Cultured OEC versus cultured SC and versus native OEC
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ABSTRACT: This microarray study aimed at identifying the differences in the global gene expression growth program of adult cultured olfactory ensheathing cells (cOEC) (from the olfactory bulb) versus adult cultured Schwann cells (SC) (from the sciatic nerve) and versus adult OEC directly dissected from the olfactory nerve layer (nOEC). The aim of the comparison between cOEC and SC is to define intrinsic molecular differences that distinguish cOEC from SC (both cell types support neuronal regeneration). The aim of the comparison between cOEC and nOEC is to determine the transcriptional responses that are induced in OEC during culturing. Three-condition experiment: cOEC vs. SC, cOEC vs. nOEC, SC vs. nOEC. Biological replicates: 3 cOEC-SC replicates, 3 cOEC-nOEC replicates, 3 SC-nOEC replicates. A factorial design was used consisting of direct comparisons between the three cell types (Glonek and Solomon, 2004 (PMID 14744830).
Project description:This microarray study aimed at identifying the differences in the global gene expression growth program of adult cultured olfactory ensheathing cells (cOEC) (from the olfactory bulb) versus adult cultured Schwann cells (SC) (from the sciatic nerve) and versus adult OEC directly dissected from the olfactory nerve layer (nOEC). The aim of the comparison between cOEC and SC is to define intrinsic molecular differences that distinguish cOEC from SC (both cell types support neuronal regeneration). The aim of the comparison between cOEC and nOEC is to determine the transcriptional responses that are induced in OEC during culturing.
Project description:Olfactory ensheathing cells (OECs) are neural crest-derived glia that ensheath bundles of olfactory axons from their peripheral origins in the olfactory epithelium to their central targets in the olfactory bulb. We took an unbiased laser microdissection and differential RNA-seq approach, validated by in situ hybridisation, to identify candidate molecular mechanisms underlying mouse OEC development and differences with the neural crest-derived Schwann cells developing on other peripheral nerves. We identified 25 novel markers for developing OECs in the olfactory mucosa and/or the olfactory nerve layer surrounding the olfactory bulb, of which 15 were OEC-specific, i.e., not expressed by Schwann cells. One pan-OEC-specific gene, Ptprz1, encodes a receptor-like tyrosine phosphatase that blocks oligodendrocyte differentiation. Mutant analysis suggests Ptprz1 may also act as a brake on OEC differentiation, and that its loss disrupts olfactory axon targeting. Overall, our results provide new insights into OEC development and the diversification of neural crest-derived glia.
Project description:Olfactory ensheathing cells (OECs) are the only glial cells that support the olfactory sensory neurons which undergo adult neurogenesis and continually project their axons to glomeruli in the olfactory bulbs. We used single cell RNA sequencing to study the gene expression programs of OECs and to determine the diversity of purified OECs previously shown to promote spinal cord injury repair. Our analyses revealed five subtypes of OECs, each expressing unique marker genes and pathways indicative of progenitor, axonal regeneration, migration, or microglia-like functions. As expected, we found substantial overlap of OEC genes with those of Schwann cells, but also with astrocytes, oligodendrocytes and microglia. We experimentally confirmed the classic marker genes of the OEC subtypes and provide evidence that Reelin and Connective Tissue Growth Factors are secreted by multiple OEC subtypes. Our results support that adult OECs are a hybrid glia including some with progenitor characteristics and that they likely carry out diverse functions related to injury repair and axonal regeneration.
