Neurotensin and the neurotensin receptor-3 in microglial cells

Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. The neuropeptide neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol-3 kinase (PI3 kinase) and mitogen-activated protein (MAP) kinases pathways. The effect of neurotensin on cell migration was blocked by the neurotensin receptor-3 propeptide, a selective ligand of this receptor. The type I neurotensin receptor-3 was the only known neurotensin receptor expressed in these microglial cells, and its activation led to the phosphorylation of both extracellular signaling-regulated kinases Erk1/2 and Akt. Furthermore, the effect of neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by neurotensin were totally blocked by selective inhibitors of MAP kinases or PI3 kinase pathways. In the murine microglial cell line N11, the neurotensin receptor-3 is also the only neurotensin receptor expressed, and its activation by neurotensin leads to the phosphorylation of both Erk1/2 and Akt. In these cells, neurotensin induces the gene expression of several cytokines/chemokines, including MIP-2, MCP-1, interleukin-1beta and tumor necrosis factor-alpha. This induction is dependent on both protein kinases pathways. We observed that the effect of neurotensin on the cytokine/chemokine expression is also inhibited by the neurotensin receptor-3 propeptide. This is the demonstration that the neurotensin receptor-3 is functional and mediates both the migratory action of neurotensin and its induction of chemokines/cytokines expression.     

TH1902, a new docetaxel-peptide conjugate for the treatment of sortilin-positive triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of cancers which lacks the expression and/or amplification of targetable biomarkers (ie, estrogen receptor, progestrogen receptor, and human epidermal growth factor receptor 2), and is often associated with the worse disease-specific outcomes than other breast cancer subtypes. Here, we report that high expression of the sortilin (SORT1) receptor correlates with the decreased survival in TNBC patients, and more importantly in those bearing lymph node metastases. By exploiting SORT1 function in ligand internalization, a new anticancer treatment strategy was designed to target SORT1-positive TNBC-derived cells both in vitro and in two in vivo tumor xenografts models. A peptide (TH19P01), which requires SORT1 for internalization and to which many anticancer drugs could be conjugated, was developed. In vitro, while the TH19P01 peptide itself did not exert any antiproliferative or apoptotic effects, the docetaxel-TH19P01 conjugate (TH1902) exerted potent antiproliferative and antimigratory activities when tested on TNBC-derived MDA-MB-231 cells. TH1902 triggered faster and more potent apoptotic cell death than did unconjugated docetaxel. The apoptotic and antimigratory effects of TH1902 were both reversed by two SORT1 ligands, neurotensin and progranulin, and on siRNA-mediated silencing of SORT1. TH1902 also altered microtubule polymerization and triggered the downregulation of the anti-apoptotic Bcl-xL biomarker. In vivo, both i.p. and i.v. administrations of TH1902 led to greater tumor regression in two MDA-MB-231 and HCC-70 murine xenograft models than did docetaxel, without inducing neutropenia. Altogether, the data demonstrates the high in vivo efficacy and safety of TH1902 against TNBC through a SORT1 receptor-mediated mechanism. This property allows for selective treatment of SORT1-positive TNBC and makes TH1902 a promising avenue for personalized therapy with the potential of improving the therapeutic window of cytotoxic anticancer drugs such as docetaxel.     

炎性痛致大鼠脊髓后角ProBDNF及其受体的表达变化

目的:探讨完全弗氏佐剂(complete Freund’s Adjuvant,CFA)致炎性疼痛后大鼠脊髓后角内ProBDNF及受体P75NTR和Sortilin的表达变化及意义。方法:大鼠随机分为正常组和实验组,实验组大鼠左侧足底皮下注射CFA和生理盐水混合溶剂100μl,建立炎性疼痛模型,实验组包括注射CFA后6 h、1、3、7和14 d组。采用Von Frey纤维测定不同时间点大鼠机械缩足阈值(pawwithdrawal threshold,PWT)的变化;采用免疫组织化学方法检测ProBDNF及P75NTR、Sortilin在脊髓后角的表达变化。结果:足底注射CFA后1 h PWT即下降,并在6 h后达到最低值,3 d后逐渐上调,至14 d仍低于基础值。ProBDNF在正常脊髓后角可见阳性表达;注射CFA后1 d注射侧脊髓后角较对侧其ProBDNF的表达明显上调,上调持续到7 d左右,至14 d逐渐恢复。正常大鼠脊髓后角P75NTR有较弱表达,主要集中在后角I、II层纤维;注射CFA后6 h开始,注射侧脊髓后角内P75NTR的表达明显上调,III-V层的阳性产物也逐渐增加,这种上调持续到7 d左右达高峰,至14 d逐渐下降。Sortilin在正常脊髓后角浅层仅有较弱阳性产物表达,注射CFA后不同时间点脊髓后角Sortilin的表达无明显差异。结论:CFA能诱导大鼠产生为期2周以上的炎性痛病程;脊髓后角ProBDNF和P75NTR的表达上调可能与炎性疼痛中外周痛觉信号的传导和中枢敏化有关。     

Organ of Corti explants direct tonotopically graded morphology of spiral ganglion neurons in vitro

