ShcC proteins: Brain aging and beyond

To date, most studies of Shc family of signaling adaptor proteins have been focused on the near-ubiquitously expressed ShcA, indicating its relevance to age-related diseases and longevity. Although the role of the neuronal ShcC protein is much less investigated, accumulated evidence suggests its importance for neuroprotection against such aging-associated conditions as brain ischemia and oxidative stress. Here, we summarize more than decade of studies on the ShcC expression and function in normal brain, age-related brain pathologies and immune disorders with a focus on the interactions of ShcC with signaling proteins/pathways, and the possible implications of these interactions for changes associated with aging.     

Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn

Aim:

In vascular strips, the adjacent endothelial cells modulate the contraction of vascular smooth muscle cells (VSMCs) induced by sphingosylphosphorylcholine (SPC) through nitric oxide (NO). The aim of this study was to elucidate the mechanisms by which vascular endothelial cells (VECs) reduce the SPC-induced contraction of VSMCs in a co-culture system.

Methods:

Human umbilical VECs and VSMCs were co-cultured. The VECs were transfected with integrin β4- or Fyn-specific siRNA. The areas of VSMCs that are involved in cell contractility were quantified using the Leica confocal software and collagen contractility assay. The production of NO in VECs was measured in the cell supernatants using NO Detection Kit. The levels of integrin β4 and Fyn in VECs and the levels of Rho kinase (ROCK) in VSMC were detected using immunofluorescence assays or Western blots.

Results:

Co-culture with VECs reduced the contraction of VSMCs induced by SPC (30 μmol/L). The down-regulation of integrin β4 or Fyn in VECs by the specific siRNA (20 nmol/L) was able to counteract the effects of VECs on the SPC-induced VSMC contractions. Furthermore, the integrin β4-specific siRNA (20 and 40 nmol/L) significantly reduced the level of Fyn protein and the production of NO in VECs, while increased the level of ROCK in VSMCs that had been stimulated by SPC.

Conclusion:

The VECs reduced the SPC-induced contraction of VSMCs in the co-culture system through integrin β4 and Fyn proteins. In this process, NO may be the factor downstream of integrin β4 in VECs, while ROCK may be the key protein regulating the contraction of VSMCs.     

Downregulation of the microtubule associated protein Tau impairs process outgrowth and myelin basic protein mRNA transport in oligodendrocytes

Oligodendrocytes, the myelin forming cells of the CNS, are characterized by their numerous membranous extensions, which enwrap neuronal axons and form myelin sheaths. During differentiation oligodendrocytes pass different morphological stages, downregulate the expression of the proteoglycan NG2, and acquire major myelin specific proteins, such as myelin basic proteins (MBP) and proteolipid protein. MBP mRNA is transported in RNA granules along the microtubules (MTs) to the periphery and translated locally. MTs participate in the elaboration and stabilization of the myelin forming extensions and are essential for cellular sorting processes. Their dynamic properties are regulated by microtubule associated proteins (MAPs). The MAP tau is present in oligodendrocytes and involved in the regulation and stabilization of the MT network. To further elucidate the functional significance of tau in oligodendrocytes, we have downregulated tau by siRNA technology and studied the effects on cell differentiation and neuron‐glia contact formation. The data show that tau knockdown impairs process outgrowth and leads to a decrease in MBP expression. Furthermore, MBP mRNA transport to distant cellular extensions is impaired and cells remain in the NG2 stage. In myelinating cocultures with dorsal root ganglion neurons, oligodendrocyte precursor cells after tau miR RNA lentiviral knockdown develop into NG2 positive cells with very long and thin processes, contacting axons loosely, but fail to form internodes. This demonstrates that tau is important for MBP mRNA transport and involved in process formation. The disturbance of the balance of tau leads to abnormalities in oligodendrocyte differentiation, neuron‐glia contact formation and the early myelination process. GLIA 2015;63:1621–1635     

Role of the Fyn −93A>G polymorphism (rs706895) in acute rejection after liver transplantation