Project description:Olfactory ensheathing cells are one of the few central nervous system regenerative cells discovered so far. It is characterized by its lifelong nerve regeneration function, and it can also release a variety of neurotrophic factors and neural adhesion molecules. It is considered to be the glial cell with the strongest myelination ability. Olfactory ensheathing cells and Schwann cells have phenotypes in common, they can promote axon regeneration(R. Doucette, 1995). Olfactory ensheathing cells have the characteristics of Schwann cells and astrocytes, but the overall performance tends to be the former, which has two unique characteristics. First, it exists not only in the peripheral nerves (Schwann cells), but also in the central nervous system (astroglia); second, the olfactory mucosa has the ability to regenerate life-long, including human olfactory ensheathing cells(J. C. Bartolomei and C. A. Greer, 2000). Regeneration is a process in which olfactory ensheathing cells participate in efficient regulation, although the specific mechanism is not yet clear. Olfactory ensheathing cells are different from astrocytes and Schwann cells, but at the same time have the characteristics of these two cells(S. C. Barnett, 2004), like Schwann cells help axon growth, but more than Schwann cells It can make axons grow long distances, that is, it has stronger migration(A. Ramon-Cueto et al., 1998); there are also astrocytes that have a nutritional effect on the survival of neurons and the growth of axons, but olfactory ensheathing cells can also wrap neurons forms myelin sheath to support the growth of nerve processes(R. Devon and R. Doucette, 1992; J. Gu et al., 2019). There are two characteristics that make olfactory ensheathing cells the best choice for the treatment of neurological diseases(S. C. Chiu et al., 2009; J. Kim et al., 2018; M. Abdel-Rahman et al., 2018). Olfactory ensheathing cells are gradually used to treat spinal cord injuries and have shown amazing effects(J. C. Bartolomei and C. A. Greer, 2000; K. J. Liu et al., 2010; R. Yao et al., 2018). Olfactory ensheathing cells that have been used in research are usually derived from the olfactory bulb(E. H. Franssen et al., 2007), but it is easier to obtain olfactory ensheathing cells from the olfactory mucosa in clinical practice(M. Ryszard et al., 2006), so the difference between the olfactory ensheathing cells from the olfactory bulb and the olfactory mucosa There are more and more studies(B. M. U. et al., 2007), and previous studies have shown that they not only have many similar functions, but also have many differences(M. W. Richter et al., 2005; L. Wang et al., 2014; K. E. Smith et al., 2020). Because olfactory ensheathing cells derived from the olfactory bulb are not easy to obtain, olfactory ensheathing cells derived from the olfactory mucosa have become the focus of attention. Although we know that olfactory ensheathing cells from two sources have nerve repair functions, it is not clear why the two different sources of olfactory ensheathing cells have different therapeutic effects. Nicolas G. once studied that the genetic difference between the two cells and found that there are many genes related to wound repair and nerve regeneration(G. Nicolas et al., 2010). We have reason to guess that olfactory ensheathing cells from these two sources will also have a large difference in protein level. Our research group wants to use the current mature transcriptome and proteomic sequencing technologies to explore the difference between olfactory ensheathing cells from the olfactory bulb and olfactory mucosa, and explain why the two sources of olfactory ensheathing cells shows different therapeutic effects, hope to provide a new theoretical basis for future clinical treatment.
Project description:Peripheral nerve injuries due to physical insults or chronic diseases are quite common, yet no pharmacological therapies are available for the effective repair of injured nerves. The slow growth rate of adult nerves and insufficient access to growth factors pose major hurdles in timely reinnervating target tissues and restoring functions after nerve injuries. A better understanding of the molecular changes that occur during the immediate regenerative reprogramming of neurons, stated here as in vivo priming, following nerve injury may reveal ideal candidates for future therapies. Hence, molecular profiling of neuronal soma within the first week of nerve injury has been the gold standard for revealing molecular candidates critical for nerve regeneration. A complementary in vitro regenerative priming approach was recently shown to induce enhanced outgrowth in adult sensory neurons. In this work, we exploited the in vitro priming model to reveal novel candidates for adult nerve regeneration. We performed the whole tissue proteomics analysis of in vitro primed DRGs and compared their molecular profile with that of the in vivo primed, and control DRGs. Through this approach, we identified several commonly and uniquely altered molecules in the in vitro and in vivo primed DRGs that have the potential to modulate adult nerve regrowth. We further validated the growth inducing potential of mesencephalic astrocyte-derived neurotrophic factor (MANF), one of the hits identified in our proteomics analysis, in primary adult sensory neurons. Overall, this study showed that in vitro priming partially reproduces the molecular features in in vivo primed adult sensory neurons. The shortlisted candidates presented here from the two priming approaches may serve as potential therapeutic targets for adult nerve regeneration.
