ADAM10 Negatively Regulates Neuronal Differentiation during Spinal Cord Development

Members of the ADAM (a disintegrin and metalloprotease) family are involved in embryogenesis and tissue formation via their proteolytic function, cell-cell and cell-matrix interactions. ADAM10 is expressed temporally and spatially in the developing chicken spinal cord, but its function remains elusive. In the present study, we address this question by electroporating ADAM10 specific morpholino antisense oligonucleotides (ADAM10-mo) or dominant-negative ADAM10 (dn-ADAM10) plasmid into the developing chicken spinal cord as well as by in vitro cell culture investigation. Our results show that downregulation of ADAM10 drives precocious differentiation of neural progenitor cells and radial glial cells, resulting in an increase of neurons in the developing spinal cord, even in the prospective ventricular zone. Remarkably, overexpression of the dn-ADAM10 plasmid mutated in the metalloprotease domain (dn-ADAM10-me) mimics the phenotype as found by the ADAM10-mo transfection. Furthermore, in vitro experiments on cultured cells demonstrate that downregulation of ADAM10 decreases the amount of the cleaved intracellular part of Notch1 receptor and its target, and increases the number of βIII-tubulin-positive cells during neural progenitor cell differentiation. Taken together, our data suggest that ADAM10 negatively regulates neuronal differentiation, possibly via its proteolytic effect on the Notch signaling during development of the spinal cord.     

Protein Tyrosine Phosphatase Receptor Type Z Negatively Regulates Oligodendrocyte Differentiation and Myelination

Background

Fyn tyrosine kinase-mediated down-regulation of Rho activity through activation of p190RhoGAP is crucial for oligodendrocyte differentiation and myelination. Therefore, the loss of function of its counterpart protein tyrosine phosphatase (PTP) may enhance myelination during development and remyelination in demyelinating diseases. To test this hypothesis, we investigated whether Ptprz, a receptor-like PTP (RPTP) expressed abuntantly in oligodendrocyte lineage cells, is involved in this process, because we recently revealed that p190RhoGAP is a physiological substrate for Ptprz.

Methodology/Principal Findings

We found an early onset of the expression of myelin basic protein (MBP), a major protein of the myelin sheath, and early initiation of myelination in vivo during development of the Ptprz-deficient mouse, as compared with the wild-type. In addition, oligodendrocytes appeared earlier in primary cultures from Ptprz-deficient mice than wild-type mice. Furthermore, adult Ptprz-deficient mice were less susceptible to experimental autoimmune encephalomyelitis (EAE) induced by active immunization with myelin/oligodendrocyte glycoprotein (MOG) peptide than were wild-type mice. After EAE was induced, the tyrosine phosphorylation of p190RhoGAP increased significantly, and the EAE-induced loss of MBP was markedly suppressed in the white matter of the spinal cord in Ptprz-deficient mice. Here, the number of T-cells and macrophages/microglia infiltrating into the spinal cord did not differ between the two genotypes after MOG immunization. All these findings strongly support the validity of our hypothesis.

Conclusions/Significance

Ptprz plays a negative role in oligodendrocyte differentiation in early central nervous system (CNS) development and remyelination in demyelinating CNS diseases, through the dephosphorylation of substrates such as p190RhoGAP.     

TAM Receptors Support Neural Stem Cell Survival,Proliferation and Neuronal Differentiation

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III⁺) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.     

