07-476 Sigma-AldrichAnti-p75NTR (Neurotrophin Receptor) Antibody

Class III β-tubulin (TUBB3)-positive cells from the hair follicle bulge are thought to be neuronal cells derived from a local neural crest stem cell. However, TUBB3 has recently been shown to be expressed in the melanocytic lineage. To evaluate the neural-crest-associated immunophenotype of TUBB3-positive cells from hair follicle bulge explants, we dissected hair follicle bulges out from mouse whisker pads and cultured for 1 month and assessed outgrowing cells by means of immunocytochemistry using the biomarkers TUBB3, nestin, NGFR, SOX9, TYRP1 and laminin. Large amounts of TUBB3-positive cells could be cultured that co-expressed nestin, NGFR, SOX9 and, to a lesser degree, TYRP1, matching a melanoglial phenotype. In addition, a small population of TUBB3-negative but laminin-positive cells was found, which presumably are of glial origin. It can be concluded that cells of melanoglial origin can easily be obtained from hair follicle bulge explants. These cells may be of use in experimental animal or human disease and wound healing models. Notably, the TUBB3-positive cells are of melanoglial rather than neuronal origin.

The communication between vascular endothelial cells (ECs) and pericytes in the microvasculature is fundamental for vascular growth and homeostasis; however, these processes are disrupted by diabetes. Here we show that modulation of p75(NTR) expression in ECs exposed to high glucose activates transcription of miR-503, which negatively affects pericyte function. p75(NTR) activates NF-κB to bind the miR-503 promoter and upregulate miR-503 expression in ECs. NF-κB further induces activation of Rho kinase and shedding of endothelial microparticles carrying miR-503, which transfer miR-503 from ECs to vascular pericytes. The integrin-mediated uptake of miR-503 in the recipient pericytes reduces expression of EFNB2 and VEGFA, resulting in impaired migration and proliferation. We confirm operation of the above mechanisms in mouse models of diabetes, in which EC-derived miR-503 reduces pericyte coverage of capillaries, increased permeability and impaired post-ischaemic angiogenesis in limb muscles. Collectively, our data demonstrate that miR-503 regulates pericyte-endothelial crosstalk in microvascular diabetic complications.

Neural crest cells (NCCs) migrate from different regions along the anterior-posterior axis of the neural tube (NT) to form different structures. Defective NCC development causes congenital neurocristopathies affecting multiple NCC-derived tissues in human. Perturbed Hoxb5 signaling in vagal NCC causes enteric nervous system (ENS) defects. This study aims to further investigate if perturbed Hoxb5 signaling in trunk NCC contributes to defects of other NCC-derived tissues besides the ENS. We perturbed Hoxb5 signaling in NCC from the entire NT, and investigated its impact in the development of tissues derived from these cells in mice. Perturbation of Hoxb5 signaling in these NCC resulted in Sox9 downregulation, NCC apoptosis, hypoplastic sympathetic and dorsal root ganglia, hypopigmentation and ENS defects. Mutant mice with NCC-specific Sox9 deletion also displayed some of these phenotypes. In vitro and in vivo assays indicated that the Sox9 promoter was bound and trans-activated by Hoxb5. In ovo studies further revealed that Sox9 alleviated apoptosis induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Sox9 expression in chick NT. This study demonstrates that Hoxb5 regulates Sox9 expression in NCC and disruption of this signaling causes Sox9 downregulation, NCC apoptosis and multiple NCC-developmental defects. Phenotypes such as ENS deficiency, hypopigmentation and some of the neurological defects are reported in patients with Hirschsprung disease (HSCR). Whether dysregulation of Hoxb5 signaling and early depletion of NCC contribute to ENS defect and other neurocristopathies in HSCR patients deserves further investigation.

The adult human cochlea contains various types of peripheral glial cells that envelop or myelinate the three different domains of the spiral ganglion neurons: the central processes in the cochlear nerve, the cell bodies in the spiral ganglia, and the peripheral processes in the osseous spiral lamina. Little is known about the distribution, lineage separation and maturation of these peripheral glial cells in the human fetal cochlea. In the current study, we observed peripheral glial cells expressing SOX10, SOX9 and S100B as early as 9 weeks of gestation (W9) in all three neuronal domains. We propose that these cells are the common precursor to both mature Schwann cells and satellite glial cells. Additionally, the peripheral glial cells located along the peripheral processes expressed NGFR, indicating a phenotype distinct from the peripheral glial cells located along the central processes. From W12, the spiral ganglion was gradually populated by satellite glial cells in a spatiotemporal gradient. In the cochlear nerve, radial sorting was accomplished by W22 and myelination started prior to myelination of the peripheral processes. The developmental dynamics of the peripheral glial cells in the human fetal cochlea is in support of a neural crest origin. Our study provides the first overview of the distribution and maturation of peripheral glial cells in the human fetal cochlea from W9 to W22.