Project description:The remarkable plasticity of Schwann cells (SCs) is essential for nerve regeneration but also contributes to neuropathies and cancer progression. It has not yet been investigated whether the adaptive potential of SCs is manifested in stromal, tumor associated SCs characteristically found within a benign subtype of neuroblastic tumors (NBTs). We here performed transcriptome profiling of human NBTs, rich and poor in SC stroma, as well as human injured nerves, rich in repair SCs, revealing that stromal SCs exhibit a repair SC characteristic gene expression signature. In turn, primary repair SCs had a pro-differentiating and anti-proliferative effect on NBT cell lines after direct and trans-well co-culture. Within the pool of secreted stromal/repair SC factors, we identified EGFL8, a matricellular protein with so far undescribed function, to induce neuronal differentiation of aggressive NBT cells. EGFL8 expression further correlated with favorable tumor stage and increased patient survival. Our findings suggest that stromal SCs exert nerve repair associated functions in the tumor-environment and underline the therapeutic value of SC-derived factors for aggressive, SC stroma-poor NBTs.
Project description:An insulating myelin sheath ensures saltatory conduction of mechanosensory A afferents. Myelin damage results in the electrical instability of A fibers and the ability to generate pain in response to light touch/pressure (mechanical allodynia). We have hypothesized and then established that the release of T cell epitopes of myelin basic protein (MBP) enables nociceptive circuitry in myelinated fibers. Thus, mass spectrometry analysis of the rat sciatic nerve proteome followed by bioinformatics examination of the datasets revealed a loss of MBP and activation of T-helper cell signaling in the nerves undergoing chronic constriction injury (CCI). Matrix metalloproteinase-9 (MMP-9) proteolysis resulted in the MBP digest peptides, including the MBP84-104 and MBP68-86 regions, which exhibit prominent immunogenic epitopes. Myelin-forming Schwann cells and paranodal areas accumulated MHCII, MMP-9 and the degraded MBP at the sciatic nerve injury site. Administration of the immunodominant MBP84-104 and MBP68-86 peptides but not of the control peptides in a naïve rat sciatic nerve produced robust mechanical allodynia. Allodynia was accompanied by the T cell infiltration and an increase in MHCII, IL-17A and TNF- levels at the nerve injection site and the segmental ganglia. The pro-nociceptive activity of the synthetic MBP84-104 diminished in athymic nude rats lacking T cells. SB-3CT, an antagonist of MMP-9, inhibited mechanical allodynia, neuroinflammation and spinal sensitization after CCI. Collectively, our novel data implicate, for the first time, MMP-mediated cleavage of MBP and the resulting MBP digest fragments as a major cause of neuropathic pain. Gene extression profiling of total RNAs extracted from rat sciatic nerves, dorsal root ganglion and spinal cords after MBP84-104 peptide injection
Project description:Nowadays, drug abuse and addiction are serious public health problems in the USA. Methamphetamine (METH) is one of the most abused drugs, which is known to cause brain damage from repeated exposure on human. Herein, a proteomic study was applied to evaluate METH-induced brain protein dynamics following a two-week chronic regimen of escalating dose of METH exposure. Proteins were extracted from rat brain hippocampal and olfactory bulb tissues and subjected to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Both shotgun and targeted proteomic analysis were performed. Protein quantitation was initially based on comparing the spectral counts between METH exposed animals and their control counterparts. Quantitative differences were further confirmed through multiple reaction monitoring (MRM) LC-MS/MS experiments. According to the quantitative results, the expression of 18 proteins (11 in hippocampal proteome, 7 in olfactory bulb proteome) were shown a significant alteration as a result of exposure of rats to METH. 13 of these proteins were up-regulated after METH exposure while 5 of were down-regulated. The altered proteins belonging to different structural and functional families were involved in processes such as cell death, inflammation, oxidation, and apoptosis.