Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the... more Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the NF1 tumor suppressor gene in the Schwann cell lineage. To understand whether neurofibroma formation is possible after birth, we induced Nf1 loss of function with an inducible proteolipid protein Cre allele. Perinatal loss of Nf1 resulted in the development of small plexiform neurofibromas late in life, whereas loss in adulthood caused large plexiform neurofibromas and morbidity beginning 4 months after onset of Nf1 loss. A conditional EGFP reporter allele identified cells showing recombination, including peripheral ganglia satellite cells, peripheral nerve S100bþ myelinating Schwann cells, and peripheral nerve p75þ cells. Neurofibromas contained cells with Remak bundle disruption but no recombination within GFAPþ nonmyelinating Schwann cells. Extramedullary lymphohematopoietic expansion was also observed in PlpCre;Nf1fl/fl mice. These tumors contained EGFPþ/Sca-1þ stromal cells among EGFP-negative lympho-hematopoietic cells indicating a noncell autonomous effect and unveiling a role of Nf1-deleted microenvironment on lympho-hematopoietic proliferation in vivo. Together these findings define a tumor suppressor role for Nf1 in the adult and narrow the range of potential neurofibromainitiating cell populations. Cancer Res; 71(13); 4675-85. Ó2011 AACR.
There are pluripotent stem cells in the adult brain that might not be very different from those f... more There are pluripotent stem cells in the adult brain that might not be very different from those found in bone marrow. Recent and profound advances in the field of neuropoiesis, which often rely on insights from studies of hematopoiesis and in some instances use cross-paradigms with this field, have already revealed that bone marrow has much in common with so-called 'brain marrow'. Proliferative primogenitors and developmentally regulated molecules are hallmarks of both neuropoiesis and hematopoiesis. This article will focus on recent advances in neuropoiesis within a central core of the mature brain that is referred to as brain marrow, discussing its pluripotency and proliferative capacity, in vitro and molecular assays used in its study, and markers of neuropoietic stem/progenitor cells. As hematopoiesis research has led to the discovery of numerous morphogenetic factors, it is anticipated that studies of neuropoiesis should also uncover many new factors and genes that affect the growth and differentiation of neural cells. Recent breakthroughs in the stem-cell field prompt an inclusion of rationale for the persistence of normal stem/progenitor cells even in the aged brain. By analogy with hematopoiesis research, a thorough investigation of brain marrow should provide basic insights into developmental and persistent neurogenesis while anticipating cell-transplant and gene therapies for debilitating neurological diseases.
Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes 30-50 % of affected i... more Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes 30-50 % of affected individuals to develop plexiform neurofibromas. We found that macrophage infiltration of both mouse and human neurofibromas correlates with disease progression. Macrophages accounted for almost half of neurofibroma cells, leading us to hypothesize that nerve macrophages are inflammatory effectors in neurofibroma development and/or growth. We tested the effects of PLX3397, a dual kit/fms kinase inhibitor that blocks macrophage infiltration, in the Dhh-Cre; Nf1 flox/flox mouse model of GEM grade I neurofibroma. In mice aged 1-4 months, prior to development of nerve pathology and neurofibroma formation, PLX3397 did not impair tumor initiation and increased tumor volume compared to controls. However, in mice aged 7-9 months, after tumor establishment, a subset of mice demonstrating the largest reductions in macrophages after PLX3397 exhibited cell death and tumor volume regression. Macrophages are likely to provide an initial line of defense against developing tumors. Once tumors are established, they become tumor permissive. Macrophage depletion may result in impaired tumor maintenance and represent a therapeutic strategy for neurofibroma therapy.