The spiral ganglion is a compelling model system to examine how morphological form contributes to sensory function. While the ganglion is composed mainly of a single class of type I neurons that make simple one‐to‐one connections with inner hair cell sensory receptors, it has an elaborate overall morphological design. Specific features, such as soma size and axon outgrowth, are graded along the spiral contour of the cochlea. To begin to understand the interplay between different regulators of neuronal morphology, we cocultured neuron explants with peripheral target tissues removed from distinct cochlear locations. Interestingly, these “hair cell microisolates” were capable of both increasing and decreasing neuronal somata size, without adversely affecting survival. Moreover, axon characteristics elaborated de novo by the primary afferents in culture were systematically regulated by the sensory endorgan. Apparent peripheral nervous system (PNS)‐like and central nervous system (CNS)‐like axonal profiles were established in our cocultures allowing an analysis of putative PNS/CNS axon length ratios. As predicted from the in vivo organization, PNS‐like axon bundles elaborated by apical cocultures were longer than their basal counterparts and this phenotype was methodically altered when neuron explants were cocultured with microisolates from disparate cochlear regions. Thus, location‐dependent signals within the organ of Corti may set the “address” of neurons within the spiral ganglion, allowing them to elaborate the appropriate tonotopically associated morphological features in order to carry out their signaling function. J. Comp. Neurol. 524:2182–2207, 2016. © 2015 Wiley Periodicals, Inc.     

Comprehensive genetic testing can save lives in hereditary hearing loss

The newly‐synthesized lysosomal enzymes travel to the trans‐Golgi network (TGN) and are then driven to the acidic organelle. While the best‐known pathway for TGN‐to‐endosome transport is the delivery of soluble hydrolases by the M6P receptors (MPRs), additional pathways do exist, as showed by the identification of two alternative receptors: LIMP‐2, implicated in the delivery of β‐glucocerebrosidase; and sortilin, involved in the transport of the sphingolipid activator proteins prosaposin and GM2AP, acid sphingomyelinase and cathepsins D and H. Disruption of the intracellular transport and delivery pathways to the lysosomes may result in lysosomal dysfunction, predictably leading to a range of clinical manifestations of lysosomal storage diseases. However, for a great percentage of patients presenting such manifestations, no condition is successfully diagnosed. To analyse if, in this group, phenotypes could be determined by impairments in the known M6P‐independent receptors, we screened the genes that encode for LIMP‐2 and sortilin. No pathogenic mutations were identified. Other approaches will be needed to clarify whether sortilin dysfunction may cause disease.     

人脑出血血肿周围组织中神经生长因子前体-神经营养因子受体p75-sortilin通路与细胞凋亡

This study demonstrated that brain areas surrounding the site of hematoma following intracerebral hemorrhage are characterized by significantly increased apoptosis and expression of neurotrophin receptor p75 and sortilin. However, as detected by terminal deoxynucleotidyl transferase dUTP nick end labeling and immunohistochemical staining, there was no significant change in nerve growth factor precursor expression levels. The appearance of neurotrophin receptor p75 expressing cells was positively correlated with cells that were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling. These findings confirm that the nerve growth factor precursor-neurotrophin receptor p75-sortilin heterotrimeric complex-mediated apoptosis pathway may play an important role in cellular apoptosis following intracerebral hemorrhage.     

神经生长因子p75受体与sortilin在皮肤及瘢痕成纤维细胞中的表达**

背景：既往研究发现神经生长因子在创面愈合过程中发挥重要作用,但对于成纤维细胞中神经生长因子低亲和力受体 p75及 sortilin 的研究较少,p75及 sortilin 在瘢痕组织成纤维细胞及正常皮肤组织成纤维细胞中的表达量是否有差异目前未见报道。目的：比较神经生长因子低亲和力受体 p75及 soritilin 在瘢痕疙瘩成纤维细胞及正常皮肤成纤维细胞的中表达的差异。方法：体外培养瘢痕疙瘩成纤维细胞及正常皮肤成纤维细胞,并以人永生化上皮细胞 HaCaT 为阳性对照。通过实时定量 PCR 在 mRNA 水平检测 p75及 sortilin 在瘢痕疙瘩成纤维细胞及正常皮肤成纤维细胞的表达及差异,进一步通过 Western-blot 及细胞免疫化学方法观察 p75神经营养因子受体及 sortilin 在蛋白水平的表达及差异。结果与结论：实时定量 PCR 及 Western blot 结果可见,在 mRNA 及蛋白水平瘢痕疙瘩成纤维细胞及正常皮肤成纤维细胞中 p75,sortilin 均呈阳性表达,其中瘢痕疙瘩成纤维细胞及正常皮肤成纤维细胞中 p75及 sortilin在 mRNA 及蛋白水平表达量明显少于 HaCaT,且 p75在瘢痕疙瘩成纤维细胞及正常皮肤成纤维细胞中表达差异无显著性意义,sortilin 表达量在瘢痕疙瘩成纤维细胞中明显低于正常皮肤成纤维细胞(P <0.05)。免疫细胞化学结果显示 p75及 sortilin 在瘢痕疙瘩成纤维细胞及正常皮肤成纤维细胞中的表达部位均位于胞膜及胞浆。由于神经生长因子前体与 p75受体高亲和力结合并在 sortilin 协助下发挥促进细胞凋亡的作用,sortilin在瘢痕成纤维细胞的表达明显低于正常皮肤成纤维细胞,可能与其高增殖状态有关,该结果为病理性瘢痕的防治提供了新的靶点。