Expression Of Olig2 Research Articles

Association between brain [18F]FDG‐PET signal and genetic cell markers in the brain tissue

AbstractBackgroundBrain glucose hypometabolism, indexed by [18F]FDG‐PET, is considered a biomarker of neurodegeneration in Alzheimer’s disease (AD). However, other brain cells, such as astrocytes and microglia, also consume considerable amounts of glucose and may contribute to the [18F]FDG‐PET signal in the brain. Thus, the cellular source of the [18F]FDG‐PET signal remains controversial. Here, we aimed to evaluate whether canonical markers of different brain cell types (neuron, oligodendrocyte, astrocyte, and microglia) associate with brain [18F]FDG‐PET signal in humans and a rat model of human amyloidosis.Method[18F]FDG‐PET images were acquired in ten‐month‐old APP/PS1 (TgF344‐AD, n = 8) and wild‐type (WT, n = 6) rats. Next, we evaluated the gene expression of NeuN, Olig2, GFAP, S100B, and IBA1 in the frontal and temporoparietal cortices and cerebellum. Association maps integrating mRNA with brain [18F]FDG‐PET signal were conducted at the voxel level using RMINC. [18F]FDG‐PET and brain tissue mRNA (GFAP, ACSA2, S100B, GLAST, IBA1, CSF1R, TREM2, Olig2, Cldn11, NeuN) of cognitively unimpaired patients were obtained from ADNI and Allen Brain Atlas database, respectively. In humans, mean regional [18F]FDG‐PET SUVr (reference region: pons) and mRNA expression were used to evaluate their association using a gaussian model. Differences were considered statistically significant at p<0.05 (t>2).ResultGFAP and S100B mRNA levels in the temporoparietal and frontal cortices positively correlated with brain [18F]FDG‐PET signal (local maxima, GFAPt(13) = 9.4, S100Bt(13) = 6.6; Fig1B‐C). No associations were found with TREM2, Olig2 and NeuN (t(13)<2; Fig1D‐F). We expanded our analysis by comparing gene astrocyte markers and astrocyte gene cell markers in humans. At the regional level, we found that FDG‐PET SUVr and astrocyte mRNA were positively associated: S100B (R‐squared = 0.34), GFAP (R‐squared = 0.33), GLAST (R‐squared = 0.31) and ACSA‐2 (R‐squared = 0.31; Fig1G).ConclusionOur findings suggest astrocyte mRNA markers are more closely associated with brain [18F]FDG‐PET signal than neuronal, oligodendrocyte, and microglial mRNA markers.

Association between brain [18F]FDG‐PET signal and genetic cell markers in the brain tissue

AbstractBackgroundBrain glucose hypometabolism, indexed by [18F]FDG‐PET, is considered a biomarker of neurodegeneration in Alzheimer’s disease (AD). However, other brain cells, such as astrocytes and microglia, also consume considerable amounts of glucose and may contribute to the [18F]FDG‐PET signal in the brain. Thus, the cellular source of the [18F]FDG‐PET signal remains controversial. Here, we aimed to evaluate whether canonical markers of different brain cell types (neuron, oligodendrocyte, astrocyte, and microglia) associate with brain [18F]FDG‐PET signal in humans and a rat model of human amyloidosis.Method[18F]FDG‐PET images were acquired in ten‐month‐old APP/PS1 (TgF344‐AD, n = 8) and wild‐type (WT, n = 6) rats. Next, we evaluated the gene expression of NeuN, Olig2, GFAP, S100B, and IBA1 in the frontal and temporoparietal cortices and cerebellum. Association maps integrating mRNA with brain [18F]FDG‐PET signal were conducted at the voxel level using RMINC. [18F]FDG‐PET and brain tissue mRNA (GFAP, ACSA2, S100B, GLAST, IBA1, CSF1R, TREM2, Olig2, Cldn11, NeuN) of cognitively unimpaired patients were obtained from ADNI and Allen Brain Atlas database, respectively. In humans, mean regional [18F]FDG‐PET SUVr (reference region: pons) and mRNA expression were used to evaluate their association using a gaussian model. Differences were considered statistically significant at p<0.05 (t>2).ResultGFAP and S100B mRNA levels in the temporoparietal and frontal cortices positively correlated with brain [18F]FDG‐PET signal (local maxima, GFAPt(13) = 9.4, S100Bt(13) = 6.6; Fig1B‐C). No associations were found with TREM2, Olig2 and NeuN (t(13)<2; Fig1D‐F). We expanded our analysis by comparing gene astrocyte markers and astrocyte gene cell markers in humans. At the regional level, we found that FDG‐PET SUVr and astrocyte mRNA were positively associated: S100B (R‐squared = 0.34), GFAP (R‐squared = 0.33), GLAST (R‐squared = 0.31) and ACSA‐2 (R‐squared = 0.31; Fig1G).ConclusionOur findings suggest astrocyte mRNA markers are more closely associated with brain [18F]FDG‐PET signal than neuronal, oligodendrocyte, and microglial mRNA markers.

