Human-induced pluripotent stem cell-derived neural stem/progenitor cell ex vivo gene therapy with synaptic organizer CPTX for spinal cord injury.

The transplantation of neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise in spinal cord injury (SCI) model animals. Establishing a functional synaptic connection between the transplanted and host neurons is crucial for motor function recovery. To boost therapeutic outcomes, we developed an ex vivo gene therapy aimed at promoting synapse formation by expressing the synthetic excitatory synapse organizer CPTX in hiPSC-NS/PCs. Using an immunocompromised transgenic rat model of SCI, we evaluated the effects of transplanting CPTX-expressing hiPSC-NS/PCs using histological and functional analyses. Our findings revealed a significant increase in excitatory synapse formation at the transplantation site. Retrograde monosynaptic tracing indicated extensive integration of transplanted neurons into the surrounding neuronal tracts facilitated by CPTX. Consequently, locomotion and spinal cord conduction significantly improved. Thus, ex vivo gene therapy targeting synapse formation holds promise for future clinical applications and offers potential benefits to individuals with SCI.

Endogenous chondroitin extends the lifespan and healthspan in C. elegans.

Chondroitin, a class of glycosaminoglycan polysaccharides, is found as proteoglycans in the extracellular matrix, plays a crucial role in tissue morphogenesis during development and axonal regeneration. Ingestion of chondroitin prolongs the lifespan of C. elegans. However, the roles of endogenous chondroitin in regulating lifespan and healthspan mostly remain to be investigated. Here, we demonstrate that a gain-of-function mutation in MIG-22, the chondroitin polymerizing factor (ChPF), results in elevated chondroitin levels and a significant extension of both the lifespan and healthspan in C. elegans. Importantly, the remarkable longevity observed in mig-22(gf) mutants is dependent on SQV-5/chondroitin synthase (ChSy), highlighting the pivotal role of chondroitin in controlling both lifespan and healthspan. Additionally, the mig-22(gf) mutation effectively suppresses the reduced healthspan associated with the loss of MIG-17/ADAMTS metalloprotease, a crucial for factor in basement membrane (BM) remodeling. Our findings suggest that chondroitin functions in the control of healthspan downstream of MIG-17, while regulating lifespan through a pathway independent of MIG-17.

Inhibitory effects of estetrol on the invasion and migration of immortalized human endometrial stromal cells.

Endometriosis, a common gynecological disorder characterized by the growth of endometrial gland and stroma outside the uterus, causes several symptoms such as dysmenorrhea, hypermenorrhea, and chronic abdominal pain. 17ß estradiol (E2) stimulates the growth of endometriotic lesions. Although estetrol (E4), produced by human fetal liver, is also a natural estrogen, it may have the opposite effects on endometriotic cells. We investigated different effects of E4 and E2 on the invasion and migration of immortalized human endometrial stromal cells (HESCs) and evaluated whether E4 affects the expression of Wiskott-Aldrich syndrome protein (WASP) family member 1 (WASF-1). We measured the invasion of HESCs by a Matrigel chamber assay. Cell migration was measured by wound healing assay and cell tracking analysis. The expression of WASF-1 was confirmed by independent real-time PCR analysis. Transfection of cells with siRNAs was carried out to knock down the expression of WASF-1 in HESCs. E4 significantly inhibited E2-induced invasion and migration of HESCs. WASF-1 was found to be a potential mediator based on metastasis PCR array. WASF-1 was upregulated by E2 and downregulated by E4. Knockdown of WASF-1 inhibited migration. Our results suggest that E4 may inhibit E2-induced growth of endometriotic lesions. Downregulation of WASF-1 is involved in the inhibitory effects of E4 on migration. The use of E4 combined with progestins as combined oral contraceptives may cause endometriotic lesions to regress in women with endometriosis.

Protocol for highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence in rodents.

