MAB1406 Sigma-AldrichAnti-Thy-1.1 Antibody, clone OX-7

The prion protein (PrPC) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC between rafts and non-raft membranes. We investigated PrPC/ganglioside relationships and their influence on PrPC localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrPC conformations coexist: in lipid rafts PrPC is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrPSc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrPC conformation and interaction with lipid bilayers, without modifying PrPC distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.

Bone marrow mesenchymal stem cells (BMSCs) transplants have been approved for treating central nervous system (CNS) injuries and diseases; however, their clinical applications are limited. Here, we model the therapeutic potential of dermal papilla cells (DPCs) in vitro. DPCs were isolated from rat vibrissae and characterized by immunocytofluorescence, RT-PCR, and multidifferentiation assays. We examined whether these cells could secrete neurotrophic factors (NTFs) by using cocultures of rat pheochromocytoma cells (PC12) with conditioned medium and ELISA assay. DPCs expressed Sox10, P75, Nestin, Sox9, and differentiated into adipocytes, osteoblasts, smooth muscle cells, and neurons under specific inducing conditions. The DPC-conditioned medium (DPC-CM) induced neuronal differentiation of PC12 cells and promoted neurite outgrowth. Results of ELISA assay showed that compared to BMSCs, DPCs secreted more brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Moreover, we observed that, compared with the total DPC population, sphere-forming DPCs expressed higher levels of Nestin and P75 and secreted greater amounts of GDNF. The DPCs from craniofacial hair follicle papilla may be a new and promising source for treating CNS injuries and diseases.

In neurons, the plasma membrane is functionally separated into several distinct segments. Neurons form these domains by delivering selected components to and by confining them within each segment of the membrane. Although some mechanisms of the delivery are elucidated, that of the confinement is unclear. We show here that 1-oleoyl-2-palmitoyl-phosphatidylcholine (OPPC), a unique molecular species of phospholipids, is concentrated at the protrusion tips of several neuronal culture cells and the presynaptic area of neuronal synapses of the mouse brain. In PC12 cells, NGF-stimulated neuronal differentiation induces a phospholipase A1 activity at the protrusion tips, which co-localizes with the OPPC domain. Inhibition of the phospholipase A1 activity leads to suppression of phospholipid remodeling in the tip membrane and results in disappearance of the OPPC at the tips. In these cells, confinement of dopamine transporter and Gαo proteins to the tip was also disrupted. These findings link the lateral distribution of the molecular species of phospholipids to the formation of functional segments in the plasma membrane of neurons and to the mechanism of protein confinement at the synapse.

This study aimed to evaluate the neuroprotective effect of EPO in the presence of N-methyl-d-aspartate (NMDA)-, trophic factor withdrawal (TFW)-, and tumor necrosis factor-alpha (TNF-α)-induced toxicity on total, small, and large retinal ganglion cells (RGCs).Retinal cells from adult rats were cultured in a medium containing brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), and forskolin. Expression of RGC markers and EPOR was examined using immunocytochemistry. RGCs were classified according to their morphological properties. Cytotoxicity was induced by NMDA, TFW, or TNF-α. RGC survival was assessed by counting thy-1 and neurofilament-l double-positive cells.EPO offered dose-dependent (EC₅₀ = 5.7 ng/mL) protection against NMDA toxicity for small RGCs; protection was not significant for large RGCs. Time-course analysis showed that the presence of EPO either before or after NMDA exposure gave effective protection. For both small and large RGCs undergoing trophic factor withdrawal, EPO at concentrations of 1, 10, or 100 ng/mL improved survival. However, EPO had to be administered soon after the onset of injury to provide effective protection. For TNF-α-induced toxicity, survival of small RGCs was seen only for the highest examined concentration (100 ng/mL) of EPO, whereas large RGCs were protected at concentrations of 1, 10, or 100 ng/mL of EPO. Time-course analysis showed that pretreatment with EPO provided protection only for large RGCs; early post-treatment with EPO protected both small and large RGCs. Inhibitors of signal transduction and activators of transcription such as (STAT)-5, mitogen-activated protein kinases (MAPK)/extracellular-regulated kinase (ERK), and phosphatidyl inositol-3 kinase (PI3K)/Akt impaired the protective effect of EPO on RGCs exposed to different insults.EPO provided neuroprotection to cultured adult rat RGCs; however, the degree of protection varied with the type of toxic insult, RGC subtype, and timing of EPO treatment.

Hypoxia-induced retinal ganglion cell (RGC) apoptosis has been implicated in many optic neuropathies. Insulin-like growth factor-1 (IGF-1) is important in maintaining neuronal survival, proliferation, and differentiation. The purpose of this study is to explore whether IGF-1 can protect RGCs from hypoxia-induced apoptosis and to determine the precise mechanisms that regulate this process.Purified RGC cultures were obtained from the retinas of neonatal Sprague Dawley (SD) rats using a two-step panning method. Primary cultured RGCs were cultured in a closed hypoxic chamber (5% O2, 5% CO2, and 90% N2) for 12 h with or without IGF-1. The degree of apoptosis in the RGCs was detected by caspase-3 expression and TUNEL and JC-1 staining assays. The expression and phosphorylation of protein kinase B (Akt), p44/42 mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase-1/2 [Erk-1/2]), Bad, and caspase-3 was investigated with immunoblot analysis.Hypoxia induces apoptosis in primary Sprague Dawley rat RGCs, as detected by caspase-3 expression and TUNEL and JC-1 staining assays, and that IGF-1 treatment could significantly reduce this effect in RGCs. Interestingly, pretreatment of RGCs with AG1024 (an IGF-1 inhibitor), U0126 (an Erk-1/2 inhibitor), and LY294002 (an Akt inhibitor) markedly attenuated the effects of IGF-1 treatment. Furthermore, western blot analysis suggested that the Erk-1/2 and Akt signaling pathways play a role in the protective effects of IGF-1 on RGCs exposed to hypoxia.These data indicate that IGF-1 can protect primary cultured RGCs against hypoxia-induced apoptosis via the Erk-1/2 and Akt signaling pathways, suggesting that IGF-1 treatment is a potential therapeutic approach for treating hypoxia-induced neurodegeneration in the retina.

