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Chronic inflammation plays crucial roles in obesity-induced metabolic diseases. Protein tyrosine phosphatase receptor type O (PTPRO) is a member of the R3 subfamily of receptor-like protein tyrosine phosphatases. We previously suggested a role for PTPRO in the inactivation of the insulin receptor. The present study aimed to elucidate the involvement of PTPRO in the control of glucose and lipid metabolism as well as in obesity-induced systemic inflammation.Lipid accumulation in adipose tissue and the liver, the expression of inflammatory cytokines, and insulin resistance associated with systemic inflammation were investigated in hyper-obese Ptpro-KO mice by feeding a high-fat/high-sucrose diet (HFHSD). The effects of the administration of AKB9778, a specific inhibitor of PTPRO, to ob/ob mice and cultured 3T3-L1 preadipocyte cells were also examined.Ptpro was highly expressed in visceral white adipose tissue and macrophages. Ptpro-KO mice fed HFHSD were hyper-obese, but did not have ectopic fat accumulation in the liver, dysfunctional lipid and glucose homeostasis, systemic inflammation, or insulin resistance. The administration of AKB9778 reproduced "the healthy obese phenotypes" of Ptpro-KO mice in highly obese ob/ob mice. Furthermore, the inhibition of PTPRO promoted the growth of lipid droplets in adipocytes through an increase in the phosphorylation of Tyr(117) in vimentin.Healthy systemic conditions with the attenuation of inflammation in hyper-obese Ptpro-KO mice were associated with the expansion of adipose tissue and low activation of NF-κb. Therefore, PTPRO may be a promising target to ameliorate hepatic steatosis and metabolic dysfunction.

Background Children receiving home medical care need special attention to prevent unexpected death. The aim of this study was to clarify the factors contributing to death in children receiving home medical care from the child death review database. Methods Children receiving home medical care were enrolled from the child death review database from 2014 to 2016 in Aichi prefecture, Japan, with a population of one million children. Types of medical care and factors contributing to death were examined. Results Of the 631 children who died, 40 children (6%) were receiving home medical care (21: tracheostomy; 19: ventilator; 26: suctioning of naso-oral secretions; 19: oxygen inhalation; 32: tube feeding; 6: urethral catheterization; and 1: peritoneal dialysis). The death rate was 50 times that in the general population of children. Ten children had contributory factors that seemed to be preventable. In four children, the families could not replace the tracheostomy tubes during an accident. In three, oxygen saturation or ventilator alarms were not set appropriately. In two, an oxygen cylinder became empty. One child fell down from a seat in a car. Conclusions Improvement of devices and correct guidance to caregivers may reduce the death rate in children receiving home medical care. Impact Children receiving home medical care, such as tracheostomy care, mechanical ventilation, or tube feeding, need special attention to prevent unexpected death. In this population-based child death review, 6% of children received home medical care, and it was estimated that 1 of 100 children receiving home medical care died per year. One-quarter of the deaths could be preventable by caregiver education or development of devices.

Chronic inflammation plays crucial roles in obesity-induced metabolic diseases. We herein demonstrated that mice lacking the protein tyrosine phosphatase receptor type O (PTPRO) exhibited the hyper-obese phenotype when fed a high-fat/high-sucrose diet. However, Ptpro-KO mice with hyperobesity showed the markedly small accumulation of ectopic fat in the liver, improvements in lipid and glucose homeostasis, and low-grade systemic inflammation associated with low macrophage activation. Expression of protein tyrosine phosphatase 1b (Ptp1b), an enzyme which is known to be implicated in metabolic disorders, was also suppressed in Ptpro-KO mice. The administration of AKB9778, a specific inhibitor of PTPRO, to highly obese ob/ob mice reproduced the phenotypes of Ptpro-KO mice along with the amelioration of inflammation. We revealed that an increase in the phosphorylation of Tyr(117) in vimentin, a component of intermediate filaments, by the inhibition of PTPRO promoted the growth of lipid droplets in adipocytes. The improvement in metabolic conditions with the attenuation of inflammation in Ptpro-KO mice was explained by the low activation of NFκb, a key transcription factor for inflammatory response, in adipose tissue. This is the first study to show that PTPRO is a promising target to ameliorate hepatic steatosis and metabolic disorders.

Extracellular fluids, including blood, lymphatic fluid, and cerebrospinal fluid, are collectively called body fluids. The Na

Olfactory ensheathing cells (OECs) are neural crest-derived glia that ensheath bundles of olfactory axons from their peripheral origins in the olfactory epithelium to their central targets in the olfactory bulb. We took an unbiased laser microdissection and differential RNA-seq approach, validated by in situ hybridization, to identify candidate molecular mechanisms underlying mouse OEC development and differences with the neural crest-derived Schwann cells developing on other peripheral nerves. We identified 25 novel markers for developing OECs in the olfactory mucosa and/or the olfactory nerve layer surrounding the olfactory bulb, of which 15 were OEC-specific (that is, not expressed by Schwann cells). One pan-OEC-specific gene, Ptprz1, encodes a receptor-like tyrosine phosphatase that blocks oligodendrocyte differentiation. Mutant analysis suggests Ptprz1 may also act as a brake on OEC differentiation, and that its loss disrupts olfactory axon targeting. Overall, our results provide new insights into OEC development and the diversification of neural crest-derived glia.

