We previously identified SPARC-related protein-containing immunoglobulin domains 1 (SPIG1, also known as Follistatin-like protein 4) as one of the dorsal-retina-specific molecules expressed in the ...developing chick retina. We here demonstrated that the knockdown of SPIG1 in the retinal ganglion cells (RGCs) of developing chick embryos induced the robust ectopic branching of dorsal RGC axons and failed to form a tight terminal zone at the proper position on the tectum. The knockdown of SPIG1 in RGCs also led to enhanced axon branching in vitro. However, this was canceled by the addition of a neutralizing antibody against brain-derived neurotrophic factor (BDNF) to the culture medium. SPIG1 and BDNF were colocalized in vesicle-like structures in cells. SPIG1 bound with the proform of BDNF (proBDNF) but very weakly with mature BDNF in vitro. The expression and secretion of mature BDNF were significantly decreased when SPIG1 was exogenously expressed with BDNF in HEK293T or PC12 cells. The amount of mature BDNF proteins as well as the tyrosine phosphorylation level of the BDNF receptor, tropomyosin-related kinase B (TrkB), in the hippocampus were significantly higher in SPIG1-knockout mice than in wild-type mice. Here the spine density of CA1 pyramidal neurons was consistently increased. Together, these results suggest that SPIG1 negatively regulated BDNF maturation by binding to proBDNF, thereby suppressing axonal branching and spine formation.
► SAP97 stabilizes Nax in the plasma membrane. ► SAP97 binds to Nax through the PDZ-binding domain. ► Disruption of this interaction reduces the cell-surface expression of Nax. ► The reduction in ...cell-surface expression is attenuated by inhibitors of endocytosis.
Nax is a sodium-level sensor for body fluids expressed in the circumventricular organs in the brain. Nax has a putative PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at the carboxyl (C)-terminus. Here we found that several PDZ proteins bind to Nax by PDZ-array overlay assay. Among them, synapse-associated protein 97 (SAP97/DLG1) was coexpressed with Nax in the subfornical organ. In C6 glioblastoma cells, destruction of the PDZ-binding motif of Nax or depletion of SAP97 resulted in a decrease in cell-surface Nax, which was attenuated with inhibitors of endocytosis. These results indicate that SAP97 contributes to the stabilization of Nax channels at the plasma membrane.
Naxphysically interacts with SAP97 by anti tag coimmunoprecipitation (View interaction) CNRasGEFbinds to Nax by protein array (View interaction) Nax and SAP97colocalize by fluorescence microscopy (View interaction) GIPC1binds to Nax by protein array (View interaction) ZO-1binds to Nax by protein array (View interaction) SAP97binds to Nax by protein array (View interaction) Densin-180binds to Nax by protein array (View interaction) Beta-1-syntrophinbinds to Nax by protein array (View interaction) ERBINbinds to Nax by protein array (View interaction) Naxphysically interacts with SAP97 by pull down (View interaction) Lnx1binds to Nax by protein array (View interaction) nNOSbinds to Nax by protein array (View interaction)
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.
After brain ischemia, significant amounts of adenosine 5'-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of ...P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y(1) receptor (P2Y(1)R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y(1)R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y(1)R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y(1)R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y(1)R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-κB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y(1)R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-κB pathway.
Na.sub.x is a sodium-concentration (Na.sup.+ )-sensitive Na channel with a gating threshold of ~150 mM for extracellular Na.sup.+ (Na.sup.+ .sub.o) in vitro. We previously reported that Na.sub.x was ...preferentially expressed in the glial cells of sensory circumventricular organs including the subfornical organ, and was involved in Na.sup.+ sensing for the control of salt-intake behavior. Although Na.sub.x was also suggested to be expressed in the neurons of some brain regions including the amygdala and cerebral cortex, the channel properties of Na.sub.x have not yet been adequately characterized in neurons. We herein verified that Na.sub.x was expressed in neurons in the lateral amygdala of mice using an antibody that was newly generated against mouse Na.sub.x . To investigate the channel properties of Na.sub.x expressed in neurons, we established an inducible cell line of Na.sub.x using the mouse neuroblastoma cell line, Neuro-2a, which is endogenously devoid of the expression of Na.sub.x . Functional analyses of this cell line revealed that the Na.sup.+ -sensitivity of Na.sub.x in neuronal cells was similar to that expressed in glial cells. The cation selectivity sequence of the Na.sub.x channel in cations was revealed to be Na.sup.+ almost equal to Li.sup.+ > Rb.sup.+ > Cs.sup.+ for the first time. Furthermore, we demonstrated that Na.sub.x bound to postsynaptic density protein 95 (PSD95) through its PSD95/Disc-large/ZO-1 (PDZ)-binding motif at the C-terminus in neurons. The interaction between Na.sub.x and PSD95 may be involved in promoting the surface expression of Na.sub.x channels because the depletion of endogenous PSD95 resulted in a decrease in Na.sub.x at the plasma membrane. These results indicated, for the first time, that Na.sub.x functions as a Na.sup.+ -sensitive Na channel in neurons as well as in glial cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nax
physically interacts with
SAP97
by anti tag coimmunoprecipitation (View interaction)
CNRasGEF
binds to
Nax
by protein array (View interaction)
Nax
and
SAP97
colocalize by fluorescence microscopy ...(View interaction)
GIPC1
binds to
Nax
by protein array (View interaction)
ZO‐1
binds to
Nax
by protein array (View interaction)
SAP97
binds to
Nax
by protein array (View interaction)
Densin‐180
binds to
Nax
by protein array (View interaction)
Beta‐1‐syntrophin
binds to
Nax
by protein array (View interaction)
ERBIN
binds to
Nax
by protein array (View interaction)
Nax
physically interacts with
SAP97
by pull down (View interaction)
Lnx1
binds to
Nax
by protein array (View interaction)
nNOS
binds to
Nax
by protein array (View interaction)
► SAP97 stabilizes Na
x
in the plasma membrane. ► SAP97 binds to Na
x
through the PDZ‐binding domain. ► Disruption of this interaction reduces the cell‐surface expression of Na
x
. ► The reduction in cell‐surface expression is attenuated by inhibitors of endocytosis.