We present a protocol to induce Cre-dependent transgene expression in specific cell types in the rat brain, suppressing a leak expression in off-target cells, by using a flip-excision switch system with a unilateral spacer sequence. We describe steps for construction of transfer plasmids, preparation of adeno-associated viral vectors, intracranial injection, and detection of transgene expression. Our protocol provides a useful strategy for a better understanding of the structure and function of specific cell types in the complex neural circuit. For complete details on the use and execution of this protocol, please refer to Matsushita et al.1.

Inhibition of MLCK­mediated migration and invasion in human endometriosis stromal cells by NF­κB inhibitor DHMEQ.

Endometriosis is initiated by the movement of endometrial cells in the uterus to the fallopian tubes, the ovaries and the peritoneal cavity after the shedding of the uterus lining. To cause endometriosis, it is often necessary for these endometrial cells to migrate, invade and grow at the secondary site. In the present study, immortalized human endometriosis stromal cells (HESC) were employed to look for the inhibitors of migration and invasion. Using a chemical library of bioactive metabolites, it was found that an NF­κB inhibitor, DHMEQ, inhibited the migration and invasion of HESC. Both whole­genome array and metastasis PCR array analyses suggested the involvement of myosin light chain kinase (MLCK) in the mechanism of inhibition. DHMEQ was confirmed to inhibit the expression of MLCK and small inhibitory RNA knockdown of MLCK reduced cellular migration and invasion. The addition of DHMEQ to the knockdown cells did not further inhibit migration and invasion. DHMEQ is particularly effective in suppressing disease models by intraperitoneal (IP) administration and this therapy is being developed for the treatment of inflammation and cancer. DHMEQ IP therapy may also be useful for the treatment of endometriosis.

Inhibition of Transglutaminase 2 Reduces Peritoneal Injury in a Chlorhexidine-Induced Peritoneal Fibrosis Model.

Long-term peritoneal dialysis (PD) is often associated with peritoneal dysfunction leading to withdrawal from PD. The characteristic pathologic features of peritoneal dysfunction are widely attributed to peritoneal fibrosis and angiogenesis. The detailed mechanisms remain unclear, and treatment targets in clinical settings have yet to be identified. We investigated transglutaminase 2 (TG2) as a possible novel therapeutic target for peritoneal injury. TG2 and fibrosis, inflammation, and angiogenesis were investigated in a chlorhexidine gluconate (CG)-induced model of peritoneal inflammation and fibrosis, representing a noninfectious model of PD-related peritonitis. Transforming growth factor (TGF)-ß type I receptor (TGFßR-I) inhibitor and TG2-knockout mice were used for TGF-ß and TG2 inhibition studies, respectively. Double immunostaining was performed to identify cells expressing TG2 and endothelial-mesenchymal transition (EndMT). In the rat CG model of peritoneal fibrosis, in situ TG2 activity and protein expression increased during the development of peritoneal fibrosis, as well as increases in peritoneal thickness and numbers of blood vessels and macrophages. TGFßR-I inhibitor suppressed TG2 activity and protein expression, as well as peritoneal fibrosis and angiogenesis. TGF-ß1 expression, peritoneal fibrosis, and angiogenesis were suppressed in TG2-knockout mice. TG2 activity was detected by α-smooth muscle actin-positive myofibroblasts, CD31-positive endothelial cells, and ED-1-positive macrophages. CD31-positive endothelial cells in the CG model were α-smooth muscle actin-positive, vimentin-positive, and vascular endothelial-cadherin-negative, suggesting EndMT. In the CG model, EndMT was suppressed in TG2-knockout mice. TG2 was involved in the interactive regulation of TGF-ß. As inhibition of TG2 reduced peritoneal fibrosis, angiogenesis, and inflammation associated with TGF-ß and vascular endothelial growth factor-A suppression, TG2 may provide a new therapeutic target for ameliorating peritoneal injuries in PD.

Highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence.