Nax is a brain [Na+] sensor expressed in the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT) in the brain. We previously demonstrated that Nax signals are involved in the control of water intake behavior through the Nax/TRPV4 pathway. Nax gene knockout mice showed significantly attenuated water intake after an intracerebroventricular (ICV) injection of a hypertonic NaCl solution; however, the induction of a certain amount of water intake still remained, suggesting that another unknown [Na+]-dependent pathway besides the Nax/TRPV4 pathway contributes to water intake. In the present study, we screened for novel [Na+] sensors involved in water intake control and identified SLC9A4 (also called sodium (Na+)/hydrogen (H+) exchanger 4 (NHE4)). SLC9A4 is expressed in angiotensin II (Ang II) receptor type 1a (AT1a)-positive neurons in the OVLT. Sodium-imaging experiments using cultured cells transfected with slc9a4 revealed that SLC9A4 was activated by increases in extracellular [Na+] ([Na+]o), but not osmolality. Moreover, the firing activity of SLC9A4-positive neurons was enhanced by increases in [Na+]o and Ang II. slc9a4 knockdown in the OVLT reduced water intake induced by increases in [Na+], but not osmolality, in the cerebrospinal fluid. ICV injection experiments of a specific inhibitor suggested that the increase in extracellular [H+] caused by SLC9A4 activation next stimulates acid-sensing channel 1a (AS1C1a) to induce water intake. Our results thus indicate that SLC9A4 in the OVLT functions as a [Na+] sensor for the control of water intake and that the SLC9A4 signal is independent of the Nax/TRPV4 pathway.

Nax is a [Na+] sensor expressed in specific glial cells in the sensory circumventricular organs (sCVOs) in the brain. We recently demonstrated that Nax signals are involved in the control of not only salt intake but also water intake behavior. Our pharmacological experiments suggested that Nax signals led to activation of neurons bearing TRPV4 by using epoxyeicosatrienoic acids (EETs) as gliotransmitters to stimulate water intake. In the present study, we performed selective lesions of individual sCVOs in wild-type (WT) mice and the site-directed rescue of Nax expression in Nax-gene knockout (Nax-KO) mice. These experiments revealed that the Nax channel in the organum vasculosum laminae terminalis (OVLT) functions as a [Na+] sensor for the control of water intake behavior. Direct measurements of 5,6-EET and 8,9-EET in the OVLT demonstrated that EET levels were indeed increased two-fold by water deprivation for two days in WT, but not Nax-KO mice, indicating that EETs were Nax-dependently produced in the OVLT in response to increases in [Na+] in body fluids. More importantly, intracerebroventricular injection of 5,6-EET at the same level was effective to induce water intake. Double staining revealed that Nax-positive cells also expressed Cyp2c44, a cytochrome P450 epoxygenase, to generate EETs. Collectively, these results indicate that Nax-positive glial cells produce EETs to activate TRPV4-positive neurons which may stimulate water intake, in response to increases in [Na+] of body fluids.

Protein-tyrosine phosphatase (PTPase) receptor type Z (PTPRZ) has two receptor isoforms, PTPRZ-A and -B, containing tandem intracellular PTP-D1 and -D2 domains, with only D1 being active. Pleiotrophin (PTN) binding to the extracellular PTPRZ region leads to inactivation of its PTPase activity, thereby facilitating oligodendrocyte precursor cell (OPC) differentiation and myelination in the central nervous system. However, the mechanisms responsible for PTN-induced PTPRZ inactivation remain unclear. We herein report that the crystal structure of the intracellular region of PTPRZ (PTPRZ-ICR) shows a “head-to-toe”-type dimer conformation, with D2 masking the catalytic site of D1. MS analyses revealed that PTPRZ-ICR proteins remain in monomer-dimer equilibrium in aqueous solution and that a substrate-derived inhibitory peptide or competitive inhibitor (SCB4380) specifically bind to the monomer form in a 1:1 ratio. A D2 deletion (ΔD2) or dimer interface mutation (DDKK) disrupted dimer formation, but SCB4380 binding was maintained. Similar to WT PTPRZ-B, monomer-biased PTPRZ-B-ΔD2 and PTPRZ-B-DDKK variants efficiently dephosphorylated p190RhoGAP at Tyr-1105 when co-expressed in BHK-21 cells. The catalytic activities of these variants were not suppressed by PTN treatment, but were inhibited by the cell-permeable PTPase inhibitor NAZ2329. Of note, the PTN treatment did not enhance OPC differentiation in primary cultured glial cells from ΔD2 or PTPase-inactive PTPRZ-B (CS) mutant knock-in mice. Our results thus indicate that PTN-induced PTPRZ inactivation results from dimer formation of the intracellular tandem PTP domains in a head-to-toe configuration, which is physiologically relevant to the control of OPC differentiation in vivo.

