage mapping using a (DBA/2J × C57BL/6J) F1 × DBA/2J backcross mice. Moreover, we showed that the contribution of Fscn2(ahl8) to early-onset hearing loss with 32-kHz stimuli is extremely low and raised the possibility of effects from the Cdh23(ahl) allele and another dominant quantitative trait locus (loci) for hearing loss at this ultrasonic frequency. Therefore, our results suggested that frequency-specific QTLs control early-onset hearing loss in DBA/2J mice.

Transgenic mouse model for the study of enterovirus 71 neuropathogenesis.

Enterovirus 71 (EV71) typically causes mild hand-foot-and-mouth disease in children, but it can also cause severe neurological disease. Recently, epidemic outbreaks of EV71 with significant mortality have been reported in the Asia-Pacific region, and EV71 infection has become a serious public health concern worldwide. However, there is little information available concerning EV71 neuropathogenesis, and no vaccines or anti-EV71 drugs have been developed. Previous studies of this disease have used monkeys and neonatal mice that are susceptible to some EV71 strains as models. The monkey model is problematic for ethical and economical reasons, and mice that are more than a few weeks old lose their susceptibility to EV71. Thus, the development of an appropriate small animal model would greatly contribute to the study of this disease. Mice lack EV71 susceptibility due to the absence of a receptor for this virus. Previously, we identified the human scavenger receptor class B, member 2 (hSCARB2) as a cellular receptor for EV71. In the current study, we generated a transgenic (Tg) mouse expressing hSCARB2 with an expression profile similar to that in humans. Tg mice infected with EV71 exhibited ataxia, paralysis, and death. The most severely affected cells were neurons in the spinal cord, brainstem, cerebellum, hypothalamus, thalamus, and cerebrum. The pathological features in these Tg mice were generally similar to those of EV71 encephalomyelitis in humans and experimentally infected monkeys. These results suggest that this Tg mouse could represent a useful animal model for the study of EV71 infection.

Generation of mouse models for type 1 diabetes by selective depletion of pancreatic beta cells using toxin receptor-mediated cell knockout.

By using the toxin receptor-mediated cell knockout (TRECK) method, we have generated two transgenic (Tg) murine lines that model type 1 (insulin-dependent) diabetes. The first strain, C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg, carries the diphtheria toxin receptor (hDTR) driven by the human insulin gene promoter, while the other strain, C57BL/6-ins2(BAC)-TRECK-Tg, expresses hDTR cDNA under the control of the mouse insulin II gene promoter. With regard to the C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg strain, only one of three Tg strains exhibited proper expression of hDTR in pancreatic ß cells. By contrast, hDTR was expressed in the pancreatic ß cells of all four of the generated C57BL/6-ins2(BAC)-TRECK-Tg strains. Hyperglycemia, severe ablation of pancreatic ß cells and depletion of serum insulin were observed within 3days after the administration of diphtheria toxin (DT) in these Tg mice. Subcutaneous injection of a suitable dosage of insulin was sufficient for recovery from hyperglycemia in all of the examined strains. Using the C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg model, we tried to perform regenerative therapeutic approaches: allogeneic transplantation of pancreatic islet cells from C57BL/6 and xenogeneic transplantation of CD34(+) human umbilical cord blood cells. Both approaches successfully rescued C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg mice from hyperglycemia caused by DT administration. The high specificity with which DT causes depletion in pancreatic ß cells of these Tg mice is highly useful for diabetogenic research.

Novel basophil- or eosinophil-depleted mouse models for functional analyses of allergic inflammation.

Basophils and eosinophils play important roles in various host defense mechanisms but also act as harmful effectors in allergic disorders. We generated novel basophil- and eosinophil-depletion mouse models by introducing the human diphtheria toxin (DT) receptor gene under the control of the mouse CD203c and the eosinophil peroxidase promoter, respectively, to study the critical roles of these cells in the immunological response. These mice exhibited selective depletion of the target cells upon DT administration. In the basophil-depletion model, DT administration attenuated a drop in body temperature in IgG-mediated systemic anaphylaxis in a dose-dependent manner and almost completely abolished the development of ear swelling in IgE-mediated chronic allergic inflammation (IgE-CAI), a typical skin swelling reaction with massive eosinophil infiltration. In contrast, in the eosinophil-depletion model, DT administration ameliorated the ear swelling in IgE-CAI whether DT was administered before, simultaneously, or after, antigen challenge, with significantly lower numbers of eosinophils infiltrating into the swelling site. These results confirm that basophils and eosinophils act as the initiator and the effector, respectively, in IgE-CAI. In addition, antibody array analysis suggested that eotaxin-2 is a principal chemokine that attracts proinflammatory cells, leading to chronic allergic inflammation. Thus, the two mouse models established in this study are potentially useful and powerful tools for studying the in vivo roles of basophils and eosinophils. The combination of basophil- and eosinophil-depletion mouse models provides a new approach to understanding the complicated mechanism of allergic inflammation in conditions such as atopic dermatitis and asthma.