The flip-excision switch (FLEX) system with an adeno-associated viral (AAV) vector allows expression of transgenes in specific cell populations having Cre recombinase. A significant issue with this system is non-specific expression of transgenes in tissues after vector injection. We show here that Cre-independent recombination events in the AAV genome carrying the FLEX sequence occur mainly during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress the recombination in the viral genome, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our FLEX/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

Increased urinary albumin leakage is related to injuries of glomerular glycocalyx and podocytes, and associated with tubular dysfunction in preeclampsia.

OBJECTIVE: The pathogenesis of preeclampsia (PE) is known to be endothelial cell damage; however, the existence of dysfunction in glomerular endothelial glycocalyx, podocytes and tubules remains unclear. The glomerular endothelial glycocalyx, basement membrane, podocytes, and tubules are permeability barriers against albumin excretion. This study aimed to assess the relationship between urinary albumin leakage and injuries of the glomerular endothelial glycocalyx, podocytes, and tubules in patients with PE. METHODS: A total of 81 women with uncomplicated pregnancies (control, n = 22), PE (PE, n = 36), or gestational hypertension (GH) (GH, n = 23) were enrolled. We assessed urinary albumin and serum hyaluronan for glycocalyx injuries, podocalyxin for podocytes injuries, and urinary N-acetyl-ß-d-glucosaminidase (NAG) and liver-type fatty acid-binding protein (l-FABP) for renal tubular dysfunctions. RESULTS: The serum hyaluronan and the urinary podocalyxin levels were higher in the PE and GH groups. The urinary NAG and l-FABP levels were higher in the PE group. Urinary NAG and l-FABP levels positively correlated with urinary albumin excretion. CONCLUSIONS: Our findings suggest that increased urinary albumin leakage is related to injuries of the glycocalyx and podocytes, and associated with tubular dysfunction in pregnant women with PE. The clinical trial described in this paper was registered at the UMIN Clinical Trials Registry under registration number UMIN000047875. URL of registration: https://centre6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000054437.

Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology.

BACKGROUND: Cell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. NEW METHOD: We aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. RESULTS: We detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH protein levels and catecholamine accumulation in the brain regions, as well as motor, reward-related, and anxiety-like behaviors, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. CONCLUSIONS: TH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.

Role of chondroitin sulfate in the developmental and healing process of the dental pulp in mice.

Chondroitin sulfate proteoglycan (CSPG), one of the major extracellular matrices, plays an important part in organogenesis. Its core protein and chondroitin sulfate (CS) chain have a specific biological function. To elucidate the role of CS in the developmental and healing process of the dental pulp, we performed an experimental tooth replantation in CS N-acethylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. We also performed cell proliferation assay and qRT-PCR analysis for the WT and T1KO primary dental pulp cells using T1-siRNA technique and external CS. During tooth development, CS was diffusely expressed in the dental papilla, and with dental pulp maturation, CS disappeared from the differentiated areas, including the odontoblasts. In fully developed molars, CS was restricted to the root apex region colocalizing with Gli1-positive cells. In the healing process after tooth replantation, CD31-positive cells accumulated in the CS-positive stroma in WT molars. In T1KO molars, the appearance of Ki67- and Gli1-positive cells in the dental pulp was significantly fewer than in WT molars in the early healing stage, and collagen I-positive reparative dentin formation was not obvious in T1KO mice. In primary culture experiments, siRNA knockdown of T1 gene significantly suppressed cell proliferation in WT dental pulp cells, and the mRNA expression of cyclin D1 and CD31 was significantly upregulated by external CS in T1KO dental pulp cells. These results suggest that CS is involved in the cell proliferation and functional differentiation of dental pulp constituent cells, including vascular cells, in the healing process of dental pulp tissue after tooth injury.

Bone marrow derived mast cells injected into the osteoarthritic knee joints of mice induced by sodium monoiodoacetate enhanced spontaneous pain through activation of PAR2 and action of extracellular ATP.