Myeloid cell mediated mechanisms regulate synovial joint inflammation. IL-34, a macrophage (Mø) growth and differentiation molecule, is markedly expressed in neutrophil and Mø-rich arthritic synovium. IL-34 engages a newly identified independent receptor, protein-tyrosine phosphatase, receptor-type, zeta (PTPRZ), that we find is expressed by Mø. As IL-34 is prominent in rheumatoid arthritis, we probed for the IL-34 and PTPRZ-dependent myeloid cell mediated mechanisms central to arthritis using genetic deficient mice in K/BxN serum-transfer arthritis. Unanticipatedly, we now report that IL-34 and PTPRZ limited arthritis as intra-synovial pathology and bone erosion were more severe in IL-34 and PTPRZ KO mice during induced arthritis. We found that IL-34 and PTPRZ: (i) were elevated, bind, and induce downstream signaling within the synovium in arthritic mice and (ii) were upregulated in the serum and track with disease activity in rheumatoid arthritis patients. Mechanistically, IL-34 and PTPRZ skewed Mø toward a reparative phenotype, and enhanced Mø clearance of apoptotic neutrophils, thereby decreasing neutrophil recruitment and intra-synovial neutrophil extracellular traps. With fewer neutrophils and neutrophil extracellular traps in the synovium, destructive inflammation was restricted, and joint pathology and bone erosion diminished. These novel findings suggest that IL-34 and PTPRZ-dependent mechanisms in the inflamed synovium limit, rather than promote, inflammatory arthritis.

Adipsic hypernatremia is a rare disease presenting as persistent hypernatremia with disturbance of thirst regulation and hypothalamic dysfunction. As a result of congenital disease, tumors, or inflammation, most cases are accompanied by structural abnormalities in the hypothalamic-pituitary area. While cases with no hypothalamic-pituitary structural lesion have been reported, their etiology has not been elucidated. Recently, we reported three patients with adipsic hypernatremia whose serum-derived immunoglobulin (Ig) specifically reacted with mouse subfornical organ (SFO) tissue. As one of the circumventricular organs (CVOs) that form a sensory interface between the blood and brain, the SFO is a critical site for generating physiological responses to dehydration and hypernatremia. Intravenous injection of the patient’s Ig fraction induced hypernatremia in mice, along with inflammation and apoptosis in the SFO. These results support a new autoimmunity-related mechanism for inducing adipsic hypernatremia without demonstrable hypothalamic-pituitary structural lesions. In this review, we aim to highlight the characteristic clinical features of these patients, in addition to etiological mechanisms related to SFO function. These findings may be useful for diagnosing adipsic hypernatremia caused by an autoimmune response to the SFO, and support development of new strategies for prevention and treatment.

Body fluid conditions are continuously monitored in the brain to regulate thirst and salt-appetite sensations. Angiotensin II drives both thirst and salt appetite; however, the neural mechanisms underlying selective water- and/or salt-intake behaviors remain unknown. Using optogenetics, we show that thirst and salt appetite are driven by distinct groups of angiotensin II receptor type 1a-positive excitatory neurons in the subfornical organ. Neurons projecting to the organum vasculosum lamina terminalis control water intake, while those projecting to the ventral part of the bed nucleus of the stria terminalis control salt intake. Thirst-driving neurons are suppressed under sodium-depleted conditions through cholecystokinin-mediated activation of GABAergic neurons. In contrast, the salt appetite-driving neurons were suppressed under dehydrated conditions through activation of another population of GABAergic neurons by Nax signals. These distinct mechanisms in the subfornical organ may underlie the selective intakes of water and/or salt and may contribute to body fluid homeostasis.

Protein tyrosine phosphatase receptor type Z (PTPRZ) is preferentially expressed in the central nervous system as two transmembrane receptor isoforms PTPRZ-A/B and one secretory isoform PTPRZ-S. Ptprz-knockout mice lacking the expression of all three isoforms show behavioral, learning, and neurological abnormalities, including increased exploratory activities to novelty, deficits in spatial and contextual learning, and reduced responses to methamphetamine, relative to wild-type mice. To investigate whether PTPRZ isoforms play distinct physiological roles, we herein performed behavioral studies on two knock-in mouse lines: One expresses the catalytically inactive Cys-1930 to Ser (CS) mutants of PTPRZ-A/B, while the other generated in the present study expresses catalytically active mutants of PTPRZ-A/B lacking the negative regulatory PTP-D2 domain and C-terminal PDZ-binding motif (ΔD2) instead of wild-type PTPRZ-A/-B. In contrast to Ptprz-knockout mice, neither increased responses to novelty in the open field nor memory impairments in the inhibitory-avoidance task were observed in Ptprz-CS or Ptprz-ΔD2 mice. However, the effects of methamphetamine on locomotor activity were significantly weaker in Ptprz-KO mice and CS mutant mice than in wild-type mice, but were normal in ΔD2 mutant mice. Furthermore, microdialysis experiments revealed that methamphetamine-evoked dopamine release in the nucleus accumbens was reduced in Ptprz-KO mice and CS mutant mice. These results suggest that the extracellular region of PTPRZ, including the secretory isoform, is crucial for behavioral responses to novelty and the formation of aversive memories, whereas the PTPase activities of PTPRZ receptor isoforms are involved in regulating the dopaminergic system.