ApoER2 and the neurotrophin receptors Trk and p75(NTR) are expressed in the CNS and regulate key functional aspects of neurons, including development, survival, and neuronal function. It is known that both ApoER2 and p75(NTR) are processed by metalloproteinases, followed by regulated intramembrane proteolysis. TrkA activation by nerve growth factor (NGF) increases the proteolytic processing of p75(NTR) mediated by ADAM17. Reelin induces the sheeding of ApoER2 ectodomain depending on metalloproteinase activity. However, it is not known if there is a common regulation mechanism for processing these receptors.We found that TrkA activation by NGF in PC12 cells induced ApoER2 processing, which was dependent on TrkA activation and metalloproteinases. NGF-induced ApoER2 proteolysis was independent of mitogen activated protein kinase activity and of phosphatidylinositol-3 kinase activity. In contrast, the basal proteolysis of ApoER2 increased when both kinases were pharmacologically inhibited. The ApoER2 ligand reelin regulated the proteolytic processing of its own receptor but not of p75(NTR). Finally, in primary cortical neurons, which express both ApoER2 and TrkB, we found that the proteolysis of ApoER2 was also regulated by brain-derived growth factor (BDNF).Our results highlight a novel relationship between neurotrophins and the reelin-ApoER2 system, suggesting that these two pathways might be linked to regulate brain development, neuronal survival, and some pathological conditions.

We present a toolbox for the study of molecular interactions occurring between NGF and its receptors. By means of a suitable insertional mutagenesis method we show the insertion of an 8 amino acid tag (A4) into the sequence of NGF and of 12 amino acid tags (A1 and S6) into the sequence of TrkA and P75NTR NGF-receptors. These tags are shortened versions of the acyl and peptidyl carrier proteins; they are here covalently conjugated to the biotin-substituted arm of a coenzyme A (coA) substrate by phosphopantetheinyl transferase enzymes (PPTases). We demonstrate site-specific biotinylation of the purified recombinant tagged neurotrophin, in both the immature proNGF and mature NGF forms. The resulting tagged NGF is fully functional: it can signal and promote PC12 cells differentiation similarly to recombinant wild-type NGF. Furthermore, we show that the insertion of A1 and S6 tags into human TrkA and P75NTR sequences leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Crucially, the two tags are labeled selectively by two different PPTases: this is exploited to reach orthogonal fluorolabeling of the two receptors co-expressed at low density in living cells. We describe the protocols to obtain the enzymatic, site-specific biotinylation of neurotrophins and their receptors as an alternative to their chemical, nonspecific biotinylation. The present strategy has three main advantages: i) it yields precise control of stoichiometry and site of biotin conjugation; ii) the tags used can be functionalized with virtually any small probe that can be carried by coA substrates, besides (and in addition to) biotin; iii) above all it makes possible to image and track interacting molecules at the single-molecule level in living systems.

The stereotyped arrangement of cochlear sensory and supporting cells is critical for auditory function. Our previous studies showed that Muenke syndrome model mice (Fgfr3P244R/+) have hearing loss associated with a supporting cell fate transformation of two Deiters' cells to two pillar cells. We investigated the developmental origins of this transformation and found that two prospective Deiters' cells switch to an outer pillar cell-like fate sequentially between embryonic day 17.5 (E17.5) and postnatal day 3 (P3). Unexpectedly, the Fgfr3P244R/+ hearing loss and supporting cell fate transformation are not rescued by genetically reducing fibroblast growth factor 8 (FGF8), the FGF receptor 3c (FGFR3c) ligand required for pillar cell differentiation. Rather, reducing FGF10, which normally activates FGFR2b or FGFR1b, is sufficient for rescue of cochlear form and function. Accordingly, we found that the P244R mutation changes the specificity of FGFR3b and FGFR3c such that both acquire responsiveness to FGF10. Moreover, Fgf10 heterozygosity does not block the Fgfr3P244R/+ supporting cell fate transformation but instead allows a gradual reversion of fate-switched cells toward the normal phenotype between P5 and at least P14. This study indicates that Deiters' and pillar cells can reversibly switch fates in an FGF-dependent manner over a prolonged period of time. This property might be exploited for the regulation of sensory cell regeneration from support cells.

