Main Research Findings

1) In both in vitro and in vivo study models, FGL was found to protect hippocampal neurons from damage induced by ischemic insult.

2) Treatment with FGL was found to reduce changes in the glia induced by increased levels of lipopolysaccharide inflammation.

 

Selected Data

1) The study conducted by researchers Skibo et al investigated the neuroprotective capabilities of a synthetic NCAM-derived peptide, FGL, against ischemic insult, employing both in vitro and in vivo models. The experimental design encompassed preparation of biological systems, controlled induction of ischemia, precise application of the therapeutic peptide, and multi-faceted assessment of outcomes [1].

For the in vitro experiments, two primary models were utilized: dissociated rat hippocampal neurons and hippocampal organotypic slice cultures. Dissociated neurons were prepared from postnatal day 1 Wistar rat pups. This involved decapitation, careful dissociation of hippocampal tissue, and plating the neurons at a density of 20,000 cells/cm² on poly-L-lysine coated tissue culture chambers. These cultures were maintained for 11-12 days in vitro (DIV) in Neurobasal medium, supplemented with HEPES, penicillin, streptomycin, bovine serum albumin (BSA), and B27, with medium changes every three days. Hippocampal organotypic slice cultures were derived from 7-day-old Wistar rat pups. Following decapitation and brain removal, hippocampi were rapidly isolated and sectioned transversally into 350-400 µm thick slices using a McIllwain tissue chopper. These slices were then transferred to porous Millicell membrane inserts in six-well plates, cultured in a specific medium containing MEM, horse serum, Tris, NaHCO3, HEPES, glucose, HBSS, penicillin, and streptomycin. Medium was changed the day after preparation and then twice weekly, with experiments typically performed on slices cultured for 12-14 DIV [1].

The ischemic insult in vitro was simulated using an oxygen-glucose deprivation (OGD) protocol. This involved first rinsing the cultures with a deoxygenated, glucose-free, and serum-free OGD solution (comprising PBS, HEPES, and sucrose). The medium was then replaced with fresh OGD solution, and cultures were transferred to an air-tight anaerobic chamber where the atmosphere was replaced with a 95% N2 and 5% CO2 gas mixture. The duration of OGD was tailored to the model: 20 minutes for dissociated cell cultures and 10 minutes for slice cultures. Following OGD, cultures were rinsed with regular culture medium and returned to normoxic incubator conditions. Outcomes were typically assessed at 1, 4, and 24 hours post-OGD.

The therapeutic agent, FGL, is a pentadecapeptide derived from the F3 module of NCAM. A control peptide, FGLala, with two glutamine residues substituted by alanines, was also used. FGL was employed in dimeric (FGLlpa) and tetrameric (FGLd) forms, with FGLlpa being used for in vivo and FGLd for in vitro experiments. For in vitro treatments, FGL or FGLala was used at a final concentration of 20 µg/mL. Three distinct treatment paradigms were explored including: pretreatment (FGL added 24 hours prior to OGD), immediate post-treatment (FGL added immediately after OGD), and delayed post-treatment (FGL added 4 hours after OGD). Control cultures received vehicle (0.5% BSA in PBS). To elucidate the mechanism of action, a specific fibroblast growth factor receptor (FGFR) inhibitor, SU5402, was added at 25 µM in some experiments [1].

Several assays were employed to evaluate the in vitro neuroprotective effects. The MTS reduction assay quantified mitochondrial metabolic activity, serving as a measure of cell viability/proliferation. Cultures were incubated with MTS in phenol-free HBSS for 40 minutes, and optical density at 450 nm was measured using a multiwell photometer. Propidium iodide (PI) staining was used to estimate cell viability and detect dying cells with damaged membranes. PI at a concentration of 2 µM was added to the medium 24 hours before OGD and during reoxygenation. Images were captured using a confocal inverted fluorescence microscope, and the percentage of PI-positive area in the hippocampal CA1 subfield was calculated using PrAverB software. FM1-43 staining assessed presynaptic function and synaptic vesicle recycling in dissociated hippocampal neurons. Synapses were loaded with FM1-43 under depolarization, and destaining rates, induced by further KCl stimulation in FM1-43-free solution, were quantified by imaging pixel intensities using a laser scanning confocal microscope [1].