Protein tyrosine phosphatase receptor type Z (PTPRZ) has two receptor isoforms (PTPRZ-A and -B) containing tandem PTP-D1 and -D2 domains intracellularly, with only D1 being active. Pleiotrophin (PTN) binding to the extracellular region of PTPRZ leads to the inactivation of PTPase, thereby inducing oligodendrocyte precursor cell (OPC) differentiation and myelination in the CNS. However, the mechanisms responsible for the ligand-induced inactivation of PTPRZ remain unclear. We herein revealed that the crystal structure of the intracellular region of PTPRZ (PTPRZ-ICR) showed the “head-to-toe”-type dimer conformation, with D2 masking the catalytic site of D1. Mass spectrometry (MS) revealed that PTPRZ-ICR proteins remained in monomer-dimer equilibrium in aqueous solution, and a substrate-derived inhibitory peptide or competitive inhibitor (SCB4380) specifically bound to the monomer form in a 1:1 stoichiometric ratio, supporting the “head-to-toe dimerization” model for inactivation. A D2 deletion (ΔD2) or dimer interface mutation (DDKK) disrupted dimer formation, while the binding of SCB4380 was maintained. Similar to wild-type PTPRZ-B, monomer-biased PTPRZ-B-ΔD2 and PTPRZ-B-DDKK mutants efficiently dephosphorylated p190RhoGAP at Tyr-1105 when co-expressed in BHK-21 cells. The catalytic activities of these mutants were not suppressed by a treatment with PTN, but were inhibited by the cell-permeable PTPase inhibitor NAZ2329. The PTN treatment did not enhance OPC differentiation in primary cultured glial cells prepared from ΔD2 or catalytically-inactive CS mutant knock-in mice. Our results indicate that PTN-induced PTPRZ inactivation is attained by dimer formation of the intracellular tandem PTP domains in the head-to-toe configuration, which is physiologically relevant to the control of OPC differentiation in vivo.

Protein tyrosine phosphatase receptor type Z (PTPRZ) maintains oligodendrocyte precursor cells (OPCs) in an undifferentiated state. The inhibition of PTPase by its ligand pleiotrophin (PTN) promotes OPC differentiation; however, the substrate molecules of PTPRZ involved in the differentiation have not yet been elucidated in detail. We herein demonstrated that the tyrosine phosphorylation of AFAP1L2, paxillin, ERBB4, GIT1, p190RhoGAP, and NYAP2 was enhanced in OPC-like OL1 cells by a treatment with PTN. AFAP1L2, an adaptor protein involved in the PI3K-AKT pathway, exhibited the strongest response to PTN. PTPRZ dephosphorylated AFAP1L2 at tyrosine residues in vitro and in HEK293T cells. In OL1 cells, the knockdown of AFAP1L2 or application of a PI3K inhibitor suppressed cell differentiation as well as the PTN-induced phosphorylation of AKT and mTOR. We generated a knock-in mouse harboring a catalytically inactive Cys to Ser (CS) mutation in the PTPase domain. The phosphorylation levels of AFAP1L2, AKT, and mTOR were higher, and the expression of oligodendrocyte markers, including myelin basic protein (MBP) and myelin regulatory factor (MYRF), was stronger in CS knock-in brains than in wild-type brains on postnatal day 10; however, these differences mostly disappeared in the adult stage. Adult CS knock-in mice exhibited earlier remyelination after cuprizone-induced demyelination through the accelerated differentiation of OPCs. These phenotypes in CS knock-in mice were similar to those in Ptprz-deficient mice. Therefore, we conclude that the PTN-PTPRZ signal stimulates OPC differentiation partly by enhancing the tyrosine phosphorylation of AFAP1L2 in order to activate the PI3K-AKT pathway.

Congenital nystagmus, involuntary oscillating small eye movements, is commonly thought to originate from aberrant interactions between brainstem nuclei and foveal cortical pathways. Here we investigated whether nystagmus associated with congenital stationary nightblindness (CSNB) can result from primary deficits in the retina. We found that CSNB patients as well as an animal model (nob mice), both of which lack functional nyctalopin protein (NYX, nyx) in ON bipolar cells (ON-BC) at their synapse with photoreceptors, showed oscillating eye movements at a frequency of 4-7Hz. nob ON direction selective ganglion cells (ON-DSGC), which detect global motion and project to the accessory optic system (AOS), oscillated with the same frequency as their eyes. In the dark, individual ganglion cells (GC) oscillated asynchronously, but their oscillations became synchronized by light stimulation. Likewise, both patient and nob mice oscillating eye movements were only present in the light. Retinal pharmacological manipulations that blocked nob ON-DSGC oscillations also eliminated their oscillating eye movements, and retinal pharmacological manipulations that reduced oscillation frequency of nob ON-DSGCs also reduced oscillation frequency of their eye movements. We conclude that, in nob mice, oscillations of retinal ON-DSGCs cause nystagmus with properties similar to those associated with CSNB in humans. These results show that the nob mouse is the first animal model for a form of congenital nystagmus paving the way for development of therapeutic strategies.