Conditions that resemble osteoarthritis (OA) were produced by injection of sodium monoiodoacetate (MIA) into the knee joints of mice. Bone marrow derived mast cells (BMMCs) injected into the OA knee joints enhanced spontaneous pain. Since no spontaneous pain was observed when BMMCs were injected into the knee joints of control mice that had not been treated with MIA, BMMCs should be activated within the OA knee joints and release some pain-inducible factors. Protease activated receptor-2 (PAR2) antagonist (FSLLRY-NH2) almost abolished the pain-enhancing effects of BMMCs injected into the OA knee joints, suggesting that tryptase, a mast cell protease that is capable of activating PAR2, should be released from the injected BMMCs and enhance pain through activation of PAR2. When PAR2 agonist (SLIGKV-NH2) instead of BMMCs was injected into the OA knee joints, it was also enhanced pain. Apyrase, an ATP degrading enzyme, injected into the OA knee joints before BMMCs suppressed the pain enhanced by BMMCs. We showed that purinoceptors (P2X4 and P2X7) were expressed in BMMCs and that extracellular ATP stimulated the release of tryptase from BMMCs. These observations suggest that ATP may stimulate degranulation of BMMCs and thereby enhanced pain. BMMCs injected into the OA knee joints stimulated expression of IL-1ß, IL-6, TNF-α, CCL2, and MMP9 genes in the infrapatellar fat pads, and PAR2 antagonist suppressed the stimulatory effects of BMMCs. Our study suggests that intermittent pain frequently observed in OA knee joints may be due, at least partly, to mast cells through activation of PAR2 and action of ATP, and that intraarticular injection of BMMCs into the OA knee joints may provide a useful experimental system for investigating molecular mechanisms by which pain is induced in OA knee joints.

Chondroitin sulfate N-acetylgalactosyltransferase-1 knockout shows milder phenotype in experimental autoimmune encephalomyelitis than in wild type.

Proteoglycans (PGs) are one of the main components in the extracellular matrix of the central nervous system. Chondroitin sulfate (CS) is a glycosaminoglycan (GAG), which is composed of major PGs. Similar to keratin sulfate (KS), another GAG, CS inhibits axon regeneration. However, the influence of these GAGs on the pathogenicity of neuroimmunological diseases is unclear. Here, we induced experimental autoimmune encephalomyelitis (EAE) in mice lacking CS N-acetylgalactosaminyltransferase-1 (CSGalNAcT1-KO), an important enzyme for CS synthesis. In our study, CSGalNAcT1-KO mice showed milder EAE symptoms than those in wild-type (WT) mice. The recall response of antigen-specific lymphocytes showed that CSGalNAcT1-KO-derived lymphocytes had a milder cell proliferation response than that in WT-derived lymphocytes. These results suggest that CS contributes toward the induction phase of EAE. We previously performed EAE experiments in GlcNAc-6-O-sulfotransferase KO (GlcNAc6ST-KO) and C6ST1-KO mice, which had reduced KS and reduced CS-C, respectively. EAE in CSGalNAcT1-KO mice was more similar to that in GlcNAc6ST-KO mice than in C6ST1-KO mice. In conclusion, the distinct GAG sugar chains are associated with severe or mild phenotypes of EAE and are therefore potential new therapeutic targets for neuroimmunological diseases, including multiple sclerosis.

A synthetic synaptic organizer protein restores glutamatergic neuronal circuits.

Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory-inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer's disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.

Isozyme-Specific Role of SAD-A in Neuronal Migration During Development of Cerebral Cortex.

SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.

Craniofacial abnormality with skeletal dysplasia in mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase-1.