Anterior mediastinal neuroblastoma in an adult: an additional case of a rare tumor in an unusual location with review of the literature

Neuroblastoma is rare in the adult population, especially in thoracic or mediastinal locations, with only 25 previously reported cases. We report an additional example of primary thymic neuroblastoma in a previously asymptomatic 71-year-old man with an anterior mediastinal mass who underwent robotic excision with pericardium and adjacent lung. The tumor was a 5.2 cm partially encapsulated, white-tan and rubbery mass with grossly identifiable areas of necrosis (25%) and hemorrhage. Histologically, the specimen showed a rim of adipose tissue and residual thymic tissue with areas of cystic thymic epithelium and prominent lymphoid tissue containing Hassall’s corpuscles. The tumor was composed of uniform, round cells with scant cytoplasm and small nuclei with inconspicuous nucleoli set within a background of conspicuous neuropil. Mitotic figures were easily found. By immunohistochemistry, the tumor cells expressed synaptophysin, chromogranin, NKX2.2 (diffuse, nuclear), GFAP (patchy), SMI31 (neurofilament) (focal, cytoplasmic), and TdT (diffuse, nuclear), while lacking expression of CD99, TTF-1, CK 20, MCPyV, PHOX2B, Olig2, OCT3/4, CD45, CD3 and PAX5. S100 protein was negative in the neuroblastic cells, with scattered positive cells in a vague sustentacular-like pattern. Fluorescence in situ hybridization for isochromosome 12p and EWSR1 gene rearrangement were negative. As thymic neuroblastoma is extremely rare in adults, a neuroblastic tumor of germ cell origin (either primary or metastatic) or spread from a sinonasal tract tumor should be excluded because of differing treatments and prognoses. The properties of these rare neoplasms appear similar to olfactory neuroblastoma rather than pediatric-type neuroblastoma.

STEM-15. STAT5-OLIG2 TRANSCRIPTIONAL COMPLEX MEDIATES GLIOMA STEM CELL SELF RENEWAL AND INVASION

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, despite current treatment options. One of the major contributing factors to this dismal outcome is a subset of cells called glioma stem cells (GSCs) that are highly tumorigenic, invasive, and resistant to conventional treatments. Thus, studies that focus on the properties of GSC biology are needed to develop novel therapies to improve overall survival. Here we report a novel role of STAT5 in GSC stemness and survival. In GBM tumors, STAT5 has been shown to be active in the infiltrative, residual population of cells remaining post-surgery. Knockdown of STAT5 results in the decrease in GSC stemness and therapeutic resistance. Interestingly, we find that expression of OLIG2, a key cell fate factor important for the tumorigenic potential of GSCs, affects STAT5 activation with a positive correlation between OLIG2 and STAT5 mRNA expression in GBM tumors. Furthermore, we find that in GSCs expressing EGFRvIII, STAT5 interacts with OLIG2 and pharmacological inhibition of STAT5 activation or knockdown of STAT5 expression results in decrease of OLIG2-mediated GSC invasion. Together, these data suggest a novel transcriptional complex, STAT5-OLIG2, in the potential role for GSC survival, maintenance, and invasion.