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Congenital nystagmus, involuntary oscillating small eye movements, is commonly thought to originate from aberrant interactions between brainstem nuclei and foveal cortical pathways. Here, we investigated whether nystagmus associated with congenital stationary night blindness (CSNB) results from primary deficits in the retina. We found that CSNB patients as well as an animal model (nob mice), both of which lacked functional nyctalopin protein (NYX, nyx) in ON bipolar cells (BCs) at their synapse with photoreceptors, showed oscillating eye movements at a frequency of 4–7 Hz. nob ON direction-selective ganglion cells (DSGCs), which detect global motion and project to the accessory optic system (AOS), oscillated with the same frequency as their eyes. In the dark, individual ganglion cells (GCs) oscillated asynchronously, but their oscillations became synchronized by light stimulation. Likewise, both patient and nob mice oscillating eye movements were only present in the light when contrast was present. Retinal pharmacological and genetic manipulations that blocked nob GC oscillations also eliminated their oscillating eye movements, and retinal pharmacological manipulations that reduced the oscillation frequency of nob GCs also reduced the oscillation frequency of their eye movements. We conclude that, in nob mice, synchronized oscillations of retinal GCs, most likely the ON-DCGCs, cause nystagmus with properties similar to those associated with CSNB in humans. These results show that the nob mouse is the first animal model for a form of congenital nystagmus, paving the way for development of therapeutic strategies., Nystagmus (involuntary eye movements) in cases of congenital stationary night blindness arises from light-induced synchronization of oscillating retinal ganglion cells, revealing that some forms of nystagmus can have a retinal origin, rather than originating from aberrant interactions between brainstem nuclei and foveal cortical pathways, as commonly thought.

Increases in sodium concentrations ([Na+]) in body fluids elevate blood pressure (BP) by enhancing sympathetic nerve activity (SNA). However, the mechanisms by which information on increased [Na+] is translated to SNA have not yet been elucidated. We herein reveal that sympathetic activation leading to BP increases is not induced by mandatory high salt intakes or the intraperitoneal/intracerebroventricular infusions of hypertonic NaCl solutions in Nax-knockout mice in contrast to wild-type mice. We identify Nax channels expressed in specific glial cells in the organum vasculosum lamina terminalis (OVLT) as the sensors detecting increases in [Na+] in body fluids and show that OVLT neurons projecting to the paraventricular nucleus (PVN) are activated via acid-sensing ion channel 1a (ASIC1a) by H+ ions exported from Nax-positive glial cells. The present results provide an insight into the neurogenic mechanisms responsible for salt-induced BP elevations.

Adipsic (or essential) hypernatremia is a rare hypernatremia caused by a deficiency in thirst regulation and vasopressin release. In 2010, we reported a case in which autoantibodies targeting the sensory circumventricular organs (sCVOs) caused adipsic hypernatremia without hypothalamic structural lesions demonstrable by magnetic resonance imaging (MRI); sCVOs include the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), which are centers for the monitoring of body-fluid conditions and the control of water and salt intakes, and harbor neurons innervating hypothalamic nuclei for vasopressin release. We herein report three newly identified patients (3- to 8-year-old girls on the first visit) with similar symptoms. The common features of the patients were extensive hypernatremia without any sensation of thirst and defects in vasopressin response to serum hypertonicity. Despite these features, we could not detect any hypothalamic structural lesions by MRI. Immunohistochemical analyses using the sera of the three patients revealed that antibodies specifically reactive to the mouse SFO were present in the sera of all cases; in one case, the antibodies also reacted with the mouse OVLT. The immunoglobulin (Ig) fraction of serum obtained from one patient was intravenously injected into wild-type mice to determine whether the mice developed similar symptoms. Mice injected with a patient's Ig showed abnormalities in water/salt intake, vasopressin release, and diuresis, which resultantly developed hypernatremia. Prominent cell death and infiltration of reactive microglia was observed in the SFO of these mice. Thus, autoimmune destruction of the SFO may be the cause of the adipsic hypernatremia. This study provides a possible explanation for the pathogenesis of adipsic hypernatremia without demonstrable hypothalamus-pituitary lesions.

Protein-tyrosine phosphatase receptor type Z (PTPRZ) is predominantly expressed in the developing brain as a CS proteoglycan. PTPRZ has long (PTPRZ-A) and short type (PTPRZ-B) receptor forms by alternative splicing. The extracellular CS moiety of PTPRZ is required for high-affinity binding to inhibitory ligands, such as pleiotrophin (PTN), midkine, and interleukin-34; however, its functional significance in regulating PTPRZ activity remains obscure. We herein found that protein expression of CS-modified PTPRZ-A began earlier, peaking at approximately postnatal days 5-10 (P5-P10), and then that of PTN peaked at P10 at the developmental stage corresponding to myelination onset in the mouse brain. Ptn-deficient mice consistently showed a later onset of the expression of myelin basic protein, a major component of the myelin sheath, than wild-type mice. Upon ligand application, PTPRZ-A/B in cultured oligodendrocyte precursor cells exhibited punctate localization on the cell surface instead of diffuse distribution, causing the inactivation of PTPRZ and oligodendrocyte differentiation. The same effect was observed with the removal of CS chains with chondroitinase ABC but not polyclonal antibodies against the extracellular domain of PTPRZ. These results indicate that the negatively charged CS moiety prevents PTPRZ from spontaneously clustering and that the positively charged ligand PTN induces PTPRZ clustering, potentially by neutralizing electrostatic repulsion between CS chains. Taken altogether, these data indicate that PTN-PTPRZ-A signaling controls the timing of oligodendrocyte precursor cell differentiation in vivo, in which the CS moiety of PTPRZ receptors maintains them in a monomeric active state until its ligand binding.