Chondroitin sulfate (CS) proteoglycan is a major component of the extracellular matrix and plays an important part in organogenesis. To elucidate the roles of CS for craniofacial development, we analyzed the craniofacial morphology in CS N-acetylgalactosaminyltransferase-1 (T1) gene knockout (KO) mice. T1KO mice showed the impaired intramembranous ossification in the skull, and the final skull shape of adult mice included a shorter face, higher and broader calvaria. Some of T1KO mice exhibited severe facial developmental defect, such as eye defects and cleft lip and palate, causing embryonic lethality. At the postnatal stages, T1KO mice with severely reduced CS amounts showed malocclusion, general skeletal dysplasia and skin hyperextension, closely resembling Ehlers-Danlos syndrome-like connective tissue disorders. The production of collagen type 1 was significantly downregulated in T1KO mice, and the deposition of CS-binding molecules, Wnt3a, was decreased with CS in extracellular matrices. The collagen fibers were irregular and aggregated, and connective tissues were dysorganized in the skin and calvaria of T1KO mice. These results suggest that CS regulates the shape of the craniofacial skeleton by modulating connective tissue organization and that the remarkable reduction of CS induces hypoplasia of intramembranous ossification and cartilage anomaly, resulting in skeletal dysplasia.

Growth Cone Phosphoproteomics Reveals that GAP-43 Phosphorylated by JNK Is a Marker of Axon Growth and Regeneration.

Neuronal growth cones are essential for nerve growth and regeneration, as well as for the formation and rearrangement of the neural network. To elucidate phosphorylation-dependent signaling pathways and establish useful molecular markers for axon growth and regeneration, we performed a phosphoproteomics study of mammalian growth cones, which identified >30,000 phosphopeptides of ∼1,200 proteins. The phosphorylation sites were highly proline directed and primarily MAPK dependent, owing to the activation of JNK, suggesting that proteins that undergo proline-directed phosphorylation mediate nerve growth in the mammalian brain. Bioinformatics analysis revealed that phosphoproteins were enriched in microtubules and the cortical cytoskeleton. The most frequently phosphorylated site was S96 of GAP-43 (growth-associated protein 43-kDa), a vertebrate-specific protein involved in axon growth. This previously uncharacterized phosphorylation site was JNK dependent. S96 phosphorylation was specifically detected in growing and regenerating axons as the most frequent target of JNK signaling; thus it represents a promising new molecular marker for mammalian axonal growth and regeneration.

Increased Synthesis of Chondroitin Sulfate Proteoglycan Promotes Adult Hippocampal Neurogenesis in Response to Enriched Environment.

Chondroitin sulfate proteoglycan (CSPG) is a candidate regulator of embryonic neurogenesis. The aim of this study was to specify the functional significance of CSPG in adult hippocampal neurogenesis using male mice. Here, we showed that neural stem cells and neuronal progenitors in the dentate gyrus were covered in part by CSPG. Pharmacological depletion of CSPG in the dentate gyrus reduced the densities of neuronal progenitors and newborn granule cells. 3D reconstruction of newborn granule cells showed that their maturation was inhibited by CSPG digestion. The novel object recognition test revealed that CSPG digestion caused cognitive memory impairment. Western blot analysis showed that expression of ß-catenin in the dentate gyrus was decreased by CSPG digestion. The amount of CSPG in the dentate gyrus was increased by enriched environment (EE) and was decreased by forced swim stress. In addition, EE accelerated the recovery of CSPG expression in the dentate gyrus from the pharmacological depletion and promoted the restoration of granule cell production. Conversely, the densities of newborn granule cells were also decreased in mice that lacked chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT1), a key enzyme for CSPG synthesis (T1KO mice). The capacity of EE to promote granule cell production and improve cognitive memory was impaired in T1KO mice. These findings indicate that CSPG is involved in the regulation of adult hippocampal neurogenesis and suggest that increased synthesis of CSPG by CSGalNacT1 may mediate promotion of granule cell production and improvement of cognitive memory in response to EE.SIGNIFICANCE STATEMENT Chondroitin sulfate proteoglycan (CSPG) is a candidate regulator of embryonic neurogenesis. Here, we specified the role of CSPG in adult neurogenesis in the mouse hippocampus. Digestion of CSPG in the dentate gyrus impaired granule cell production and cognitive memory. Enriched environment (EE) promoted the recovery of CSPG expression and granule cell production from the CSPG digestion. Additionally, adult neurogenesis was impaired in mice that lacked a key enzyme for CSPG synthesis (T1KO mice). The capacity of EE to promote granule cell production and cognitive memory was impaired in T1KO mice. Altogether, these findings indicate that CSPG underlies adult hippocampal neurogenesis and suggest that increased synthesis of CSPG may mediate promotion of granule cell production in response to EE.

