The potential of COVID-19 patients' sera to cause antibody-dependent enhancement of infection and IL-6 production.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many vaccine trials have been initiated. An important goal of vaccination is the development of neutralizing antibody (Ab) against SARS-CoV-2. However, the possible induction of antibody-dependent enhancement (ADE) of infection, which is known for other coronaviruses and dengue virus infections, is a particular concern in vaccine development. Here, we demonstrated that human iPS cell-derived, immortalized, and ACE2- and TMPRSS2-expressing myeloid cell lines are useful as host cells for SARS-CoV-2 infection. The established cell lines were cloned and screened based on their function in terms of susceptibility to SARS-CoV-2-infection or IL-6 productivity. Using the resulting K-ML2 (AT) clone 35 for SARS-CoV-2-infection or its subclone 35-40 for IL-6 productivity, it was possible to evaluate the potential of sera from severe COVID-19 patients to cause ADE and to stimulate IL-6 production upon infection with SARS-CoV-2.

Mice deficient in protein tyrosine phosphatase receptor type Z (PTPRZ) show reduced responsivity to methamphetamine despite an enhanced response to novelty.

Methamphetamine (METH), a commonly abused drug, elevates extracellular dopamine (DA) levels by inducing DA efflux through the DA transporter (DAT). Emerging evidence in rodent models suggests that locomotor responses to a novel inescapable open field may predict behavioral responses to abused drugs; METH produces more potent stimulant effects in high responders to novelty than in low responders. We herein found that mice deficient in protein tyrosine phosphatase receptor type Z (Ptprz-KO) exhibited an enhanced behavioral response to novelty; however, METH-induced hyperlocomotion was significantly lower in Ptprz-KO than in wild-type mice when METH was administered at a non-toxic dose of 1 mg per kg body weight (bdw). Single-cell RT-PCR revealed that the majority of midbrain DA neurons expressed PTPRZ. No histological alterations were observed in the mesolimbic or nigrostriatal dopaminergic pathways in Ptprz-KO brains; however, a significant decrease was noted in brain DA turnover, suggesting functional alterations. In vivo microdialysis experiments revealed that METH-evoked DA release in the nucleus accumbens was significantly lower in Ptprz-KO mice than in wild-type mice. Consistent with this result, Ptprz-KO mice showed significantly fewer cell surface DAT as well as weaker DA uptake activity in striatal synaptosomes prepared 1 hr after the administration of METH than wild-type mice, while no significant differences were observed in the two groups treated with saline. These results indicate that the high response phenotype of Ptprz-KO mice to novelty may not be simply attributed to hyper-dopaminergic activity, and that deficits in PTPRZ reduce the effects of METH by reducing DAT activity.

A head-to-toe dimerization has physiological relevance for ligand-induced inactivation of protein tyrosine receptor type Z.

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.

Behavioral and neurological analyses of adult mice carrying null and distinct loss-of-receptor function mutations in protein tyrosine phosphatase receptor type Z (PTPRZ).

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.

The PTN-PTPRZ signal activates the AFAP1L2-dependent PI3K-AKT pathway for oligodendrocyte differentiation: Targeted inactivation of PTPRZ activity in mice.

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.

Enhanced extinction of aversive memories in mice lacking SPARC-related protein containing immunoglobulin domains 1 (SPIG1/FSTL4).

Brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity related to learning and memory. We previously reported that SPARC-related protein containing immunoglobulin domains 1 (SPIG1, also known as Follistatin-like protein 4, FSTL4) binds to pro-BDNF and negatively regulates BDNF maturation; however, its neurological functions, particularly in learning and memory, have not yet been elucidated. We herein examined the electrophysiological and behavioral phenotypes of Spig1-knockout (Spig1-KO) mice. Adult Spig1-KO mice exhibited greater excitability and facilitated long-term potentiation (LTP) in the CA1 region of hippocampal slices than age- and sex-matched wild-type (WT) mice. Facilitated LTP was reduced to the level of WT by the bath application of an anti-BDNF antibody to hippocampal slices. A step-through inhibitory avoidance learning paradigm revealed that the extinction of aversive memories was significantly enhanced in adult Spig1-KO mice, while they showed the normal acquisition of aversive memories; besides, spatial reference memory formation was also normal in the standard Morris water maze task. An intracerebroventricular (icv) injection of anti-BDNF in the process of extinction learning transiently induced the recurrence of aversive memories in Spig1-KO mice, but exerted no effects in WT mice. These results indicate a critical role for SPIG1 in BDNF-mediated synaptic plasticity in extinction of inhibitory avoidance memory.