MODL-12. REGULATION OF OLIGODENDROGLIAL PROGENITOR CELL (OPC) CELL FATE IN DIFFUSE MIDLINE GLIOMA (DMG) BY THE HOMEOBOX TRANSCRIPTION FACTOR DLX2

Abstract Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. 80% of patients with DMG harbor K27M mutations in canonical histone variants H3.3 and H3.1, leading to global epigenetic dysregulation and an oligodendroglial progenitor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. Our lab has previously identified DLX2 transcription factor occupancy of genes required for OPC differentiation in vivo (Olig2, Nkx2.2, Myt1, Plp1), and shown that co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. Likewise, increased expression of Olig2, Nkx2.2, and Plp-1 are observed in a Dlx1/Dlx2 double knockout (DKO) mouse model. Transient overexpression of a Dlx2-GFP construct into cells of a genetically engineered mouse model (GEMM) of DMG resulted in increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, and a reduction in migration, invasion, and colony formation in vitro. Inverse expression of DLX2 and OLIG1/2 was observed in a bulk RNAseq database of 45 human DMG cell lines. We have established a pipeline to evaluate cell fate changes following changes in DLX2 expression levels. OLIG1/2high/DLX2low DMG expressing cell lines underwent stable transfection of a DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone marks (H3K27me3, H3K27ac), K27M, and OPC signature genes will be presented with comparison of altered OPC genes from the DKO mouse as determined by RNA-seq. The manipulation of DLX2 expression in human DMG cells in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.

BIOM-10. KI-67 IS ASSOCIATED WITH PROGRESSION-FREE SURVIVAL IN PATIENTS WITH GLIOBLASTOMA

Abstract Identifying predictive and prognostic markers for glioblastoma (GBM) is an area of intense investigation. The objective of this study was to evaluate OLIG2 and Ki-67 as prognostic biomarkers. A tissue microarray with clinical data was created for 70 patients diagnosed with GBM between 2002-2007 at a single institution. Immunohistochemistry was performed to evaluate expression of OLIG2 and Ki-67. Overall survival and progression free survival (PFS) were calculated using the Kaplan-Meier method. Univariable (UVA) and multivariable (MVA) analysis was performed using Cox proportional hazards modeling. The median age of diagnosis was 63, 66% of patients were male, 80% had a KPS ≥70, 64% had a subtotal resection or biopsy, and 36% had a gross total resection. Median OS and PFS were 13.3 months and 9.5 months respectively. For patients who received resection plus chemoradiation, median OS and PFS were 16.7 months and 9.5 months respectively. 36.4% of patient samples had Ki-67 expression fraction > 20%. 18.0% had heavy OLIG2 staining. On MVA, lower KPS was associated with shorter OS and radiation therapy was associated with longer OS. OLIG2 was not associated with OS or PFS. Patients who had a Ki-67 < 20% had a shorter PFS, but not OS on MVA. Our data demonstrate higher Ki-67 levels correlate with improved PFS without an OS benefit. This association persisted for patients receiving standard of care treatment. OLIG2 levels did not correlate with PFS or OS in either cohort. Our study indicates that further investigation is needed to determine whether Ki-67 is useful in predicting recurrence risk and potentially changing treatment strategies for these patients, as well as the mechanism behind the associated improvement seen with higher Ki-67 levels. These data are currently limited by lack of IDH and MGMT status but suggest Ki-67 should be further investigated as a biomarker for progression.

CELF2 Sustains a Proliferating/OLIG2+ Glioblastoma Cell Phenotype via the Epigenetic Repression of SOX3.

Glioblastomas (GBs) are incurable brain tumors. The persistence of aggressive stem-like tumor cells after cytotoxic treatments compromises therapeutic efficacy, leading to GBM recurrence. Forcing the GBM cells to irreversibly abandon their aggressive stem-like phenotype may offer an alternative to conventional cytotoxic treatments. Here, we show that the RNA binding protein CELF2 is strongly expressed in mitotic and OLIG2-positive GBM cells, while it is downregulated in differentiated and non-mitotic cells by miR-199a-3p, exemplifying GBM intra-tumor heterogeneity. Using patient-derived cells and human GBM samples, we demonstrate that CELF2 plays a key role in maintaining the proliferative/OLIG2 cell phenotype with clonal and tumorigenic properties. Indeed, we show that CELF2 deficiency in patient-derived GSCs drastically reduced tumor growth in the brains of nude mice. We further show that CELF2 promotes TRIM28 and G9a expression, which drive a H3K9me3 epigenetic profile responsible for the silencing of the SOX3 gene. Thus, CELF2, which is positively correlated with OLIG2 and Ki67 expression in human GBM samples, is inversely correlated with SOX3 and miR-199a-3p. Accordingly, the invalidation of SOX3 in CELF2-deficient patient-derived cells rescued proliferation and OLIG2 expression. Finally, patients expressing SOX3 above the median level of expression tend to have a longer life expectancy. CELF2 is therefore a crucial target for the malignant potential of GBM and warrants attention when developing novel anticancer strategies.