Roles of CSGalNAcT1, a key enzyme in regulation of CS synthesis, in neuronal regeneration and plasticity.

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan composed of a long chain of repeating disaccharide units that are attached to core proteins, resulting in CS proteoglycans (CSPGs). In the mature brain, CS is concentrated in perineuronal nets (PNNs), which are extracellular structures that surround synapses and regulate synaptic plasticity. In addition, CS is rapidly synthesized after CNS injury to create a physical and chemical barrier that inhibits axon growth. Most previous studies used a bacterial CS-degrading enzyme to investigate the physiological roles of CS. Recent studies have shown that CS is synthesized by more than 15 enzymes, all of which have been characterized in vitro. Here we focus on one of those enzymes, CSGalNAcT1 (T1). We produced T1 knockout mice (KO), which show extensive axon regeneration following spinal cord injury, as well as the loss of onset of ocular dominance plasticity. These results from T1KO mice suggest important roles for extracellular CS in the brain regarding neuronal plasticity and axon regeneration.

Abnormalities in perineuronal nets and behavior in mice lacking CSGalNAcT1, a key enzyme in chondroitin sulfate synthesis.

Chondroitin sulfate (CS) is an important glycosaminoglycan and is mainly found in the extracellular matrix as CS proteoglycans. In the brain, CS proteoglycans are highly concentrated in perineuronal nets (PNNs), which surround synapses and modulate their functions. To investigate the importance of CS, we produced and precisely examined mice that were deficient in the CS synthesizing enzyme, CSGalNAcT1 (T1KO). Biochemical analysis of T1KO revealed that loss of this enzyme reduced the amount of CS by approximately 50% in various brain regions. The amount of CS in PNNs was also diminished in T1KO compared to wild-type mice, although the amount of a major CS proteoglycan core protein, aggrecan, was not changed. In T1KO, we observed abnormalities in several behavioral tests, including the open-field test, acoustic startle response, and social preference. These results suggest that T1 is important for plasticity, probably due to regulation of CS-dependent PNNs, and that T1KO is a good model for investigation of PNNs.

Chondroitin Sulfate Is Required for Onset and Offset of Critical Period Plasticity in Visual Cortex.

Ocular dominance plasticity is easily observed during the critical period in early postnatal life. Chondroitin sulfate (CS) is the most abundant component in extracellular structures called perineuronal nets (PNNs), which surround parvalbumin-expressing interneurons (PV-cells). CS accumulates in PNNs at the critical period, but its function in earlier life is unclear. Here, we show that initiation of ocular dominance plasticity was impaired with reduced CS, using mice lacking a key CS-synthesizing enzyme, CSGalNAcT1. Two-photon in vivo imaging showed a weaker visual response of PV-cells with reduced CS compared to wild-type mice. Plasticity onset was restored by a homeoprotein Otx2, which binds the major CS-proteoglycan aggrecan and promotes its further expression. Continuous CS accumulation together with Otx2 contributed bidirectionally to both onset and offset of plasticity, and was substituted by diazepam, which enhances GABA function. Therefore, CS and Otx2 may act as common inducers of both onset and offset of the critical period by promoting PV-cell function throughout the lifetime.