Interaction of PDK1 with Phosphoinositides Is Essential for Neuronal Differentiation but Dispensable for Neuronal Survival

3-Phosphoinositide-dependent protein kinase 1 (PDK1) operates in cells in response to phosphoinositide 3-kinase activation and phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P₃] production by activating a number of AGC kinases, including protein kinase B (PKB)/Akt. Both PDK1 and PKB contain pleckstrin homology (PH) domains that interact with the PtdIns(3,4,5)P₃ second messenger. Disrupting the interaction of the PDK1 PH domain with phosphoinositides by expressing the PDK1 K465E knock-in mutation resulted in mice with reduced PKB activation. We explored the physiological consequences of this biochemical lesion in the central nervous system. The PDK1 knock-in mice displayed a reduced brain size due to a reduction in neuronal cell size rather than cell number. Reduced BDNF-induced phosphorylation of PKB at Thr308, the PDK1 site, was observed in the mutant neurons, which was not rate limiting for the phosphorylation of those PKB substrates governing neuronal survival and apoptosis, such as FOXO1 or glycogen synthase kinase 3 (GSK3). Accordingly, the integrity of the PDK1 PH domain was not essential to support the survival of different embryonic neuronal populations analyzed. In contrast, PKB-mediated phosphorylation of PRAS40 and TSC2, allowing optimal mTORC1 activation and brain-specific kinase (BRSK) protein synthesis, was markedly reduced in the mutant mice, leading to impaired neuronal growth and differentiation.     

RAGE,Receptor of Advanced Glycation Endoproducts,Negatively Regulates Chondrocytes Differentiation

RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.     

Nox4-Mediated Cell Signaling Regulates Differentiation and Survival of Neural Crest Stem Cells

The function of reactive oxygen species (ROS) as second messengers in cell differentiation has been demonstrated only for a limited number of cell types. Here, we used a well-established protocol for BMP2-induced neuronal differentiation of neural crest stem cells (NCSCs) to examine the function of BMP2-induced ROS during the process. We first show that BMP2 indeed induces ROS generation in NCSCs and that blocking ROS generation by pretreatment of cells with diphenyleneiodonium (DPI) as NADPH oxidase (Nox) inhibitor inhibits neuronal differentiation. Among the ROS-generating Nox isozymes, only Nox4 was expressed at a detectable level in NCSCs. Nox4 appears to be critical for survival of NCSCs at least in vitro as down-regulation by RNA interference led to apoptotic response from NCSCs. Interestingly, development of neural crest-derived peripheral neural structures in Nox4−/− mouse appears to be grossly normal, although Nox4−/− embryos were born at a sub-Mendelian ratio and showed delayed over-all development. Specifically, cranial and dorsal root ganglia, derived from NCSCs, were clearly present in Nox4−/− embryo at embryonic days (E) 9.5 and 10.5. These results suggest that Nox4-mediated ROS generation likely plays important role in fate determination and differentiation of NCSCs, but other Nox isozymes play redundant function during embryogenesis.     

Surface Trafficking of APP and BACE in Live Cells

Amyloid‐β (Aβ)‐peptide, the major constituent of the plaques that develop during Alzheimer's disease, is generated via the cleavage of Aβ precursor protein (APP) by β‐site APP‐cleaving enzyme (BACE). Using live‐cell imaging of APP and BACE labeled with pH‐sensitive proteins, we could detect the release events of APP and BACE and their distinct kinetics. We provide kinetic evidence for the cleavage of APP by α‐secretase on the cellular surface after exocytosis. Furthermore, simultaneous dual‐color evanescent field illumination revealed that the two proteins are trafficked to the surface in separate compartments. Perturbing the membrane lipid composition resulted in a reduced frequency of exocytosis and affected BACE more strongly than APP. We propose that surface fusion frequency is a key factor regulating the aggregation of APP and BACE in the same membrane compartment and that this process can be modulated via pharmacological intervention.      

HDAC6 Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation in Renal Epithelial Cells

We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.     

