The Role of Basigin as an Immune Mediator in the CNS

Chronic inflammation is a hallmark of many neurodegenerative disorders. Although the central nervous system (CNS) can stave peripheral pathogens from crossing the blood-brain barrier (BBB) through a network of endothelia, astrocytes, and pericytes, prolonged exposure to a pathogen can comprise this barrier, leading to an infiltration of cytokines and T-cells. T-cells are lined with surface receptors that detect pathogens and elicit further immune responses, particularly toll-like receptor 4 (TLR4). TLR4 has been demonstrated to detect lipopolysaccharide (LPS), found on the outer membrane of Gram-negative bacteria. Recently, Basigin, a member of the immunoglobulin superfamily, has been shown to interact with TLR4 and is expressed on endothelial cells. The present study aims to address the expression pattern of Basigin and TLR4 in brain tissue stimulated with LPS for a variation of time to mirror acute and chronic inflammation, as well as different life stages to determine whether the expression pattern is dynamic. Total RNA and protein were purified from the isolated tissue and used in qRT-PCR and direct ELISA. The results of the study suggest expression of Basigin is elevated after 3 and 24 hours of incubation in LPS relative to control in adolescent mice, but not neonates. Expression of TLR4 did not change between age groups or incubation periods. The data suggest that expression of Basigin, but not TLR4, changes from neonate to adolescent age. This may leave the neonatal CNS more susceptible to an inflammatory response

Characterization of metabolic gene expression in the RAW 264.7 cell line

The current study is being conducted to examine transporter proteins on the surface of white blood cells (murine monocyte cell line RAW 246.7), specifically the transporter GLUT1 (an amino acid transporter) in the presence of lipopolysaccharide (LPS). The purpose of this is to analyze metabolism in monocytes because surprisingly little is known about this subject. Cellular metabolism is important because it consists of all the biochemical reactions occurring within cells and thus drives cellular processes and activity. We hypothesize that expression of the GLUT1 transporter will increase in the presence of LPS. To test this hypothesis, we plated two cell treatments – one control group with PBS and one test group treated with LPS. We analyzed MCT1 activity and GLUT1 activity within the cells by using a laser confluency microscope and fluorescent antibody tags DAPI and Alexa488. Interestingly, MCT1 activity was significantly higher than GLUT1 activity. Furthermore, in the control condition with PBS, activity of MCT1 was higher than activity of GLUT1. In the experimental condition with LPS, relative activity of the transporters was opposite, showing that MCT1 activity was higher and GLUT1 activity was lower. This supports our hypothesis that GLUT1 transporter activity will increase in the presence of LPS compared to the control PBS

Investigation of Authophagy in Activated Monocytes

The innate immune system is the body’s first line of defense against foreign molecules. When white blood cells like monocytes encounter a foreign molecule, they initiate an immune response to eliminate that molecule and further infection. Metabolism is the biochemical reactions that happen inside a cell. Typically, cell metabolism increases in active cells. It would therefore be thought that metabolic processes would increase in monocytes that have become activated through the innate immune response. A study conducted by this laboratory, however, indicated that transporter proteins responsible for delivering metabolic substrates to cells were decreased in expression in response to an immune challenge. Therefore, the purpose of the present study was to determine if activated monocytes rely on substrates already found within the cell by using a process known as autophagy or “self-eating.” It was hypothesized that proteins involved in autophagy would increase in expression during an immune response as compared to normal, non-treated conditions. The RAW 264.7 cell line was treated with 1 g/mL lipopolysaccharide (LPS), which is a component of Gram-negative bacteria or with saline. After 24 hours, RAW 264.7 cellular proteins were isolated and the expression of four proteins known to be involved in various events in the autophagy process were analyzed via an ELISA. It was determined that no significant difference in expression was observed for the autophagy-specific proteins in the LPS-treated cells when compared to those treated with saline. The data indicate that activated monocytes do not undergo autophagy to support their metabolic needs

Characterization of the expression of Basigin and Monocarboxylate Transporters 1 and 4 in aged mouse brains in response to acute and chronic inflammation

Project of Merit Winner Chronic inflammation, or prolonged activation of the immune system, is a major hallmark of many age-related diseases, including heart disease, COVID-19, and neuroinflammation. Typically, the central nervous system (CNS) is protected by a barrier that forms through the coordinated efforts of blood vessel endothelial cells, astrocytes, and pericytes. However, as that barrier breaks down because of chronic inflammation, neuroinflammation can occur. Blood vessel endothelial cells express membrane association proteins, including the cell adhesion molecule Basigin and the transporter protein Monocarboxylate transporter 1 (MCT1). A related MCT protein named MCT4 often increases in expression during chronic inflammation. Basigin associates with both MCT1 and MCT4 in the neural retina. Therefore, the expression of these three proteins (Basigin, MCT1, and MCT4) was investigated in response to chronic inflammation in aged mouse brains. It was hypothesized that the expression of Basigin and MCT1 would decrease and that of MCT4 would increase in response to chronic inflammation. The brains of 6-month-old mice were harvested and exposed to lipopolysaccharide (LPS; 10 g/mL), a known activator of inflammation, or saline control for 3, 6, 12, or 24 hours. The brain proteins were isolated and subjected to ELISA analyses designed to measure each target protein. It was determined that no significant difference in Basigin, MCT1, or MCT4 expression was observed for any treatment time. The hypothesis was not supported, and the data suggest that Basigin, MCT1, and MCT4 may not contribute to the development of neuroinflammatory diseases