Retraction Note: Effect of catalpol on remyelination through experimental autoimmune encephalomyelitis acting to promote Olig1 and Olig2 expressions in mice

Retraction Note: Effect of catalpol on remyelination through experimental autoimmune encephalomyelitis acting to promote Olig1 and Olig2 expressions in mice

Astrocyte lipocalin-2 modestly effects disease severity in a mouse model of multiple sclerosis while reducing mature oligodendrocyte protein and mRNA expression

Roles for lipocalin-2 (LCN2, also referred to as neutrophil gelatinase associated lipocalin, NGAL) in the progression of disease in multiple sclerosis and its animal models have been reported; however, the importance of astrocyte-derived LCN2, a major source of LCN2, have not been defined. We found that clinical scores in experimental autoimmune encephalomyelitis (EAE) were modestly delayed in mice with conditional knockout of LCN2 from astrocytes, associated with a small decrease in astrocyte GFAP expression. Immunostaining and qPCR of spinal cord samples showed decreased oligodendrocyte proteolipid protein and transcription factor Olig2 expression, but no changes in PDGFRα expression. These results suggest astrocyte LCN2 contributes to early events in EAE and reduces damage to mature oligodendrocytes at later times.

Evaluation of BCAS1-positive immature oligodendrocytes after cerebral ischemic stroke and SVD

Ischemic cerebrovascular disease is an important cause of physical disability and dementia. Oligodendrocytes (OLGs), which differentiate from oligodendrocyte precursor cells (OPCs), are crucial for remyelination of the damaged brain and functional recovery. Breast carcinoma amplified sequence 1 (BCAS1) has recently been shown to be highly expressed in newly formed pre-myelinating oligodendrocytes (pre-mOLGs), while its expression level is reduced in mature OLGs. In this study, we analyzed BCAS1 expression by immunohistochemical analysis of human post-mortem brain tissue from six stroke patients (death within 2 months after stroke onset) and eight small vessel disease (SVD) patients. Control post-mortem brain tissue was from eight age-matched patients without any obvious central nervous system (CNS) pathology. The Olig2 expression in the area corresponding to the same section of the BCAS1-stained slice was analyzed to determine the total oligodendrocyte lineage. The percentage of differentiating OPCs in the oligodendrocyte lineage was calculated as the ratio of BCAS1+ to Olig2+ cells (BCAS1+/Olig2+). The stroke and SVD cases showed demyelination with decreased expression of myelin basic protein (MBP, a mature OLG marker). The stroke cases showed significantly increased numbers of early-stage BCAS1+ cells with an immature morphology and Olig2+ cells (pan-oligodendrocyte lineages) in the peri-infarct areas in both the cortex and white matter, but showed no increase in the number of late-stage BCAS1+ cells with a mature morphology. In contrast, the SVD cases showed no significant increase in Olig2+ and BCAS1+ cells. These results indicated that remyelination dysfunction could be attributed to insufficient maturation of OPCs in stroke and impaired recruitment of OPCs in SVD.

Alterations of oligodendrocyte progenitor cells (OPCs) with survival time in humans following high impact brain trauma