PLPCre;Nf1fl/fl mice. Gross micrographs (left panels) show representative malignant Grade III GEM... more PLPCre;Nf1fl/fl mice. Gross micrographs (left panels) show representative malignant Grade III GEM peripheral nerve sheath tumors (PNST). Arrows in the lower left panel point to two hindlimb tumors. Rulers show 1mm markings. In panels at right, H&E staining of paraffin sections of these tumors are shown. A representative anti-S100β stain (DAB, brown reaction product) is shown in the inset on the top right. The star in the bottom right panel designates a prominent nerve bundle. Supplemental Figure 2: EGFP+ non-neural cells in heart, lung, thyroid, and spleen. EGFP immunohistochemistry was performed on a variety of paraffin embedded organs. Tissuespecific EGFP+ cells (green) within the heart (top left), lung (top right), thyroid (bottom left), and spleen (bottom right) 15 months post adult Tamoxifen exposure in PlpCre;Nf1fl/fl animals. Tissue sections were photographed at 20X magnification using 300ms exposure of FITC fluorochrome.
2809 Glioblastoma multiform (GBM) is the most common and most malignant primary brain tumor. We h... more 2809 Glioblastoma multiform (GBM) is the most common and most malignant primary brain tumor. We have reported that the expression of PAX6 is decreased in most GBM patient samples when compared to adjacent normal tissue and anaplastic astrocytomas (AAs). Lower PAX6 expression in tumor tissue is an indication of an unfavorable prognosis for patients with malignant astrocytic gliomas (Zhou et. al. Clin. Can. Res. 9:3369-75). PAX6 encodes a transcription factor critical in the development of the eye and central nervous system. In contrast to the decreased expression of PAX6 in GBMs, we observed an increase in the expression of PAX6 in the GBM cell line U251 after introducing a normal chromosome 10. Loss of heterozygosity (LOH) of chromosome 10 occurs in up to 80% of human GBM cases. Therefore, the reduced expression of PAX6 in GBMs may result from the loss of unidentified tumor suppression gene(s) in chromosome 10; however, this is not a simple side effect of LOH of chromosome 10. Overexpression of PAX6 via stable transfection of a PAX6/pRC-CMV construct in the GBM cell line U251HF suppressed anchorage-independent growth in vivo and tumor formation in vitro. This data is consistent with the hypothesis that PAX6 may have a tumor-suppressor function in GBM (Zhou et. al. J. Neuro-Oncology, in print). Microarray hybridization using Affymetrix chips revealed alterations of matrix metalloproteinase-2 (MMP2) gene expression in the suppressed U251HF PAX6-transfected cells. This data was confirmed by real-time quantitative reverse transcription PCR from cells grown in vitro and in the brain of nude mice. Moreover, there is a reverse correlation between the expression of PAX6 and MMP2 in GBMs, but not in AAs. MMP2 is a well-known protease involved in cell invasion and the malignant progression of gliomas. MMP2 was also shown to be suppressed at a functional level in gelatin zymography assays in PAX6 transfectant cells. This suppression correlates with the near abolition of the ability of all PAX6 transfectants to invade in matrigel invasion assays; however, cells expressing a dominant negative mutant form of PAX6 (PAX6-344) displayed a significant enhancement in their ability to invade in this assay. Luciferase MMP2 promoter assays have indicated that the lower expression level of MMP2 in PAX6-transfected cells is due to lower MMP2 promoter activity. In conclusion, our data indicate that PAX6 plays an important role in suppressing the formation of GBM by suppressing the expression of MMP2. Identification and functional characterization of the cis-element responsible for the suppression of the MMP2 promoter activity by PAX6 is being examined.