Regenerative medicine therapy: adipose derived extracellular vesicles in viral myocarditis

Objective: Myocarditis, inflammation of the heart muscle, is an autoimmune heart disease that can be caused by viruses, bacteria and toxins. Myocarditis can lead to dilated cardiomyopathy (DCM) and heart failure. Currently there are no disease-specific therapies for treating myocarditis or preventing progression to DCM. Adipose Extracellular Vesicles (AEVs) are lipid bilayer nanoparticles that are released into the outside environment of adipocytes and provide promising regenerative potential for inflammatory diseases like myocarditis. Methods: Lipoaspirate was obtained from women and men and AEVs isolated from the lipoaspirate using tangential flow filtration. We injected wild type male BALB/c mice with 250uL AEVs (1×10^10 EV/mL) intraperitoneally or sucrose control on day -1, 0, 1 with viral infection on day 0. Mice were harvested on day 10 post infection at the peak of myocarditis. Results: We found that male mice treated with AEVs from a female patient had a significantly higher body weight (p=0.0003), less calcification in the gut (p=0.001) and less myocardial inflammation (p=0.007) than controls. Mouse hearts analyzed by qRT-PCR revealed that AEV treated mice had significantly lower relative gene expression of cell markers for total immune cells (CD45, p=0.002), macrophages (CD11b, p=0.002, F4/80, p=0.0004); specifically M2 macrophages (Chi313, p=0.003), as well as CD3+ (p=0.007) and CD4+ T cells (p=0.01) than controls. Additionally, we found that mice treated with AEVs from a male patient also had significantly less myocardial inflammation (p=0.01). Conclusion: AEVs could provide an innovative therapy to reduce cardiac inflammation and decrease the risk of developing DCM following myocarditis

Dynamic usage of alternative splicing exons during mouse retina development

Alternative processing of pre-mRNA plays an important role in protein diversity and biological function. Previous studies on alternative splicing (AS) often focused on the spatial patterns of protein isoforms across different tissues. Here we studied dynamic usage of AS across time, during murine retina development. Over 7000 exons showed dynamical changes in splicing, with differential splicing events occurring more frequently in early development. The overall splicing patterns for exclusive and inclusive exons show symmetric trends and genes with symmetric splicing patterns that tend to have similar biological functions. Furthermore, we observed that within the retina, retina-enriched genes that are preferentially expressed at the adult stage tend to have more dynamically spliced exons compared to other genes, suggesting that genes maintaining retina homeostasis also play an important role in development via a series of AS events. Interestingly, the transcriptomes of retina-enriched genes largely reflect the retinal developmental process. Finally, we identified a number of candidate cis-regulatory elements for retinal AS by analyzing the relative occurrence of sequence motifs in exons or flanking introns. The occurrence of predicted regulatory elements showed strong correlation with the expression level of known RNA binding proteins, suggesting the high quality of the identified cis-regulatory elements

Avoiding Drama in the Lab: Using Roleplaying to Teach Research Ethics

As undergraduate students gain research experience, they may find themselves in ethically-sensitive situations for which they have no training. In an effort to get students thinking and talking about research ethics, a workshop was held at an annual undergraduate research symposium. Student actors portrayed situations while faculty moderators challenged the audience to discuss the issues. Representatives from the Office of Research Integrity were present to clarify guidelines as necessary. Commonalities and differences were found between the natural and social sciences, as well as between individuals. This pedagogical approach successfully challenged students to realize the ethically-sensitive situations they may encounter

Deletion of the Basigin gene results in reduced mitochondria in the neural retina

Basigin-null mice are characterized as blind from the time of eye opening, with degeneration of the retina beginning at 8 weeks of age, and progressing until the entire photoreceptor cell layer is destroyed. It is likely that a metabolic deficiency underlies the blindness and degeneration phenotypes, as it has been determined that Basigin-null mice do not express the transporter protein monocarboxylate transporter 1 on the membrane of photoreceptor cells and inner segments, nor Müller cells of the neural retina, as is observed in normal mice. The purpose of the present study was to assess the health of mitochondria in normal and Basigin-null mice, specifically to determine if mitochondria within the Basigin-null mouse neural retina are metabolically active. This was achieved via a measurement of cytochrome c concentration and the expression of autophagy-specific proteins via ELISA analyses. Additionally, Mitotracker dyes were used to assess the number and relative activity of mitochondria. It was determined that cytochrome C concentrations and expression of autophagy-specific proteins were not increased in Basigin-null animals, as compared to control animals. Also, while Basigin-null mice do have metabolically active mitochondria, the amount of mitochondria was greatly reduced, when compared to control animals. The results suggest that a reduction in mitochondria is a result, rather than the cause, of the metabolic deficiency observed in Basigin-null mice, and likely occurs because of reduced metabolic activity in the absence of MCT1 expression