BackgroundAs there is a lack of comprehensive literature regarding the molecular environment of the human brain emphasizing on oligodendrocyte progenitor cells (OPCs) following high impact brain trauma. The protagonist of OPCs post severe traumatic brain injury (sTBI) provides a significant thrust towards estimating time elapsed since trauma as well as developing novel therapeutic approaches. The present study was carried out to study post trauma alterations pertaining to myelin sheath and oligodendrocyte response with survival time. Materials and methodsIn the present study, victims (both male and female) of sTBI (n = 64) were recruited and contrasted with age and gender matched controls (n = 12). Post mortem brain samples from corpus callosum and grey white matter interface were collected during autopsy examination. Extent of myelin degradation and response of OPC markers Olig-2 and PDGFR-α were evaluated using immunohistochemistry and qRT-PCR. STATA 14.0 statistical software was used for data analysis with P-value<0.05 considered statistically significant. ResultsTimewise qualitative correlation with extent of demyelination performed using LFB-PAS/IHC-MBP, IHC Olig-2 and mRNA expression revealed tendency towards remyelination in both corpus callosum and grey white matter interface. Number of Olig-2 positive cells was significantly higher in sTBI group as compared to control group (P-value: 0.0001). Moreover, mRNA expression studies of Olig-2 showed significant upregulation in sTBI patients. mRNA expression of Olig-2 and PDGFR-α in sTBI patients showed significant variation with respect to survival time (p value:0.0001). ConclusionDetailed assessment of post TBI changes implementing various immunohistochemical and molecular techniques shall potentially reveal intriguing and important inferences in medicolegal practices and neurotherapeutics.

DIPG-21. LINKING DETERMINATION OF CELL FATE IN THE DEVELOPING CENTRAL NERVOUS SYSTEM TO DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. Next-generation sequencing has revealed that the majority of patients with DMG harbor K27M mutations in canonical histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and an oligodendrocyte precursor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. We identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required in OPC differentiation in vivo. Co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. The Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, as well as a reduction in migration, invasion, and colony formation in vitro. An RNAseq database containing 45 human DMG cell lines showed an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DLX2high/OLIG1/2low cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2high/DLX2low expressing cell lines underwent stable transfection of an DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone H3 marks (K27me3, K27ac), K27M, and OPC signature genes will be presented along with comparison of altered OPC genes from the Dlx1/Dlx2 DKO mouse as determined by RNAseq. The manipulation of DLX2 expression in human DMG cells both in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.

Oligodendroglia Confer Neuroprotection to NSC-34 Motor Neuronal Cells Against the Toxic Insults of Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder with multifactorial pathomechanisms affecting not only motor neurons but also glia. Both astrocytes and microglia get activated and contribute significantly to neurodegeneration. The role of oligodendroglia in such a situation remains obscure, especially in the sporadic form of ALS (SALS), which contributes to 90% of cases. Here, we have investigated the role of oligodendroglia in SALS pathophysiology using a human oligodendroglial cell line, MO3.13, by exposing the cells to cerebrospinal fluid from SALS patients (ALS-CSF; 10% v/v for 48 h). ALS-CSF significantly reduced the viability of MO3.13 cells and down-regulated the expression of oligodendroglia-specific proteins, namely, CNPase and Olig2. Furthermore, to investigate the effect of the observed oligodendroglial changes on motor neurons, NSC-34 motor neuronal cells were co-cultured/supplemented with conditioned/spent medium of MO3.13 cells upon exposure to ALS-CSF. Live cell imaging experiments revealed protection to NSC-34 cells against ALS-CSF toxicity upon co-culture with MO3.13 cells. This was evidenced by the absence of neuronal cytoplasmic vacuolation and beading of neurites, which instead resulted in better neuronal differentiation. Enhanced lactate levels and increased expression of its transporter, MCT-1, with sustained expression of trophic factors, namely, GDNF and BDNF, by MO3.13 cells hint towards metabolic and trophic support provided by the surviving oligodendroglia. Similar metabolic changes were seen in the lumbar spinal cord oligodendroglia of rat neonates intrathecally injected with ALS-CSF. The findings indicate that oligodendroglia are indeed rescuer to the degenerating motor neurons when the astrocytes and microglia turn topsy-turvy.