The LacZ gene, which encodes Escherichia coli β-galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ-positive cells in living organisms or tissues at single-cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β-galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single-cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.

Chondroitin sulfate proteoglycans (CSPGs), which are enriched in demyelinating plaques in neurodegenerative diseases, such as multiple sclerosis (MS), impair remyelination by inhibiting the migration and differentiation of oligodendrocyte precursor cells (OPCs) in the central nervous system (CNS). We herein show that protamine (PRM, also known as a heparin antagonist) effectively neutralizes the inhibitory activities of CSPGs, thereby enhancing OPC differentiation and (re)myelination in mice. Cell-based assays using mouse OPC-like OL1 cells revealed that the PRM treatment exerted masking effects on extracellular CSPGs and improved oligodendrocyte differentiation on inhibitory CSPG-coated substrates. PRM also bound to the extracellular region of protein tyrosine phosphatase receptor type Z (PTPRZ), a membrane-spanning CSPG predominantly expressed in OPCs, and functioned as a ligand mimetic of PTPRZ, thereby suppressing its negative regulatory activity on oligodendrocyte differentiation. In primary cultures, the differentiation of OPCs from wild-type and Ptprz-deficient mice was equally enhanced by PRM. Moreover, the intranasal administration of PRM accelerated myelination in the developing mouse brain, and its intracerebroventricular administration stimulated remyelination after cuprizone-induced demyelination. These results indicate that PRM has CSPG-neutralizing activity which promotes oligodendrocyte differentiation under developmental and morbid conditions.

Local regulation of synaptic efficacy is thought to be important for proper networking of neurons and memory formation. Dysregulation of global translation influences long-term memory in mice, but the relevance of the regulation specific for local translation by RNA granules remains elusive. Here, we demonstrate roles of RNG105/caprin1 in long-term memory formation. RNG105 deletion in mice impaired synaptic strength and structural plasticity in hippocampal neurons. Furthermore, RNG105-deficient mice displayed unprecedentedly severe defects in long-term memory formation in spatial and contextual learning tasks. Genome-wide profiling of mRNA distribution in the hippocampus revealed an underlying mechanism: RNG105 deficiency impaired the asymmetric somato-dendritic localization of mRNAs. Particularly, RNG105 deficiency reduced the dendritic localization of mRNAs encoding regulators of AMPAR surface expression, which was consistent with attenuated homeostatic AMPAR scaling in dendrites and reduced synaptic strength. Thus, RNG105 has an essential role, as a key regulator of dendritic mRNA localization, in long-term memory formation., eLife digest Messages pass from one nerve cell to the next across gaps called synapses. The first neuron releases chemical signals from the end of its long, thin nerve fiber. The second receives the message at receptors on branching structures known as dendrites. Each connection has a corresponding bump called a dendritic spine. As animals learn, these can grow larger, strengthening the connection. This is the basis of how memories form. To strengthen a synapse, the cell must transport the materials to the dendritic spine. The cell makes copies of the genetic instructions to strengthen the synapse in the form of messenger RNA (often shortened to mRNA). But, this happens in the body of the cell, a long way from the dendrites themselves. The mRNA travels from the cell body to the dendrites in collections of molecules referred to as ‘RNA granules’. One of the key components of the RNA granule system is a protein called RNG105/caprin1. Now, Nakayama, Ohashi et al. have engineered mice to delete the gene for RNG105/caprin1, revealing its effect on memory. Mice lacking RNG105/caprin1 struggled to make long-term memories. Unlike their normal counterparts, these mutant mice did not become accustomed to new environments or objects. They also found it more challenging to learn the position of a hidden platform in a water-based maze. Lastly, over time, the mutant mice forgot to be fearful of a dark chamber where they had received a small electric shock. Memories form in a part of the brain called the hippocampus and the dendritic spines in this region were smaller in mice lacking RNG105/caprin1. Furthermore, when the nerve cells from this part of the brain were grown in Petri dishes, they did not respond normally to stimulation. The dendritic spines of normal cells increased in size, but those on the cells lacking RNG105/caprin1 got smaller compared to normal cells. A closer look revealed that the distribution of mRNA in brain cells from mice lacking RNG105/caprin1 differed from that of normal mice. Some pieces of genetic information failed to make it from the cell body to the dendrites. This included mRNA involved in making regulators of a component of dendritic spines called the AMPA receptor. The AMPA receptor detects the chemical messenger, glutamate, and is crucial for memory formation. These findings further our understanding of long-term memory and open the way for future research into human disease. Mutations in RNA granule components, including RNG105/caprin1, have links to conditions such as amyotrophic lateral sclerosis (ALS) and autism spectrum disorder (ASD). Further investigation could reveal new targets for drug treatment.