Membrane-type Matrix Metalloproteinase-3 Regulates Neuronal Responsiveness to Myelin through Nogo-66 Receptor 1 Cleavage

Nogo-66 receptor 1 (NgR1) is a glycosylphosphatidylinositol-anchored receptor for myelin-associated inhibitors that restricts plasticity and axonal regrowth in the CNS. NgR1 is cleaved from the cell surface of SH-SY5Y neuroblastoma cells in a metalloproteinase-dependent manner; however, the mechanism and physiological consequence of NgR1 shedding have not been explored. We now demonstrate that NgR1 is shed from multiple populations of primary neurons. Through a loss-of-function approach, we found that membrane-type matrix metalloproteinase-3 (MT3-MMP) regulates endogenous NgR1 shedding in primary neurons. Neuronal knockdown of MT3-MMP resulted in the accumulation of NgR1 at the cell surface and reduced the accumulation of the NgR1 cleavage fragment in medium conditioned by cortical neurons. Recombinant MT1-, MT2-, MT3-, and MT5-MMPs promoted NgR1 shedding from the surface of primary neurons, and this treatment rendered neurons resistant to myelin-associated inhibitors. Introduction of a cleavage-resistant form of NgR1 reconstitutes the neuronal response to these inhibitors, demonstrating that specific metalloproteinases attenuate neuronal responses to myelin in an NgR1-dependent manner.     

CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking

Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking.     

A Neuronal Acetylcholine Receptor Regulates the Balance of Muscle Excitation and Inhibition in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, cholinergic motor neurons stimulate muscle contraction as well as activate GABAergic motor neurons that inhibit contraction of the contralateral muscles. Here, we describe the composition of an ionotropic acetylcholine receptor that is required to maintain excitation of the cholinergic motor neurons. We identified a gain-of-function mutation that leads to spontaneous muscle convulsions. The mutation is in the pore domain of the ACR-2 acetylcholine receptor subunit and is identical to a hyperactivating mutation in the muscle receptor of patients with myasthenia gravis. Screens for suppressors of the convulsion phenotype led to the identification of other receptor subunits. Cell-specific rescue experiments indicate that these subunits function in the cholinergic motor neurons. Expression of these subunits in Xenopus oocytes demonstrates that the functional receptor is comprised of three α-subunits, UNC-38, UNC-63 and ACR-12, and two non–α-subunits, ACR-2 and ACR-3. Although this receptor exhibits a partially overlapping subunit composition with the C. elegans muscle acetylcholine receptor, it shows distinct pharmacology. Recordings from intact animals demonstrate that loss-of-function mutations in acr-2 reduce the excitability of the cholinergic motor neurons. By contrast, the acr-2(gf) mutation leads to a hyperactivation of cholinergic motor neurons and an inactivation of downstream GABAergic motor neurons in a calcium dependent manner. Presumably, this imbalance between excitatory and inhibitory input into muscles leads to convulsions. These data indicate that the ACR-2 receptor is important for the coordinated excitation and inhibition of body muscles underlying sinusoidal movement.     

An Antimicrobial Peptide Regulates Tumor-Associated Macrophage Trafficking via the Chemokine Receptor CCR2, a Model for Tumorigenesis

Background

Tumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human β-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown.

Methodology

The relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out.

Conclusions/Findings

MCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1α (IL-1α), IL-6, IL-8, CCL18, and tumor necrosis factor-α (TNF-α) in macrophages derived from human peripheral blood monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3. Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of tumors.     

Auxin-Mediated Ribosomal Biogenesis Regulates Vacuolar Trafficking in Arabidopsis

In plants, the mechanisms that regulate the transit of vacuolar soluble proteins containing C-terminal and N-terminal vacuolar sorting determinants (VSDs) to the vacuole are largely unknown. In a screen for Arabidopsis thaliana mutants affected in the trafficking of C-terminal VSD containing proteins, we isolated the ribosomal biogenesis mutant rpl4a characterized by its partial secretion of vacuolar targeted proteins and a plethora of developmental phenotypes derived from its aberrant auxin responses. In this study, we show that ribosomal biogenesis can be directly regulated by auxins and that the exogenous application of auxins to wild-type plants results in vacuolar trafficking defects similar to those observed in rpl4a mutants. We propose that the influence of auxin on ribosomal biogenesis acts as a regulatory mechanism for auxin-mediated developmental processes, and we demonstrate the involvement of this regulatory mechanism in the sorting of vacuolar targeted proteins in Arabidopsis.