Overexpression of OLIG2 and MYT1L Transcription Factors Enhance the Differentiation Potential of Human Mesenchymal Stem Cells into Oligodendrocytes

Demyelinating diseases represent a broad spectrum of disorders and are characterized by the loss of specialized glial cells (oligodendrocytes), which eventually leads to neuronal degeneration. Stem cell-based regenerative approaches provide therapeutic options to regenerate demyelination-induced neurodegeneration. The current study aims to explore the role of oligodendrocyte-specific transcription factors (OLIG2 and MYT1L) under suitable media composition to facilitate human umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) differentiation toward oligodendrocyte for their potential use to treat demyelinating disorders. hUC-MSCs were isolated, cultured, and characterized based on their morphological and phenotypic characteristics. hUC-MSCs were transfected with OLIG2 and MYT1L transcription factors individually and in synergistic (OLIG2 + MYT1L) groups using a lipofectamine-based transfection method and incubated under two different media compositions (normal and oligo induction media). Transfected hUC-MSCs were assessed for lineage specification and differentiation using qPCR. Differentiation was also analyzed via immunocytochemistry by determining the expression of oligodendrocyte-specific proteins. All the transfected groups showed significant upregulation of GFAP and OLIG2 with downregulation of NES, demonstrating the MSC commitment toward the glial lineage. Transfected groups also presented significant overexpression of oligodendrocyte-specific markers (SOX10, NKX2.2, GALC, CNP, CSPG4, MBP, and PLP1). Immunocytochemical analysis showed intense expression of OLIG2, MYT1L, and NG2 proteins in both normal and oligo induction media after 3 and 7 days. The study concludes that OLIG2 and MYT1L have the potential to differentiate hUC-MSCs into oligodendrocyte-like cells, which is greatly facilitated by the oligo induction medium. The study may serve as a promising cell-based therapeutic strategy against demyelination-induced neuronal degeneration.

Promoting the Differentiation of Neural Progenitor Cells into Oligodendrocytes through the Induction of Olig2 Expression: A Transcriptomic Study Using RNA-seq Analysis.

Oligodendrocytes are the myelinating cells of the central nervous system that facilitate efficient signal transduction. The loss of these cells and the associated myelin sheath can lead to profound functional deficits. Moreover, oligodendrocytes also play key roles in mediating glial-neuronal interactions, which further speaks to their importance in health and disease. Neural progenitor cells (NPCs) are a promising source of cells for the treatment of oligodendrocyte-related neurological diseases due to their ability to differentiate into a variety of cell types, including oligodendrocytes. However, the efficiency of oligodendrocyte differentiation is often low. In this study, we induced the expression of the Olig2 transcription factor in tripotent NPCs using a doxycycline-inducible promoter, such that the extent of oligodendrocyte differentiation could be carefully regulated. We characterized the differentiation profile and the transcriptome of these inducible oligodendrogenic NPCs (ioNPCs) using a combination of qRT-PCR, immunocytochemistry and RNA sequencing with gene ontology (GO) and gene set enrichment analysis (GSEA). Our results show that the ioNPCs differentiated into a significantly greater proportion of oligodendrocytes than the NPCs. The induction of Olig2 expression was also associated with the upregulation of genes involved in oligodendrocyte development and function, as well as the downregulation of genes involved in other cell lineages. The GO and GSEA analyses further corroborated the oligodendrocyte specification of the ioNPCs.

Long-term Culture of Cerebral Organoid Reveals Disruption of Glial Cell Differentiation and Glutamate Excitotoxicity in Multiple Sclerosis. (P2-3.002)

<h3>Objective:</h3> Analyze differentiation and maturation of glial cells and neurons in long-term cultured cerebral organoids derived from induced pluripotent stem cells of patients with multiple sclerosis (PwMS). <h3>Background:</h3> MS is an auto-immune disease characterized by inflammation, demyelination and neural degeneration. It has been shown that environmental and genetic factors contribute to the development of MS, however, the etiology of this condition is unknown. Our previous research showed that organoids from PwMS display an exhaustion of the neural progenitor pool and an increase in neurogenesis. Here, we studied differentiation of neurons into glutamate and GABAergic neurons as glutamate excitotoxicity has been proposed to lead to inflammation and neuronal injury in MS. We also investigated glial cell differentiation and maturation in astrocytes and oligodendrocytes to determine their role in MS pathogenesis. <h3>Design/Methods:</h3> We analyzed neural cell differentiation in neuronal and glial lineages along with myelination capacity by immunofluorescence in MS cerebral organoids compared to healthy control organoids, after 120 days in vitro. <h3>Results:</h3> <i>A</i> strong decrease of Olig2 expression in MS organoids was detected highlighting a lower number of oligodendrocyte lineage cells. Moreover, no colocalization of Olig2 and APC was observed in MS, while multiple double positive cells were detected in control organoids, showing a defect of oligodendrocytes maturation. In parallel, GFAP staining showed a decrease of GFAP expression in MS organoids compared to control. We then analyzed GABAergic and glutamatergic differentiation. A strong increase of glutamatergic marker vGlut1 and a decrease of GABAergic marker GAD67 was detected at d120 in MS organoids compared to control signifying an imbalance of excitatory and inhibitory neurons. <h3>Conclusions:</h3> Mature cerebral organoids developed from PwMS represent an innovative tool to study MS and allowed us to detect a defect of astrocyte and oligodendrocyte differentiation and maturation as well as an imbalance of GABA/Glutamate release in MS organoids <b>Disclosure:</b> Mr. Mehta has nothing to disclose. Ms. McDermott has nothing to disclose. Nicolas Daviaud has nothing to disclose. Dr. Sadiq has nothing to disclose.