Development of lymphoproliferative diseases by hypoxia inducible factor-1alpha is associated with prolonged lymphocyte survival.

Hypoxia-inducible factor-1alpha (HIF-1 alpha) plays an essential role in the regulation of various genes associated with low oxygen consumption. Elevated expression of HIF-1alpha has been reported to be associated with tumor progression, invasion and metastasis in many cancers. To investigate the role of HIF-1alpha in tumor development and metastasis, we established transgenic mice constitutively expressing HIF1A gene under regulation of the cytomegalovirus gene promoter. Although HIF-1alpha protein levels varied among organs, expression of HIF1A mRNA in most organs gradually increased in an age-dependent manner. The transgenic mice showed no gross morphological abnormality up to 8 weeks after birth, although they subsequently developed tumors in the lymphoid, lung, and breast; the most prominent tumor was lymphoma appearing in the intestinal mucosa and intra-mesenchymal tissues. The prevalence of tumors reached 80% in 13 months after birth. The constitution of lymphocyte populations in the transgenic mice did not differ from that in wild-type mice. However, lymphocytes of the transgenic mice revealed prolonged survival under long-term culture conditions and revealed increased resistance to cytotoxic etoposide. These results suggest that HIF-1alpha itself is not oncogenic but it may play an important role in lymphomagenesis mediated through the prolonged survival of lymphocytes in this transgenic mouse model.

RP58 regulates the multipolar-bipolar transition of newborn neurons in the developing cerebral cortex.

Accumulating evidence suggests that many brain diseases are associated with defects in neuronal migration, suggesting that this step of neurogenesis is critical for brain organization. However, the molecular mechanisms underlying neuronal migration remain largely unknown. Here, we identified the zinc-finger transcriptional repressor RP58 as a key regulator of neuronal migration via multipolar-to-bipolar transition. RP58(-/-) neurons exhibited severe defects in the formation of leading processes and never shifted to the locomotion mode. Cre-mediated deletion of RP58 using in utero electroporation in RP58(flox/flox) mice revealed that RP58 functions in cell-autonomous multipolar-to-bipolar transition, independent of cell-cycle exit. Finally, we found that RP58 represses Ngn2 transcription to regulate the Ngn2-Rnd2 pathway; Ngn2 knockdown rescued migration defects of the RP58(-/-) neurons. Our findings highlight the critical role of RP58 in multipolar-to-bipolar transition via suppression of the Ngn2-Rnd2 pathway in the developing cerebral cortex.

Persistent cortical plasticity by upregulation of chondroitin 6-sulfation.

Cortical plasticity is most evident during a critical period in early life, but the mechanisms that restrict plasticity after the critical period are poorly understood. We found that a developmental increase in the 4-sulfation/6-sulfation (4S/6S) ratio of chondroitin sulfate proteoglycans (CSPGs), which are components of the brain extracellular matrix, leads to the termination of the critical period for ocular dominance plasticity in the mouse visual cortex. Condensation of CSPGs into perineuronal nets that enwrapped synaptic contacts on parvalbumin-expressing interneurons was prevented by cell-autonomous overexpression of chondroitin 6-sulfation, which maintains a low 4S/6S ratio. Furthermore, the increase in the 4S/6S ratio was required for the accumulation of Otx2, a homeoprotein that activates the development of parvalbumin-expressing interneurons, and for functional maturation of the electrophysiological properties of these cells. Our results indicate that the critical period for cortical plasticity is regulated by the 4S/6S ratio of CSPGs, which determines the maturation of parvalbumin-expressing interneurons.

Selective depletion of mouse kidney proximal straight tubule cells causes acute kidney injury.

The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the "toxin receptor mediated cell knockout" method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 ß-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3-7 days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery.

The Ufm1-activating enzyme Uba5 is indispensable for erythroid differentiation in mice.

Post-translational protein modifications are systems designed to expand restricted genomic information through functional conversion of target molecules. Ubiquitin-like post-translational modifiers regulate numerous cellular events through their covalent linkages to target protein(s) by an enzymatic cascade analogous to ubiquitylation consisting of E1 (activating), E2 (conjugating) and E3 (ligating) enzymes. In this study, we report the essential role of Uba5, a specific activating enzyme for the ubiquitin-like modifier, Ufm1, in erythroid development. Mice lacking Uba5 exhibited severe anaemia, followed by death in utero. Although Uba5 was dispensable for the production of erythropoietin, its genetic loss led to impaired development of megakaryocyte and erythroid progenitors from common myeloid progenitors. Intriguingly, transgenic expression of Uba5 in the erythroid lineage rescued the Uba5-deficient embryos from anaemia and prolonged their survival, demonstrating the importance of Uba5 in cell-autonomous erythroid differentiation. Our results suggest that one of the ubiquitin-like protein modification systems, the Ufm1 system, is involved in the regulation of haematopoiesis.