The membrane expression and gene promoter of the glycosylphosphatidylinositol (GPI)-anchored protein Thy1 have been widely used to examine the morphology and distribution of retinal ganglion cells in normal eyes and disease models. However, it is not known how adult mammalian retinal neurons use Thy1. Because Thy1 is not a membrane-spanning protein and, instead, complexes with structural and signaling proteins in other tissues, the aim of this study was to find protein partners of retinal Thy1.Coimmunoprecipitation, immunohistochemistry, confocal imaging, and patch-clamp recording were used to test for association of Thy1 and HCN4, a cation channel subunit, in adult rat retina.Hyperpolarization of cells immunopanned by an anti-Thy1 antibody activated HCN channels. Confocal imaging showed that individual somata in the ganglion cell layer bound antibodies against Thy1 and HCN4, that the majority of these bindings colocalized, and that some of the immunopositive cells also bound antibody against a ganglion cell marker (Brn3a). Consistent with these results, Thy1 and HCN4 were coimmunoprecipitated by magnetic beads coated with either anti-Thy1 antibody or anti-HCN4 antibody. In control experiments, beads coated with these antibodies did not immunoprecipitate a photoreceptor rim protein (ABCR) and uncoated beads did not immunoprecipitate either Thy1 or HCN4.This is the first report that Thy1 colocalizes and coimmunoprecipitates with a membrane-spanning protein in retina, that Thy1 complexes with an ion channel protein in any tissue, and that a GPI-anchored protein associates with an HCN channel subunit protein.

Sclerostin is a soluble inhibitor of osteoblast function. Sclerostin is downregulated by the parathyroid hormone (PTH). Here, it was investigated whether sclerostin levels are influenced by intact (i) PTH and whether sclerostin is associated with bone turnover, microarchitecture and mass in dialysis patients.

Olfactory ensheathing cells (OEC) have been shown to stimulate regeneration, myelination and functional recovery in different spinal cord injury models. However, recent reports from several laboratories have challenged this treatment strategy. The discrepancy in results could be attributed to many factors including variations in culture protocols. The present study investigates whether the differences in culture preparation could influence neuroprotective and growth-promoting effects of OEC after transplantation into the injured spinal cord. Primary OEC cultures were purified using method of differential cell adhesion (a-OEC) or separated with immunomagnetic beads (b-OEC). After cervical C4 hemisection in adult rats, short-term (3 weeks) or long-term (7 weeks) cultured OEC were transplanted into the lateral funiculus at 1mm rostral and caudal to the transection site. At 3-8 weeks after transplantation, labeled OEC were mainly found in the injection sites and in the trauma zone. Short-term cultured a-OEC supported regrowth of rubrospinal, raphaespinal and CGRP-positive fibers, and attenuated retrograde degeneration in the red nucleus. Short-term cultured b-OEC failed to promote axonal regrowth but increased the density of rubrospinal axons within the dorsolateral funiculus and provided significant neuroprotection for axotomized rubrospinal neurons. In addition, short-term cultured OEC attenuated sprouting of rubrospinal terminals. In contrast, long-term cultured OEC neither enhanced axonal growth nor prevented retrograde cell death. The results suggest that the age of OEC in culture and the method of cell purification could affect the efficacy of OEC to support neuronal survival and regeneration after spinal cord injury. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Optineurin is a Golgi complex-associated ubiquitous protein with high expression levels in retinal ganglion cells (RGCs). Mutations in optineurin have been observed in rare hereditary cases of primary open-angle glaucoma and in amyotrophic lateral sclerosis. We explored the possibility that optineurin deficiency will compromise neuronal exocytosis leading to a diminished secretion of neurotrophic factors that are critically required for neuronal survival. To this end, we used RNA interference to induce depletion of optineurin in RGC-5 cells derived from retinal neurons. SiRNA specific for optineurin was transiently transfected. Moreover, a stable cell line with constitutive optineurin deficiency (RGC-5 pSilencer OPTN) was generated. In addition, we investigated the subcellular localization of optineurin in primary RGCs in retinal cell cultures isolated from eyes of mature mice. In RGC-5 cells, optineurin localized to the periphery of the Golgi complex and was observed in vesicular structures throughout the cytoplasm and close to the plasma membrane. A comparable Golgi-associated localization of optineurin was observed in cultured primary RGCs that were identified by TUJ1 labeling. Optineurin deficiency caused a marked increase in the number of RGC-5 cells with fragmented Golgi complex. RGC-5 pSilencer OPTN with stable optineurin deficiency showed a pronounced increase in the number of cells undergoing apoptotic cell death. Furthermore, the amounts of secreted neurotrophin-3 (NT-3) and ciliary neurotrophic factor were significantly lower in culture medium of RGC-5 pSilencer OPTN cells when compared to controls. Adding exogenous NT-3 to the culture medium to achieve amounts seen in control cultures completely prevented the increase in apoptotic cell death. We propose that lack of neurotrophic support due to impaired secretion of neurotrophic proteins is a critical factor that causes or contributes to RGC or motor neuron death in patients with mutated optineurin.