Nose to brain delivery of ibudilast micelles for treatment of multiple sclerosis in an experimental autoimmune encephalomyelitis animal model

Multiple sclerosis is a chronic inflammatory disease of the central nervous system ultimate to neurodegeneration and demyelination. Ibudilast is a phosphodiesterase inhibitor, effective on the function of glial cells and lymphocytes, and inhibits the release of TNF-α by inflammatory cells. Dysregulation of glia is one of the most important pathological causes of MS. Therefore, ibudilast as a glial attenuator can be a useful treatment. The objective of the present study was to investigate the effect of nasal spray of polydopamine coated micelles of surfactin, a biosurfactant, loaded with ibudilast on its brain targeted delivery and effectiveness in remylination and neuroprotection in animal model of MS. In animal studies the micelles were administrated intranasally in different doses of 10, 25 and 50 mg/kg/day in C57/BL6 mice immunized by experimental autoimmune encephalomyelitis (EAE) model. The results of Luxol fast blue staining indicated increment in myelin fiber percent more significantly (p < 0.05) in the groups treated with the polydopamine coated micelles (PDAM) compared to nasal spray of free drug or oral administration. These formulations also increased expression of Mbp, Olig2 and Mog genes in the corpus callosum. These results suggest a positive outcome of polydopamine coated micelles loaded with ibudilast in active MS as an anti-inflammatory and neuroprotective agent.

Abstract 2162: Preliminary analysis of circulating biomarkers of glioblastoma response during chemoradiotherapy

Abstract Intro. Glioblastoma is the most common and aggressive adult brain cancer, with a 5-year survival of less than 15%. Treatment decisions are guided by imaging, which at early timepoints cannot distinguish tumor pseudoprogression from non-response. Our work seeks to identify circulating biomarkers that can classify response as an alternative or adjunct to imaging. Here we present preliminary data exploring the potential of circulating cytokines and tumor cells collected before, during, and after treatment with the goal to identify potential makers for treatment adaptation. Methods. After consent, patient blood was collected the week before initiating chemoradiotherapy, weekly during treatment, and three weeks after treatment. Serum was tested by multiplex bead-based immunoassay (Legendplex Human Inflammation Panel 1, Biolegend inc.) targeting 13 cytokines and ELISA targeting soluble Human GFAP (Glial fibrillary acidic protein) (Biovendor R&amp;D). The Circulogix filtration system was also used to capture cells of size consistent with circulating tumor cells (CTCs). Initial efforts focused on cells co-staining for GFAP and Olig2 and negative for CD45 (hematopoietic marker). All patient tumor specimens and cell lines were verified for GFAP and Olig2 expression by IHC. We selected 13 patients for the current analysis: 5 with growth due to non-response (NR), 6 stable disease (S) and two pseudo progression (PP). These classifications were evaluated radiologically at the first follow up after chemoradiotherapy (S or no) and based on whether changes observed stabilized or resolved spontaneously within six months or continued to progress (NR or PP). Results. The immunoassays detected approximately 70% of the analytes in our samples. Among those, IL10 and IL33 were higher before treatment on patients later determined to have stable disease (IL10 p=0.09; IL33 p=0.043 KW test; IL10 median S=22.93 ng/mL, NR=13.415ng/mL; IL33 median S= 164.10ng/mL, NR=55.57ng/mL). Interestingly, IL33 is reduced on stable patients during week 5 of treatment (PRE vs. W5 p=0.033; median PRE=164.1ng/mL, W5=57.57ng/mL). GFAP presented a slightly lower level after treatment in stable patients (S vs. NR p=0.014; median S=0.441 ng/ml, NR=0.473 ng/ml). As proof of concept, 50 GBM cells from each of U87 adherent cells, or 0913 and 0821 neurosphere cells (CD45 negative) glioblastoma cell lines were mixed in separate vials with 106 hematopoietic cells (RAJI; CD45 positive) and filtered per the procedure to identify CTCs in patient blood. We separated GBM cells from hematopoietic cells on the filtration system. Conclusion. Our preliminary data suggest that blood obtained during chemoradiotherapy can identify biomarkers associated with response. For example, IL10, IL33, GFAP, and CTCs might be used to classify treatment response at very early timepoints. We continue to recruit additional patients for additional analyses. Citation Format: Paulo Roberto Del Valle, Jonathan B. Bell, Scott M. Welford, Kaylie Kullison, Alessandro Valderrama, Gregory A. Azzam, Jessica J. Meshman, Macarena I. De La Fuente, Eric A. Mellon. Preliminary analysis of circulating biomarkers of glioblastoma response during chemoradiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2162.