After brain ischemia, significant amounts of adenosine 5'-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of ...P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y sub(1) receptor (P2Y sub(1)R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y sub(1)R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y sub(1)R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y sub(1)R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor- alpha , monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y sub(1)R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)- Kappa B inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y sub(1)R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF- Kappa B pathway.
After brain ischemia, significant amounts of adenosine 5'-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of ...P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y
1
receptor (P2Y
1
R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y
1
R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y
1
R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y
1
R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y
1
R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-κB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y
1
R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-κB pathway.
Nax is a sodium-concentration (Na+)-sensitive Na channel with a gating threshold of ~150 mM for extracellular Na+ (Na+o) in vitro. We previously reported that Nax was preferentially expressed in the ...glial cells of sensory circumventricular organs including the subfornical organ, and was involved in Na+ sensing for the control of salt-intake behavior. Although Nax was also suggested to be expressed in the neurons of some brain regions including the amygdala and cerebral cortex, the channel properties of Nax have not yet been adequately characterized in neurons. We herein verified that Nax was expressed in neurons in the lateral amygdala of mice using an antibody that was newly generated against mouse Nax. To investigate the channel properties of Nax expressed in neurons, we established an inducible cell line of Nax using the mouse neuroblastoma cell line, Neuro-2a, which is endogenously devoid of the expression of Nax. Functional analyses of this cell line revealed that the Na+-sensitivity of Nax in neuronal cells was similar to that expressed in glial cells. The cation selectivity sequence of the Nax channel in cations was revealed to be Na+ approximately Li+ > Rb+ > Cs+ for the first time. Furthermore, we demonstrated that Nax bound to postsynaptic density protein 95 (PSD95) through its PSD95/Disc-large/ZO-1 (PDZ)-binding motif at the C-terminus in neurons. The interaction between Nax and PSD95 may be involved in promoting the surface expression of Nax channels because the depletion of endogenous PSD95 resulted in a decrease in Nax at the plasma membrane. These results indicated, for the first time, that Nax functions as a Na+-sensitive Na channel in neurons as well as in glial cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A growing body of evidence indicates that P2X receptors (P2XRs), a family of ligand-gated cation channels activated by extracellular ATP, play an important role in pain signaling. In contrast to the ...role of the P2X
3
R subtype that has been extensively studied, the precise roles of others among the seven P2XR subtypes (P2X
1
R-P2X
7
R) remain to be determined because of a lack of sufficiently powerful tools to specifically block P2XR signaling in vivo. In the present study, we investigated the behavioral phenotypes of a line of mice in which the p2rx4 gene was disrupted in a series of acute and chronic pain assays. While p2rx4
−/−
mice showed no major defects in pain responses evoked by acute noxious stimuli and local tissue damage or in motor function as compared with wild-type mice, these mice displayed reduced pain responses in two models of chronic pain (inflammatory and neuropathic pain). In a model of chronic inflammatory pain developed by intraplantar injection of complete Freund's adjuvant (CFA), p2rx4
−/−
mice exhibited attenuations of pain hypersensitivity to innocuous mechanical stimuli (tactile allodynia) and also of the CFA-induced swelling of the hindpaw. A most striking phenotype was observed in a test of neuropathic pain: tactile allodynia caused by an injury to spinal nerve was markedly blunted in p2rx4
−/−
mice. By contrast, pain hypersensitivity to a cold stimulus (cold allodynia) after the injury was comparable in wild-type and p2rx4
−/−
mice. Together, these findings reveal a predominant contribution of P2X
4
R to nerve injury-induced tactile allodynia and, to the lesser extent, peripheral inflammation. Loss of P2X
4
R produced no defects in acute physiological pain or tissue damaged-induced pain, highlighting the possibility of a therapeutic benefit of blocking P2X
4
R in the treatment of chronic pain, especially tactile allodynia after nerve injury.
PDF