Diabetic retinopathy is characterised by early blood-retina barrier (BRB) breakdown and neurodegeneration. Diabetes causes imbalance of nerve growth factor (NGF), leading to accumulation of the NGF precursor (proNGF), as well as the NGF receptor, p75 neurotrophin receptor (p75(NTR)), suggesting a possible pathological role of the proNGF-p75(NTR) axis in the diabetic retina. To date, the role of this axis in diabetes-induced retinal inflammation and BRB breakdown has not been explored. We hypothesised that modulating p75(NTR) would prevent diabetes- and proNGF-induced retinal inflammation and BRB breakdown.Diabetes was induced by streptozotocin in wild-type and p75(NTR) knockout (p75KO) mice. After 5 weeks, the expression of inflammatory mediators, ganglion cell loss and BRB breakdown were determined. Cleavage-resistant proNGF was overexpressed in rodent retinas with and without p75(NTR) short hairpin RNA or with pharmacological inhibitors. In vitro, the effects of proNGF were investigated in retinal Müller glial cell line (rMC-1) and primary Müller cells.Deletion of p75(NTR) blunted the diabetes-induced decrease in retinal NGF expression and increases in proNGF, nuclear factor κB (NFκB), p-NFκB and TNF-α. Deletion of p75(NTR) also abrogated diabetes-induced glial fibrillary acidic protein expression, ganglion cell loss and vascular permeability. Inhibited expression or cleavage of p75(NTR) blunted proNGF-induced retinal inflammation and vascular permeability. In vitro, proNGF induced p75(NTR)-dependent production of inflammatory mediators in primary wild-type Müller and rMC-1 cultures, but not in p75KO Müller cells.The proNGF-p75(NTR) axis contributes to retinal inflammation and vascular dysfunction in the rodent diabetic retina. These findings underscore the importance of p75(NTR) as a novel regulator of inflammation and potential therapeutic target in diabetic retinopathy.

Facilitation of nerve growth factor (NGF) signaling by the p75 neurotrophin receptor (p75(NTR)) is critical for neuronal survival and differentiation. However, the interaction between p75(NTR) and TrkA receptors required for this activity is not understood. Here, we report that a specific 29-amino acid peptide derived from the intracellular domain fragment of p75(NTR) interacts with and potentiates binding of NGF to TrkA-expressing cells, leading to increased neurite outgrowth in sympathetic neurons as a result of enhanced Erk1/2 and Akt signaling. An endogenous intracellular domain fragment of p75(NTR) (p75(ICD)) containing these 29 amino acids is produced by regulated proteolysis of the full-length receptor. We demonstrate that generation of this fragment is a requirement for p75(NTR) to facilitate TrkA signaling in neurons and propose that the juxtamembrane region of p75(ICD) acts to cause a conformational change within the extracellular domain of TrkA. This finding provides new insight into the mechanism by which p75(NTR) and TrkA interact to enhance neurotrophic signaling.

To investigate differentially expressed genes in eyecup and retina of the ELOVL4 transgenic mouse, a model of Stargardt-like macular dystrophy (STGD3).We examined gene and protein expression in known pathways relevant to retinal degeneration using PCR arrays, Western blotting, and immunohistochemistry. Investigations were performed on ELOVL4 transgenic mice at 9 months, when 50% of rod (but no cone) photoreceptors had degenerated. Age-matched wild-type littermates served as controls.Significant expression level changes were found in only 17 of the 252 genes examined. Nine were upregulated (Fgf2, Fgfr1, Ntf5, Cbln1, Ngfr, Ntrk1, Trp53, Tlr6, and Herpud1), and eight were downregulated (Ccl22, Ccr3, Il18rap, Nf1, Ccl11, Atf6β, Rpn1, and Serp1). Overexpression of FGF2 was detected at 1 month, before rod loss onset, and was maintained at high levels until cone loss (18 months). By 9 months, FGF2 overexpression was seen in photoreceptor cell bodies. Increased glial fibrillary acidic protein (GFAP) expression due to glial cell reactivity followed the same time course. Levels of NGFR/p75NTR remained invariant. Although present in rod outer segments at 1 month, the macrophage chemoattracting chemokine CCL22 became undetectable by 9 months, a likely consequence of progressive rod outer segment truncation.At a mid-degeneration stage, major changes in gene expression in the ELOVL4 transgenic mouse retina included upregulation of Fgf2 and Fgfr1 and downregulation of Ccl22. Modulation of FGF2 occurred very early, concomitant with an increase in GFAP expression. Future studies will address which factors upstream of Fgf2 could provide potential therapeutic targets to slow photoreceptor degeneration in STGD3.