Protamine neutralizes chondroitin sulfate proteoglycan-mediated inhibition of oligodendrocyte differentiation.

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.

Mllt10 knockout mouse model reveals critical role of Af10-dependent H3K79 methylation in midfacial development.

Epigenetic regulation is required to ensure the precise spatial and temporal pattern of gene expression that is necessary for embryonic development. Although the roles of some epigenetic modifications in embryonic development have been investigated in depth, the role of methylation at lysine 79 (H3K79me) is poorly understood. Dot1L, a unique methyltransferase for H3K79, forms complexes with distinct sets of co-factors. To further understand the role of H3K79me in embryogenesis, we generated a mouse knockout of Mllt10, the gene encoding Af10, one Dot1L complex co-factor. We find homozygous Mllt10 knockout mutants (Mllt10-KO) exhibit midline facial cleft. The midfacial defects of Mllt10-KO embryos correspond to hyperterolism and are associated with reduced proliferation of mesenchyme in developing nasal processes and adjacent tissue. We demonstrate that H3K79me level is significantly decreased in nasal processes of Mllt10-KO embryos. Importantly, we find that expression of AP2α, a gene critical for midfacial development, is directly regulated by Af10-dependent H3K79me, and expression AP2α is reduced specifically in nasal processes of Mllt10-KO embryos. Suppression of H3K79me completely mimicked the Mllt10-KO phenotype. Together these data are the first to demonstrate that Af10-dependent H3K79me is essential for development of nasal processes and adjacent tissues, and consequent midfacial formation.

Targeting PTPRZ inhibits stem cell-like properties and tumorigenicity in glioblastoma cells.

The R5 subfamily of receptor-type protein tyrosine phosphatases (RPTPs) comprises PTPRZ and PTPRG. A recent study on primary human glioblastomas suggested a close association between PTPRZ1 (human PTPRZ) expression and cancer stemness. However, the functional roles of PTPRZ activity in glioma stem cells have remained unclear. In the present study, we found that sphere-forming cells from the rat C6 and human U251 glioblastoma cell lines showed high expression levels of PTPRZ-B, the short receptor isoform of PTPRZ. Stable PTPRZ knockdown altered the expression levels of stem cell transcription factors such as SOX2, OLIG2, and POU3F2 and decreased the sphere-forming abilities of these cells. Suppressive effects on the cancer stem-like properties of the cells were also observed following the knockdown of PTPRG. Here, we identified NAZ2329, a cell-permeable small molecule that allosterically inhibits both PTPRZ and PTPRG. NAZ2329 reduced the expression of SOX2 in C6 and U251 cells and abrogated the sphere-forming abilities of these cells. Tumor growth in the C6 xenograft mouse model was significantly slower with the co-treatment of NAZ2329 with temozolomide, an alkylating agent, than with the individual treatments. These results indicate that pharmacological inhibition of R5 RPTPs is a promising strategy for the treatment of malignant gliomas.

Role of Chondroitin Sulfate (CS) Modification in the Regulation of Protein-tyrosine Phosphatase Receptor Type Z (PTPRZ) Activity: PLEIOTROPHIN-PTPRZ-A SIGNALING IS INVOLVED IN OLIGODENDROCYTE DIFFERENTIATION.

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.

Small-molecule inhibition of PTPRZ reduces tumor growth in a rat model of glioblastoma.

Protein tyrosine phosphatase receptor-type Z (PTPRZ) is aberrantly over-expressed in glioblastoma and a causative factor for its malignancy. However, small molecules that selectively inhibit the catalytic activity of PTPRZ have not been discovered. We herein performed an in vitro screening of a chemical library, and identified SCB4380 as the first potent inhibitor for PTPRZ. The stoichiometric binding of SCB4380 to the catalytic pocket was demonstrated by biochemical and mass spectrometric analyses. We determined the crystal structure of the catalytic domain of PTPRZ, and the structural basis of the binding of SCB4380 elucidated by a molecular docking method was validated by site-directed mutagenesis studies. The intracellular delivery of SCB4380 by liposome carriers inhibited PTPRZ activity in C6 glioblastoma cells, and thereby suppressed their migration and proliferation in vitro and tumor growth in a rat allograft model. Therefore, selective inhibition of PTPRZ represents a promising approach for glioma therapy.