International journal of molecular biology, Nov 16, 2018
One common symptom of most neurodegenerative diseases related to aging is the presence of blood-b... more One common symptom of most neurodegenerative diseases related to aging is the presence of blood-brain barrier disruption and mitochondrial disfunction. N-acetylcysteine (NAC) is an FDA approved drug commonly utilized for a variety of neuropsychiatric and neurodegenerative diseases; however, while studies have shown that NAC can improve mitochondrial function in mouse models of some of these diseases, the mechanism(s) of action have yet to be fully defined. Our previous studies have shown that NAC treatment can alter blood-brain barrier (BBB) permeability in mice via changes in reactive oxygen species (ROS). To further examine the effects of NAC on both cell-specificity and the mitochondrial respiration of brain vascular endothelial cells and astrocytes that make up the blood-brain barrier, we utilized brain microvascular endothelial and cerebrum astrocytic cells and an in vitro mouse BBB model. We show that NAC caused time-and dose-dependent changes in BBB permeability that were correlated to significant changes in tight and gap junction proteins, change in endothelial nNOS, and downregulation of mitochondrial complex proteins in endothelial cells. Extracellular flux analysis revealed a significant reduction in endothelial cell mitochondrial maximal respiration after 48hrs. In contrast, astrocyte respiration showed no change after 48hours but was significantly increased after 24hours of NAC treatment corresponding to the decreased permeability noted at this time. These changes paralleled alterations in NOS in endothelial cells but not astrocytes. We conclude that murine astrocytes and endothelial cells respond to NAC treatment in a cell-specific fashion, altering their perspective metabolic profiles and ultimately the tight and gap junctions that determine the permeability of the BBB. Given the current theory that mitochondrial dysfunction and BBB leakiness are predisposing, possibly causative symptoms, for most neurodegenerative diseases associated with aging, our findings suggest that NAC's cell-specific effects upon both metabolism and BBB permeability may be concurrent mechanisms by which this drug is effective.
Background: Ras is a small G-protein switch, activated when GTP-bound; in the GTP-bound state Ras... more Background: Ras is a small G-protein switch, activated when GTP-bound; in the GTP-bound state Ras signals to downstream effector proteins. GTPase inactivating proteins (GAPs) including neurofibromain (Nf1) accelerate hydrolysis of Ras-GTP to inactive Ras-GDP. We showed that activation of Ras or loss of Nf1 in brain oligodendrocytes correlates with myelin decompaction, down-regulation of claudins, and down-regulation and mis-localization of connexins (Mayes et al., Cell Reports, 2013). Non-cell autonomous defects in perivascular astrocytes and blood-brain barrier (BBB) are also observed. The bloodbrain barrier becomes leaky, implicating a soluble mediator. NO and nitric oxide synthases (NOS1-3) are up-regulated in mutant white matter. Treating mice with the NOS inhibitor L-NAME or the antioxidant NAC corrected cellular phenotypes. Consistent with recent data implicating white matter changes in learning, CNP-HRasG12V mice displayed locomotor hyperactivity which could be rescued by antioxidant treatment. Results: Using flow cytometry to define reactive species in cells in sorted brain non-neuronal cells we have now identified alterations in reactive species (NO, SO and/or peroxynitrites) in oligodendrocyte precursors, oligodendrocytes, astrocytes, pericytes and endothelial cells when Ras-GTP is elevated in oligodendrocytes. We also found that many changes are absent in Nf1+/− mice, when cells in the brain in addition to oligodendrocytes are hemizygous for Nf1 loss. Mating mice in which oligodendrocyte only have activated Ras with Nf1+/− mice confirmed that effects on reactive species are mediated by hemizygosity in Nf1+/− astrocytes, pericytes and/or endothelial cells. FACs analysis showed that NO is significantly diminished in oligodendrocytes in these mutants, and shuttled to surrounding cells of the vasculature. Conclusion: We conclude that signaling between oligodendrocytes and cells of white matter blood vessels contributes to homeostasis of the blood-brain barrier. NR is supported by the NIH, the DOD Program in Neurofibromatosis and the Children's Tumor Foundation.
