The Effect of LPS on Phosphorylation of AKT Signaling in Schwann Cells

Schwann cells (SCs) are cells in the peripheral nervous system that may play a significant role in neuronal repair after an injury, however, little is known about their mechanisms on how they do so. In the lab, lipopolysaccharide (LPS), a bacterial endotoxin, is used to stimulate an injury while forskolin, a growth hormone, is used to activate the cAMP pathway. In this experiment, the cAMP pathway, which is involved in many cellular processes, and protein kinase b (AKT), a cell survival signal, where analyzed in different LPS doses (0.1, 1, and 10 µg/mL) and over different time periods (3-,6-,12-, and 24-hours). It was predicted that cells treated with LPS will increase the expression of activated phospho-AKT as dose and time increases, while cells treated with both LPS and forskolin would have a synergistic effect on the upregulation of phospho-AKT. It was discovered that, in LPS alone, from 1 µg/mL to 10 µg/mL had an increased expression in phospho-AKT across all time intervals besides 24 hours. In LPS and forskolin treatments, a synergistic effect was observed from 1 µg/mL to 10 µg/mL across all time intervals.https://digitalcommons.misericordia.edu/surf2023/1001/thumbnail.jp

The Location of NF-kB and AKAP-95 in Lipopolysaccharide Treated RT4-D6P2T Schwannoma Cells

https://digitalcommons.misericordia.edu/surf2023/1003/thumbnail.jp

The Effects of Rolipram, a Selective Phosphodiesterase Inhibitor, on Immortalized Schwann Cell Proliferation

The regulation of Schwann cell growth in vitro is facilitated by heregulin, a neuron-secreted growth factor, and an unknown mitogen that activates the cyclic adenosine monophosphate (cAMP) pathway. The quantity of cAMP available to Schwann cells can determine if they become a myelinating or proliferating phenotype. The abundance of intracellular cAMP available to the cell is widely regulated by a family of enzymes called phosphodiesterases (PDEs). PDE inhibitors such as rolipram have therapeutic potential in various disorders and function by increasing the levels of cAMP in the cell. This study was undertaken to determine the concentration of rolipram that would induce optimal Schwann cell proliferation. It was hypothesized that an increase in rolipram levels will elicit a dose-dependent rise in cell proliferation.https://digitalcommons.misericordia.edu/research_posters2020/1067/thumbnail.jp

The Role of Schwann Cells in Nerve Injury: Forskolin-Mediated cAMP Activation Upregulates TNFα Expression Despite NF-κB Downregulation in LPS-Treated Schwann Cells

Although Schwann cells are known to play a role in axonal regeneration following nerve injury and inflammation, the exact mechanism is unknown. This study explores two potential mechanisms: the NF-κB and cAMP pathways. The NF-κB pathway produces cytokines, such as TNFα, to regulate inflammation, whereas the cAMP pathway is anti-inflammatory and regulates Schwann cell proliferation via AKAP95 and cyclin D3. Although it is well-known that NF-κB and cAMP are involved in inflammation, not much is known regarding the effects of forskolin-mediated cAMP activation on LPS-mediated NF-κB activation in Schwann cells. In this study, RT4-D6P2T immortalized rat Schwann cells were treated with 0.1, 1, or 10 μg/mL of LPS, with or without 2 μM of forskolin, for 3 hours, and then an MTT viability assay and Western blot were performed. It was found that cAMP activation decreased cell viability regardless of LPS dose compared to the control. It was also found that at high doses of LPS, cAMP activation upregulated TNFα expression despite a downregulation of NF-κB, meaning cAMP may regulate TNFα through NF-κB-independent mechanisms. Furthermore, at high doses of LPS, cAMP activation downregulated AKAP95 and cyclin D3 expression and decreased cell viability, meaning that at high LPS doses, NF-κB might inhibit cAMP\u27s ability to upregulate AKAP95 and cyclin D3, decreasing cell proliferation and thus viability. A better understanding of the potential interactions between the NF-κB and cAMP pathways in Schwann cells may help to find a potential therapeutic target for the treatment of nerve injury and inflammation.https://digitalcommons.misericordia.edu/research_posters2023/1029/thumbnail.jp

The Effects of Rolipram, a Selective Phosphodiesterase Inhibitor, on Immortalized Schwann Cell Proliferation, AKAP95 and Cyclin D3 Expression