For in vivo validation, male Mongolian gerbils aged 6-week-old and weighing 65-75g, were used in a transient global ischemia model. Animals were handled in strict accordance with European ethical guidelines. Gerbils received a single suboccipital injection of 5 µg of the dimeric FGLlpa in 5 µL vehicle or vehicle alone, 24 hours prior to ischemia. Anesthesia was maintained with 2% isoflurane. Global ischemia was induced by temporarily clamping both common superior carotid arteries for 5 minutes, followed by reperfusion. Sham-operated animals underwent the same procedure without clamping. Four days after the ischemic insult, brains were perfusion-fixed, postfixed, sliced into 40 µm coronal sections, and stained with haematoxylin-eosin for histological evaluation. Neuronal survival in the hippocampal CA1 region was quantified using unbiased stereology, counting intact neurons with clear nuclear membranes and nucleoli [1].

2) This study conducted by Cox et al investigated whether the anti-inflammatory effects of the neural cell adhesion molecule (NCAM)-derived peptide, FGL, are dependent on CD200, a glycoprotein crucial for maintaining microglial quiescence. The methodological approach combined in vitro cell culture techniques with molecular and biochemical analyses to delineate FGL’s mechanism of action in the context of lipopolysaccharide (LPS)-induced inflammation in glial cells [2].

The primary cellular model employed was mixed glial cultures prepared from the brain tissue of neonatal wildtype (WT) mice and CD200-deficient (CD200-/-) mice. This comparative approach was central to determining the CD200 dependency of FGL’s effects. The mixed glial cultures were established using established protocols, ensuring a representative mixture of astrocytes and microglia, which are the main focus of this investigation. In addition to mixed glial cultures, the study also utilized isolated astrocytes and purified microglia in specific experiments to dissect the cellular interactions. Astrocytes were prepared from C57BL/6 mice and were cultured until confluent. Purified microglia were isolated and cultured separately to assess their direct response to stimuli and FGL [2].

The peptide FGL, a fibroblast growth loop peptide mimicking the FGFR-1 binding site of NCAM, was used at a concentration of 10 µg/ml. Its application regimen was crucial: FGL was typically pre-incubated with the glial cultures for 24 hours prior to the inflammatory challenge. This pre-treatment strategy was chosen based on preliminary experiments demonstrating significantly greater effects compared to co-treatment with LPS. The inflammatory stimulus used was lipopolysaccharide (LPS), a potent activator of microglia, applied at a concentration of 1 µg/ml for 24 hours after the FGL pre-incubation period. Control cultures were treated with vehicle (saline or culture medium) instead of FGL or LPS, or with LPS alone.

To quantify the cellular responses to LPS and FGL, several molecular techniques were employed. Quantitative real-time PCR (Q-PCR/RT-qPCR) was extensively used to measure the mRNA expression levels of key markers. RNA was isolated from cultured cells using an RNAII KIT and subsequently reverse transcribed into cDNA using a cDNA RT kit. Gene expression was then measured using predesigned Taqman gene expression assays for markers of microglial activation (CD11b, CD40, and intercellular adhesion molecule 1 (ICAM-1)) and inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)). Beta-actin served as the endogenous control for relative quantification, applying the 2^-DDCT method [2].

Complementary to mRNA analysis, ELISA were performed to quantify the protein concentrations of IL-1β, TNF-α, and IL-6 in the supernatant of mixed glial cultures. This involved coating 96-well plates with specific capture antibodies for each cytokine, incubating with cell supernatants or standards, followed by detection with corresponding biotinylated antibodies and streptavidin-horseradish peroxidase conjugate. The reaction was developed with a substrate solution and stopped with sulfuric acid, and absorbance was read at 450 nm. This provided a direct measure of cytokine release, reflecting the inflammatory output of the glial cells.

Additional experiments were conducted to delve deeper into the role of CD200. Flow cytometric analysis was used to determine the expression of CD200 on CD11b-negative cells in mixed glial cultures from WT and CD200-/- mice, confirming its presence on astrocytes. To investigate FGL’s direct effect on CD200 expression, purified astrocyte cultures were treated with 10 µg/ml FGL for 24 hours, and subsequent Western blot analysis using densitometric quantification against β-actin to measure changes in CD200 protein levels. Furthermore, to explore the astrocyte-microglia interaction, cell membranes were isolated from cultured astrocytes using a subcellular protein fractionation kit. These isolated astrocytic membrane preparations containing CD200 were then used to pre-incubate purified microglia for 2 hours before the addition of LPS. This particular setup aimed to mimic the cell-cell contact mediated by CD200 [2].