Sodium (Na) homeostasis is crucial for life, and the Na(+) level ([Na(+)]) of body fluids is strictly maintained at a range of 135-145 mM. However, the existence of a [Na(+)] sensor in the brain has long been controversial until Nax was identified as the molecular entity of the sensor. This review provides an overview of the [Na(+)]-sensing mechanism in the brain for the regulation of salt intake by summarizing a series of our studies on Nax. Nax is a Na channel expressed in the circumventricular organs (CVOs) in the brain. Among the CVOs, the subfornical organ (SFO) is the principal site for the control of salt intake behavior, where Nax populates the cellular processes of astrocytes and ependymal cells enveloping neurons. A local expression of endothelin-3 in the SFO modulates the [Na(+)] sensitivity for Nax activation, and thereby Nax is likely to be activated in the physiological [Na(+)] range. Nax stably interacts with Na(+)/K(+)-ATPase whereby Na(+) influx via Nax is coupled with activation of Na(+)/K(+)-ATPase associated with the consumption of ATP. The consequent activation of anaerobic glucose metabolism of Nax-positive glial cells upregulates the cellular release of lactate, and this lactate functions as a gliotransmitter to activate GABAergic neurons in the SFO. The GABAergic neurons presumably regulate hypothetic neurons involved in the control of salt intake behavior. Recently, a patient with essential hypernatremia caused by autoimmunity to Nax was found. In this case, the hypernatremia was considered to be induced by the complement-mediated cell death in the CVOs, where Nax specifically populates.

Protein tyrosine phosphatase receptor type Z (PTPRZ, also known as PTPζ or RPTPβ) is preferentially expressed in the central nervous system (CNS). PTPRZ plays important roles during development and adulthood in CNS myelination, learning and memory. Three splicing isoforms for PTPRZ have been identified to date: two receptor type isoforms, PTPRZ-A and PTPRZ-B, and one secretory isoform, PTPRZ-S. We herein identified novel PTPRZ receptor sub-isoforms without a seven-amino acid sequence encoded by exon 16. This sequence forms a part of the helix-turn-helix segment called the 'wedge' structure, which is located at the N-terminal region in the membrane-proximal protein tyrosine phosphatase domain. In contrast to conventional receptor isoforms with uniform expression, the deleted isoforms were expressed in the brain, but not in the retina, indicating the tissue-specific splicing of exon 16. Biochemical analyses of PTPRZ intracellular regions revealed differences in the characteristics of the deleted form, namely, stronger binding activity to postsynaptic density protein 95 (PSD95) and greater enrichment in the postsynaptic density fraction than the full-length form. Furthermore, the exon 16-deleted form exhibited higher catalytic efficiency in vitro. These results suggest that sub-isoforms of PTPRZ have different functions because of variations in the wedge structure.

Nax, which is preferentially expressed in the glial cells of sensory circumventricular organs in the brain, is a sodium channel that is poorly homologous to voltage-gated sodium channels. We previously reported that Naxis a sodium concentration ([Na+])-sensitive, but not a voltage-sensitive channel that is critically involved in body-fluid homeostasis. Nax-knockout mice do not stop ingesting salt even when dehydrated and transiently develop hypernatremia. [Na+] in body fluids is strictly controlled at 135 to 145 mM in mammals. Although the set point must be within this range, Naxwas shown to have a threshold value of ~150 mM for extracellular [Na+] ([Na+]o) for activation in vitro. Therefore, the [Na+]odependency of Naxin vivo is presumably modified by an as yet unidentified mechanism. We recently demonstrated that the [Na+]odependency of Naxin the subfornical organ was adjusted to the physiological range by endothelin-3. Pharmacological experiments revealed that endothelin receptor B signaling was involved in this modulation of Naxgating through protein kinase C and ERK1/2 activation. In addition, we identified a case of essential hypernatremia caused by autoimmunity to Nax. Occurrence of a ganglioneuroma composed of Schwann-like cells that robustly expressed Naxwas likely to induce the autoimmune response in this patient. An intravenous injection of the immunoglobulin fraction of the patient’s serum, which contained anti-Naxantibodies, into mice reproduced the patient’s symptoms. This review provides an overview of the physiological functions of Naxby summarizing our recent studies.

Na(x), a sodium concentration-sensitive sodium channel, is expressed in non-myelinating Schwann cells of the adult peripheral nervous system, but the pathophysiological role remains unclear. We found that functional recovery of the hind paw responses from the sciatic nerve transection was delayed in Na(x) knockout (Na(x)⁻/⁻) mice. Histological analyses showed a decrease in the number of regenerated myelinated axons in (Na(x)⁻/⁻) sciatic nerves. The delay in the recovery in Na(x)⁻/⁻ mice was improved by lactate and inhibited by a monocarboxylate transporter inhibitor. In vitro experiments using cultured Schwann cells showed that lactate release was enhanced by endothelin (ET)-1 and blocked by an ET receptor type B antagonist. Here, it is conceivable that Na(x) was activated by ET-1. The amount of lactate release by ET-1 was lower in Na(x)⁻/⁻ mice than in wild-type mice. These results indicated that Na(x) is functionally coupled to ET for lactate release via ET receptor type B and is involved in peripheral nerve regeneration.

PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.

Receptor-like protein-tyrosine phosphatases (RPTPs) are involved in various aspects of cellular functions, such as proliferation, differentiation, survival, migration, and metabolism. A small number of RPTPs have been reported to regulate activities of some cellular proteins including receptor protein-tyrosine kinases (RPTKs). However, our understanding about the roles of individual RPTPs in the regulation of RPTKs is still limited. The R3 RPTP subfamily reportedly plays pivotal roles in the development of several tissues including the vascular and nervous systems. Here, we examined enzyme-substrate relationships between the four R3 RPTP subfamily members and 21 RPTK members selected from 14 RPTK subfamilies by using a mammalian two-hybrid system with substrate-trapping RPTP mutants. Among the 84 RPTP-RPTK combinations conceivable, we detected 30 positive interactions: 25 of the enzyme-substrate relationships were novel. We randomly chose several RPTKs assumed to be substrates for R3 RPTPs, and validated the results of this screen by in vitro dephosphorylation assays, and by cell-based assays involving overexpression and knock-down experiments. Because their functional relationships were verified without exception, it is probable that the RPTKs identified as potential substrates are actually physiological substrates for the R3 RPTPs. Interestingly, some RPTKs were recognized as substrates by all R3 members, but others were recognized by only one or a few members. The enzyme-substrate relationships identified in the present study will shed light on physiological roles of the R3 RPTP subfamily.

Summary Salt homeostasis is essential to survival, but brain mechanisms for salt-intake control have not been fully elucidated. Here, we found that the sensitivity of Na x channels to [Na + ] o is dose-dependently enhanced by endothelin-3 (ET-3). Na x channels began to open when [Na + ] o exceeded ∼150 mM without ET-3, but opened fully at a physiological [Na + ] o (135–145 mM) with 1 nM ET-3. Importantly, ET-3 was expressed in the subfornical organ (SFO) along with Na x , and the level was robustly increased by dehydration. Pharmacological experiments revealed that endothelin receptor B (ET B R) signaling is involved in this modulation of Na x gating through protein kinase C and ERK1/2 activation. ET B R agonists increased the firing rate of GABAergic neurons via lactate in the SFO, and an ET B R antagonist attenuated salt aversion during dehydration. These results indicate that ET-3 expression in the SFO is tightly coupled with body-fluid homeostasis through modulation of the [Na + ] o sensitivity of Na x .

The brain monitors conditions of body fluids and levels of circulating neuroactive factors to maintain the systemic homeostasis. Unlike most regions in the brain, circumventricular organs (CVOs) lack the blood-brain barrier, and serve as the sensing center. Among the CVOs, the subfornical organ (SFO) is the sensing site of Na+ levels in body fluids to control water and salt intake. The SFO harbors neuronal cell bodies with a variety of hormone receptors and innervates many brain loci. In addition, the SFO harbors specialized glial cells (astrocytes and ependymal cells) expressing Nax, a Na+-level-sensitive sodium channel. These glial cells wrap a specific population of neurons with their processes, and control the firing activities of the neurons by gliotransmitters, such as lactate and epoxyeicosatrienoic acids (EETs), relevant to water/salt-intake behaviors. Recent advances in the understanding of physiological functions of the SFO are reviewed herein with a focus on the Na+-sensing mechanism by Nax.

Adipsic (or essential) hypernatremia is a rare hypernatremia caused by a deficiency in thirst regulation and vasopressin release. In 2010, we reported a case in which autoantibodies targeting the sensory circumventricular organs (sCVOs) caused adipsic hypernatremia without hypothalamic structural lesions demonstrable by magnetic resonance imaging (MRI); sCVOs include the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), which are centers for the monitoring of body‐fluid conditions and the control of water and salt intakes, and harbor neurons innervating hypothalamic nuclei for vasopressin release. We herein report three newly identified patients (3‐ to 8‐year‐old girls on the first visit) with similar symptoms. The common features of the patients were extensive hypernatremia without any sensation of thirst and defects in vasopressin response to serum hypertonicity. Despite these features, we could not detect any hypothalamic structural lesions by MRI. Immunohistochemical analyses using the sera of the three patients revealed that antibodies specifically reactive to the mouse SFO were present in the sera of all cases; in one case, the antibodies also reacted with the mouse OVLT. The immunoglobulin (Ig) fraction of serum obtained from one patient was intravenously injected into wild‐type mice to determine whether the mice developed similar symptoms. Mice injected with a patient's Ig showed abnormalities in water/salt intake, vasopressin release, and diuresis, which resultantly developed hypernatremia. Prominent cell death and infiltration of reactive microglia was observed in the SFO of these mice. Thus, autoimmune destruction of the SFO may be the cause of the adipsic hypernatremia. This study provides a possible explanation for the pathogenesis of adipsic hypernatremia without demonstrable hypothalamus‐pituitary lesions.