Schwann cells are a vital component of the Peripheral Nervous System and aid in the repair of axons following injury. The regulation of Schwann cell growth in vitro is facilitated by heregulin, a neuron-secreted growth factor, and an unknown mitogen that activates the cyclic adenosine monophosphate (cAMP) pathway. The abundance of intracellular cAMP is regulated by a family of enzymes called phosphodiesterases (PDEs). PDE inhibitors such as rolipram have therapeutic potential in various disorders and function by increasing the levels of intracellular cAMP. A-Kinase anchoring proteins (AKAPs), a family of scaffolding proteins that belong to the cAMP/Protein Kinase A (PKA) pathway are known to bind both PDE and PKA to regulate cAMP concentration in cardiac myocytes. Previous studies have shown that AKAP95, a nuclear AKAP, known for scaffolding cyclins, is essential for Schwann cell growth. Based on these reports, it was hypothesized that increasing the concentration of rolipram would elicit a dose-dependent increase in Schwann cell proliferation by augmenting the expression of AKAP95 and cyclin D3. Immortalized Schwann cells were cultured with no mitogens, 12.5 ng/mL heregulin, 1 µM of forskolin (a pharmacological activator of cAMP), heregulin + forskolin, and various doses of rolipram at 0, 0.5, 1, 5, 10, 25, and 50 µM for 12 or 24 hours. Using the MTT assay, preliminary results indicate that cells incubated for 12 hours and 24 hours exhibited the highest rate of proliferation at a dose of 5µM and 10 µM rolipram, respectively. Meanwhile, immunoblot analysis revealed that in cells treated with heregulin + forskolin, the expression of cyclin D3 and AKAP95 was highest when incubated with 25 µM and 50 µM of rolipram, respectively. These results suggest that increasing the concentration of cAMP by inhibiting phosphodiesterases augments Schwann cell proliferation by amplifying the expression of proteins regulating cell division.https://digitalcommons.misericordia.edu/research_posters2021/1020/thumbnail.jp

Simulation of an Inflammatory Model Using Schwann Cells

Schwann cells are a type of glial cell in the peripheral nervous system that produce the myelin sheath surrounding neuronal axons. This myelin insulates the neurons and promotes the rapid conduction of electrical impulses throughout the body. Schwann cells have also been found to play a critical role in neuron repair following nerve injury. During nerve injury, the myelin sheath is damaged, stimulating Schwann cells to release cytokines, or inflammatory mediators, that recruit immune cells to the site of injury so that the myelin debris can be cleared, and repair can take place.1 Then neuronal growth is facilitated by heregulin and an unknown growth factor that stimulates the cyclic adenosine monophosphate (cAMP) pathway.2,3 There is still yet to be known regarding the exact mechanisms by which Schwann cells mediate nerve repair. Two pathways of interest are the nuclear factor kappa B (NK-κB) and cAMP pathways. The NF-κB pathway plays a major role in inflammation through the production of cytokines like tumor necrosis factor alpha (TNF-α) and can be stimulated in vitro by treating cells with lipopolysaccharide (LPS), a cell wall immunostimulatory component of Gram-negative bacteria.1 The cAMP pathway is a key regulator of cell division2,4 and can be stimulated by treating cells with an artificial plant extract called forskolin.2 This study aims to examine proteins of the NF-κB pathway when stimulated with cAMP-activating growth factors. It was hypothesized that cells treated with LPS and growth factors express less NF-κB and TNF-α than cells treated with LPS only. A better understanding of the mechanisms underlying nerve injury and Schwann cell-mediated repair will hopefully shed light on a potential therapeutic target in the treatment of nerve injury and inflammation.https://digitalcommons.misericordia.edu/surf2022/1001/thumbnail.jp

The Effect of Creatine on Immortalized Schwann Cell Proliferation

Creatine is an important component of the high-energy phosphate transfer to regulate cellular levels of ATP. The functions of creatine supplementation are well studied in the muscular and skeletal system, however the impact of creatine in the nervous system, specifically Schwann cells, is relatively unknown. Schwann cell growth in vitro is facilitated by heregulin, a neuron secreted growth factor, and forskolin, a pharmacological agent that activates cAMP. In peripheral nervous system injuries, recovery time is based around Schwann cell’s ability to proliferate and differentiate, and it is unknown what role creatine plays in this process. It was hypothesized that increasing creatine concentrations in combination with growth factors would augment Schwann cell growth. Immortalized Schwann cell line S16 cells were treated with N2 (control), heregulin (12.5ng/mL), forskolin (2µM) and heregulin plus forskolin for 24 hours. To determine the optimal dose of creatine for the Schwann cells, doses of 200nM creatine, 2µM creatine, and 20µM creatine were added to each of the previously stated treatments, and was repeated for 1-, 6-, and 12-hour timepoints. At the 1- and 6-hour time points 200nM and 2µM showed the greatest proliferation amongst the three doses and, the cells were most influenced by the presence of heregulin (108.9±5.45), (113.3±20.1). A synergistic effect was noted between heregulin and forskolin at both time points (104.86±2.9), (107.76±20.88). At the 12- and 24-hour time points the optimal creatine dosage for proliferation was 2µM and, the cells were most influenced by the presence of forskolin (108.38±7.6), (110.52±1.60). These time points showed a synergistic effect as well. This suggests that at shorter timepoints, creatine stimulates a different pathway than at longer timepoints. In summary, these findings suggest that creatine stimulate proliferation through different pathways depending on concentration and timepoint.https://digitalcommons.misericordia.edu/research_posters2022/1006/thumbnail.jp