 

Discussion

1) The study completed by Skibo et al demonstrated that the synthetic NCAM-derived peptide, FGL, offers significant neuroprotection against ischemic insult in both in vitro and in vivo models, underscoring its potential as a therapeutic agent for brain injury. The results consistently highlighted FGL’s ability to preserve neuronal viability, maintain metabolic function, and support presynaptic activity, with its effects being dependent on fibroblast growth factor receptor (FGFR) activation [1].

In the in vitro models, particularly using hippocampal organotypic slice cultures subjected to OGD, FGL showed a potent protective effect against delayed neuronal cell death. PI staining, a marker for cell death, revealed that 10 minutes of OGD induced progressive neuronal damage, with PI-positive areas in the hippocampal CA1 region increasing significantly by 24 hours post-OGD. Remarkably, FGL administered as a pretreatment, 24 hours prior to OGD, almost completely restored the PI-positive area to baseline levels, effectively preventing OGD-induced neuronal death. Immediate post-treatment with FGL also conferred a similar protective effect. However, when FGL application was delayed until 4 hours after OGD, no neuroprotective benefit was observed, indicating a critical therapeutic window. The specificity of FGL’s action was confirmed by the absence of effects with a control peptide, FGLala. Crucially, the neuroprotective effect of FGL was abolished when co-administered with SU5402, a specific FGFR inhibitor, thereby establishing that FGL’s actions are mediated via the activation of FGFR [1].

Figure 1: A) Changes in PI-positive area defined by the percent of total CA1 area in relation to time after injection of experimental treatments and B) changes in PI-positive area defined by the percent of total CA1 area in response to experimental treatments.

Further assessment of metabolic activity using the MTS reduction assay corroborated these findings. OGD initially led to a transient increase in metabolic activity at 1 hour, followed by a significant decline below control levels by 24 hours, indicative of mitochondrial dysfunction. FGL pretreatment not only enhanced metabolic activity in normoxic conditions but also fully normalized the OGD-induced reduction in metabolic activity at all time points examined. Similar to the PI staining results, FGL applied immediately after OGD also normalized metabolic function, while delayed administration at 4 hours post-OGD had no beneficial impact. Again, the FGFR inhibitor SU5402 completely abrogated FGL’s positive effects on metabolic activity, reinforcing the FGFR-dependent mechanism.

The study also investigated FGL’s impact on presynaptic function using FM1-43 staining in dissociated hippocampal neurons. FGL pretreatment for 24 hours significantly increased the rate of high KCl-evoked FM1-43 destaining in normoxic cultures, indicating an enhancement of presynaptic activity as a measure of synaptic vesicle recycling. OGD profoundly impaired this function, leading to a slower FM1-43 destaining rate. Impressively, FGL, whether applied as a pretreatment or immediately post-OGD, completely normalized the OGD-induced impairment in FM1-43 destaining. This suggested that FGL not only protected neurons from death but also preserved or restored their critical synaptic functions. Consistent with other assays, the control peptide FGLala had no effect on presynaptic function, and SU5402 abolished FGL’s beneficial actions [1].

The neuroprotective efficacy of FGL was successfully translated and validated in vivo using a transient global ischemia model in Mongolian gerbils. A 5-minute global ischemia induced severe neuronal loss in the hippocampal CA1 region, resulting in only 12.5% neuronal survival after 4 days. However, a single suboccipital injection of 5 µg of dimeric FGLlpa 24 hours prior to the ischemic insult significantly increased the survival of CA1 neurons to 36.6%. This demonstrated a robust neuroprotective effect of FGL in a physiologically relevant model of ischemic brain injury.

Figure 2: Changes in the total number of neurons in the CA1 region following ischemic injury in response to experimental treatment.

Collectively, these results provide strong evidence that FGL, by acting as an agonist of FGFR, exerts specific and potent neuroprotective effects. Its capacity to prevent neuronal death, maintain cellular metabolic health, and preserve presynaptic function, coupled with its proven in vivo efficacy, positions FGL as a promising candidate for therapeutic intervention in conditions characterized by ischemic brain damage, such as stroke and potentially vascular dementia. The findings further highlight the importance of NCAM-mediated signaling pathways in neuronal resilience and repair processes [1].

2) The results of the experiment completed by Cox et al demonstrated that FGL attenuates LPS-induced inflammatory changes in glial cells through a CD200-dependent mechanism, highlighting the critical role of astrocyte-microglia interaction in modulating microglial activation.