The tyrosine kinase Fyn phosphorylates tyrosine residues on key targets involved in early T-cell signal transduction. T-cell signal transduction is one essential step for acute transplant rejection. The aim of this study was to evaluate the association of Fyn −93A>G single nucleotide polymorphism (SNP) (rs706895) with the susceptibility to acute rejection episodes in liver transplantation. In total, 72 liver transplant recipients with one biopsy proven acute rejection (S-BPAR), 56 with multiple BPAR (M-BPAR), 105 without BPAR (No-BPAR), and 145 healthy controls were enrolled in this case-control study. The SNP was genotyped by polymerase chain reaction–allele specific restriction enzyme analysis (PCR–ASRA) and was analyzed for a recessive and a dominant model. The Fyn −93G allele exhibits in healthy controls a statistically significant lower frequency than in liver recipients (18% vs. 24%; p = 0.046) or in liver recipients with BPAR (18% vs. 27%; p = 0.017). However, the genotype and allele frequencies of the Fyn −93A>G SNP demonstrate no significant differences between recipients with acute rejection episodes (S-BPAR and M-BPAR) and No-BPAR recipients. Thus our results provide no evidence that the Fyn −93A>G SNP contributes to the susceptibility to acute liver transplant rejection in a Caucasian population.     

Src family kinases and lipid mediators in control of allergic inflammation

Summary:  The Src family kinases Fyn and Lyn are important modulators of the molecular events initiated by engagement of the high-affinity IgE receptor (FcɛRI). These kinases control many of the early signaling events and initiate the production of several lipid metabolites that have an important role in regulating mast cell responses. The intracellular level of phosphatidylinositol (3,4,5)-trisphosphate (PIP₃), which is produced by phosphatidylinositol 3-OH kinase, plays an important role in determining the extent of a mast cells response to a stimulus. Enhanced levels lead to a hyperdegranulating phenotype (as seen in SHIP-1^−/− and Lyn^−/− mast cells), whereas decreased levels cause hypodegranulation (as seen in Fyn^−/− mast cells). Downregulation of mast cell phosphatase and tensin homologue deleted on chromosone 10 expression, a phosphatase that reduces cellular levels of PIP₃, caused constitutive cytokine production, demonstrating that this response is particularly sensitive to PIP₃ levels. Lyn and Fyn are also intimately linked to other lipid kinases, like sphingosine kinases (SphK). By producing sphingosine-1-phosphate (S1P), SphKs contribute to mast cell chemotaxis and degranulation. In vivo studies now reveal that circulating S1P as well as that found within the mast cell is important in determining mast cell responsiveness. These studies demonstrate the connection between Src protein tyrosine kinases and lipid second messengers that control mast cell function and allergic responses.     

Post-synaptic density-93 mediates tyrosine-phosphorylation of the N-methyl-D-aspartate receptors

Src family protein kinases (SFKs) -mediated tyrosine-phosphorylation regulates N-methyl-d-aspartate (NMDA) receptor synaptic function. Some members of the membrane-associated guanylate kinase (MAGUK) family of proteins bind to both SFKs and NMDA receptors, but it is unclear whether the MAGUK family of proteins is required for SFKs-mediated tyrosine-phosphorylation of the NMDA receptors. Here, we showed by co-immunoprecipitation that post-synaptic density (PSD) -93, a member of the MAGUK family of proteins, interacts with the NMDA receptor subunits NR2A and NR2B as well as with Fyn, a member of the SFKs, in mouse cerebral cortex. Using a biochemical fractionation approach to isolate subcellular compartments revealed that the expression of Fyn, but not of other members of the SFKs (Lyn, Src, and Yes), was significantly decreased in synaptosomal membrane fractions derived from the cerebral cortex of PSD-93 knockout mice. Interestingly, we found that PSD-93 disruption causes reduction of tyrosine-phosphorylated NR2A and NR2B in the same fraction. Moreover, PSD-93 deletion markedly blocked the SFKs-mediated increase in tyrosine-phosphorylated NR2A and NR2B through the protein kinase C pathway after induction with 4-phorbol 12-myristate 13-acetate in cultured cortical neurons. Our findings indicate that PSD-93 appears to mediate tyrosine-phosphorylation of the NMDA receptors and synaptic localization of Fyn.     

NO引起Fyn酪氨酸激酶巯基亚硝基化的体外研究

目的 探讨离体情况下一氧化氮（NO）对酪氨酸蛋白激酶Fyn蛋白巯基亚硝基化的影响.方法 HEK293细胞转染pcDNA3.1-Fyn质粒后,用S-亚硝基谷胱甘肽（GSNO）刺激,或共转染神经型一氧化氮合酶（nNOS）-Fyn质粒,再用A23187和7-硝基吲唑（7-NI）刺激,生物素转换法（biotin-switch method）和Western blotting检测Fyn巯基亚硝基化情况.结果 GSNO以及 nNOS在A23187存在的情况下,都能够引起Fyn巯基亚硝基化.结论外源性和内源性NO都能够使Fyn巯基亚硝基化.