Inhibition of CXCR2 enhances CNS remyelination via modulating PDE10A/cAMP signaling pathway

CXC chemokine receptor 2 (CXCR2) plays an important role in demyelinating diseases, but the detailed mechanisms were not yet clarified. In the present study, we mainly investigated the critical function and the potential molecular mechanisms of CXCR2 on oligodendrocyte precursor cell (OPC) differentiation and remyelination. The present study demonstrated that inhibiting CXCR2 significantly enhanced OPC differentiation and remyelination in primary cultured OPCs and ethidium bromide (EB)-intoxicated rats by facilitating the formation of myelin proteins, including PDGFRα, MBP, MAG, MOG, and Caspr. Further investigation identified phosphodiesterase 10A (PDE10A) as a main downstream protein of CXCR2, interacting with the receptor to regulate OPC differentiation, in that inhibition of CXCR2 reduced PDE10A expression while suppression of PDE10A did not affect CXCR2. Furthermore, inhibition of PDE10A promoted OPC differentiation, whereas overexpression of PDE10A down-regulated OPC differentiation. Our data also revealed that inhibition of CXCR2/PDE10A activated the cAMP/ERK1/2 signaling pathway, and up-regulated the expression of key transcription factors, including SOX10, OLIG2, MYRF, and ZFP24, that ultimately promoted remyelination and myelin protein biosynthesis. In conclusion, our findings suggested that inhibition of CXCR2 promoted OPC differentiation and enhanced remyelination by regulating PDE10A/cAMP/ERK1/2 signaling pathway. The present data also highlighted that CXCR2 may serve as a potential target for the treatment of demyelination diseases.

Prognostic model for assessing the human glioma cell malignancy grade based on MDM2, MELK, SOX2, CDK4, DR5 and OCT4 gene expression

We analyzed the relationship between expression of marker genes TUBB3, CD133, CDK4, CDK6, CIRBP, DR4, DR5, EGFR, FGFR, FSHR, GDNF, GFAP, L1CAM, LEF1, MAP2, MDM2, MELK, NANOG, NOTCH2, OCT4, OLIG2, PDGFRA, PDGFA, PDGFB and SOX2 and glioma cell malignancy grade, as well as created appropriate prognostic model. We analyzed expression of 25 marker genes in 22 samples of human glioma cultures using quantitative real-time PCR. Statistical analysis was performed using the IBM SPSS Statistics 26.0 software. We used the Kolmogorov-Smirnov and Shapiro-Wilk tests to assess distribution normality. Nonparametric Jonckheere-Terpstra and Spearman tests were applied. We obtained a prognostic model for assessing the grade III and IV glioma cell malignancy based on expression of marker genes MDM2, MELK, SOX2, CDK4, DR5 and OCT4. Predictive accuracy was 83% (Akaike information criterion -55.125).