Initially, the researchers characterized the inflammatory response induced by LPS in mixed glial cultures from both WT and CD200-/- mice. LPS significantly increased the mRNA expression of microglial activation markers, namely CD40, ICAM-1, and CD11b, in glia from both genotypes. However, this LPS-induced upregulation of activation markers was significantly more pronounced in CD200-/- glia compared to WT glia. Similarly, LPS treatment led to a significant increase in both mRNA expression and supernatant concentrations of inflammatory cytokines, including IL-1β, IL-6, and TNF-α, in glia from both WT and CD200-/- mice. Consistent with the activation markers, the cytokine response was markedly enhanced in CD200-/- glia, indicating a heightened inflammatory susceptibility in the absence of CD200. Interestingly, even under resting conditions, CD11b mRNA levels were already elevated in CD200-/- glia compared to WT glia, suggesting a basal state of increased activation or altered morphology in these cells [2].

The core finding regarding FGL’s modulatory effects was its differential impact on WT versus CD200-/- glial cultures. In WT mixed glial cultures, pre-treatment with FGL effectively attenuated the LPS-induced increases in mRNA expression of all microglial activation markers (CD40, ICAM-1, and CD11b). Furthermore, FGL pre-treatment significantly reduced both the mRNA expression and supernatant concentrations of the inflammatory cytokines IL-1β, IL-6, and TNF-α in WT glia. These findings unequivocally established FGL’s anti-inflammatory potential in a normal glial environment.

Crucially, when FGL was applied to CD200-/- mixed glial cultures, its anti-inflammatory effects were completely abrogated. FGL pre-treatment had no significant impact on the LPS-induced upregulation of CD40, ICAM-1, CD11b mRNA, nor on the increased mRNA and protein levels of IL-1β, IL-6, and TNF-α in CD200-/- glia. This stark difference in response between WT and CD200-/- cells provided compelling evidence that the anti-inflammatory actions of FGL are strictly dependent on the presence of CD200 [2].

Figure 3: Changes in A) CD40, B) ICAM-1, and C) CD11b in each type of animal test subject and in response to each experimental treatment

To further elucidate the role of CD200, the study explored its expression on astrocytes and its involvement in astrocyte-microglia communication. Flow cytometric analysis confirmed that CD200 is indeed expressed on CD11b-negative cells within mixed glial cultures from WT mice, but, as expected, was absent in CD200-/- cells. A key mechanistic insight emerged when purified astrocytes were treated with FGL: FGL significantly increased the protein expression of CD200 on these isolated astrocytes. This suggests a potential pathway through which FGL could exert its CD200-dependent effects by upregulating CD200 on astrocytes [2].

The study also investigated the direct communication between astrocytes and microglia. When purified microglia were pre-incubated with astrocytic membrane preparations containing CD200 prior to LPS stimulation, the LPS-induced increases in microglial IL-1β mRNA were significantly attenuated. This protective effect extended to other cytokines, as the astrocytic membrane preparations also attenuated LPS-induced increases in TNF-α and IL-6 mRNA and protein levels in microglia. These results strongly supported the hypothesis that cell-cell contact, mediated by CD200 on astrocytes interacting with its receptor, CD200R, on microglia, is a critical mechanism for modulating microglial activation.

In summary, the study concludes that FGL exerts its modulatory effects on LPS-induced microglial activation in a CD200-dependent manner. This dependence is highlighted by FGL’s ability to attenuate inflammation only in WT glia and its complete lack of effect in CD200-/- glia. Furthermore, the findings indicate that FGL can increase CD200 expression on astrocytes, and that astrocytic CD200 is essential for modulating microglial inflammatory responses through cell-cell contact. This research underscores the importance of the CD200-CD200R pathway and astrocyte-microglia interactions in regulating neuroinflammation and identifies FGL as a promising therapeutic agent that acts via this pathway [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Skibo GG, Lushnikova IV, Voronin KY, et al. A synthetic NCAM-derived peptide, FGL, protects hippocampal neurons from ischemic insult both in vitro and in vivo. Eur J Neurosci. 2005;22(7):1589-1596. doi:10.1111/j.1460-9568.2005.04345.x

[2] Cox FF, Berezin V, Bock E, Lynch MA. The neural cell adhesion molecule-derived peptide, FGL, attenuates lipopolysaccharide-induced changes in glia in a CD200-dependent manner. Neuroscience. 2013;235:141-148. doi:10.1016/j.neuroscience.2012.12.030

 

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