Simulation of an Inflammatory Model Using Schwann Cells

During peripheral nerve injury, the myelin surrounding the neuronal axons is damaged, initiating an inflammatory response to remove myelin debris. Once myelin debris is cleared, Schwann cells acquire a proliferating phenotype which allows them to grow and divide so that remyelination can occur. The neuron stimulates Schwann cell division by secreting growth factors, like heregulin, and an unknown growth factor that activates the cAMP pathway. Although the role of cAMP in axonal regeneration is well-known, not much has been explored about its function in Schwann cells during nerve injury and inflammation. To simulate an inflammatory environment, the S16 Schwann cell line (SC-2941) was activated with lipopolysaccharide (LPS), a cell-wall immunostimulatory component of Gram-negative bacteria. It was hypothesized that Schwann cells stimulated with LPS and growth factors will have higher proliferation in comparison to LPS treatment only. Schwann cells were treated for 1, 3, 12 or 24 hours with no growth factors (control media, N2), 12.5 ng/mL heregulin (H), 2mM forskolin (F) or H+F and various doses of LPS at 5, 50 or 500 ng/mL. Using the MTT proliferation assay, preliminary studies in 24-hour cultures reveal that cell proliferation, as measured by optical density, was significantly higher in cells treated with 5 ng/mL of LPS+F (0.846 ± 0.054), and H+F (1.023 ± 0.189) in comparison to cells grown with H (0.699 ± 0.057) or N2 only (0.765 ± 0.016). In contrast, cells treated for 1, 3 and 12 hours, with various concentrations of LPS revealed an overall decrease in proliferation when compared to 24-hour cultures. However, cultures treated with LPS and F or H+F, for all time points, showed an increase in cell growth when compared to N2 and H. In summary, it appears as though a combination of LPS and forskolin, with or without heregulin, may promote more Schwann cell proliferation than LPS alone. These findings also suggest that, when LPS-activated cells are treated with heregulin or forskolin, alone, they may activate two very distinct pathways to initiate opposite responses, with heregulin hindering cell division and forskolin promoting cell division. However, when heregulin and forskolin are combined, the forskolin-activated cAMP pathway appears to promote higher proliferation to offset the decrease in proliferation initiated by the heregulin pathway. Considering these results, it appears that the cAMP pathway in Schwann cells may play a major role in the inflammatory environment during nerve injury.https://digitalcommons.misericordia.edu/research_posters2022/1003/thumbnail.jp

The Effects of Neuronal Growth Factors on LPS-Activated Schwann Cells

Schwann cells (SCs) are the principal support cells of neurons in the peripheral nervous system, that both myelinate axons for the rapid conduction of electrical impulses as well as assist in axonal repair during nerve injury. During nerve injury, SCs secrete tumor necrosis factor alpha (TNF-α)1,5,6 and other proinflammatory mediators1,6, attracting macrophages to the site of injury to induce inflammation and clear myelin debris.1,6 Once the debris is cleared, the neuron stimulates SC proliferation by secreting neuronal mitogens, such as heregulin3,4, and an unknown factor that activates the cAMP pathway3, an important regulator of cell division.3,4 In vitro, SCs can be treated with an artificial plant extract called forskolin3,4 to activate the cAMP pathway. Studies show that heregulin and forskolin act synergistically to enhance SC proliferation under normal, non-inflammatory conditions.4 Although the role of cAMP in proliferation and axonal regeneration is well-known, not much has been explored about its function in SCs during nerve injury and inflammation. In vitro, inflammatory conditions can be simulated by treating SCs with lipopolysaccharide (LPS), a cell-wall immunostimulatory component of Gram-negative bacteria.1,2,6 In most mammalian cells, LPS binds to a transmembrane protein called toll-like receptor 4 (TLR4)1,2,6, activating both the mitogen-activated protein kinase (MAPK) pathway1,2,6 and the nuclear factor kappa B (NF-κB) pathway2,6, to promote the secretion of inflammatory mediators, such as TNF-α.1,2,6 With that being said, the aim of this preliminary study was to determine the role of the cAMP pathway in SCs during LPS-induced inflammation. It was hypothesized that SCs stimulated with LPS and growth factors will have higher proliferation than SCs treated with LPS only.https://digitalcommons.misericordia.edu/research_posters2022/1004/thumbnail.jp