1057 Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. High expression of ... more 1057 Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. High expression of vascular endothelial growth factor ( VEGF ) has been a hallmark of GBM. However, the regulation of VEGF expression in malignant gliomas is not well understood. We have previously reported that the transcription factor PAX6 suppresses the tumorigenecity of glioblastoma cells. In addition, there is a reverse correlation between the expression level of PAX6 and VEGF when human glioma tumors and the surrounding normal tissues were compared, suggesting that PAX6 might regulate VEGF expression. In this study, several techniques such as real-time quantitative reverse transcription PCR, enzyme-linked immunosorbent assay and immunohistochemistry were used to investigate if PAX6 regulates VEGF expression in glioma cell lines. Our results showed a significant reduction of VEGF expression in the PAX6 stable-transfected glioma cell line U251HF grown both in vitro and in subcutaneous xenografts in nude mice. We further demonstrated in two glioma cell lines (U251HF and U87) that VEGF expression was markedly suppressed by adenoviral-mediated overexpression of PAX6, as well as PTEN. Interestingly, in cells when both genes were overexpressed together, VEGF expression was further suppressed. Co-immunoprecipitation identified the existence of physical association between PAX6 and PTEN in the situation where the enhanced suppression of VEGF expression occurred. This discovery revealed a novel mechanism underlying the tumor-suppression functions of both PAX6 and PTEN in malignant gliomas. Data from this study are consistent with our previous finding in which patients with malignant gliomas have significantly more favorable outcomes when their tumors have high PAX6 and PTEN expression levels compared to patients with low expression values for either or both genes.
Glioblastoma multiforme (GBM) is the most invasive brain tumor. We have previously reported that ... more Glioblastoma multiforme (GBM) is the most invasive brain tumor. We have previously reported that the transcription factor PAX6 suppresses the tumorigenecity of GBM cells. By an in vitro Matrigel invasion assay on two GBM cell lines stably transfected with wild-type and/or two mutant forms of PAX6, this study displays the first evidence that PAX6 inhibits the invasiveness of GBM cells and that the DNA-binding domain of PAX6 is required for this function. Using real-time quantitative reverse transcription-PCR (RT-PCR), gelatin zymography, and immunohistochemistry assays, the expression of the gene encoding matrix metalloproteinase-2 (MMP2) in GBM cell lines grown in vitro or in intracranial xenografts in nude mice was shown to be repressed by either stable or adenoviralmediated overexpression of PAX6. Luciferase promoter assays revealed PAX6-mediated suppression of MMP2 promoter activity. Electrophoretic mobility shift assays showed direct binding of PAX6 to the MMP2 promoter. A significant reverse correlation (P < 0.05) occurred between PAX6 and MMP2 expression quantified by real-time quantitative RT-PCR in 41 GBMs, 43 anaplastic astrocytomas, and 7 adjacent normal tissues. Interestingly, the degree and significance of the reverse correlation increased after excluding astrocytomas, whereas it became insignificant after excluding GBMs. In GBM cells stably transfected with a dominant negative mutant PAX6 showing increased MMP2 expression and invasiveness, knockdown of MMP2 revealed that MMP2 is one of the PAX6 target genes mediating its suppression of invasion. Overall data delineated a mechanism for the suppressive function of PAX6 in GBM: suppression of cell invasion by repressing the expression of proinvasive genes such as MMP2.
Dr. Mayes explains how studying an animal model can carry over to human patients in a short inter... more Dr. Mayes explains how studying an animal model can carry over to human patients in a short interview
Supplementary Figure 2 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre ... more Supplementary Figure 2 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre Each Cause Neurofibroma Formation
Supplementary Table 1 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre E... more Supplementary Table 1 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre Each Cause Neurofibroma Formation
Supplementary Figure 1 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre ... more Supplementary Figure 1 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre Each Cause Neurofibroma Formation
Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the... more Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the NF1 tumor suppressor gene in the Schwann cell lineage. To understand whether neurofibroma formation is possible after birth, we induced Nf1 loss of function with an inducible proteolipid protein Cre allele. Perinatal loss of Nf1 resulted in the development of small plexiform neurofibromas late in life, whereas loss in adulthood caused large plexiform neurofibromas and morbidity beginning 4 months after onset of Nf1 loss. A conditional EGFP reporter allele identified cells showing recombination, including peripheral ganglia satellite cells, peripheral nerve S100β+ myelinating Schwann cells, and peripheral nerve p75+ cells. Neurofibromas contained cells with Remak bundle disruption but no recombination within GFAP+ nonmyelinating Schwann cells. Extramedullary lympho-hematopoietic expansion was also observed in PlpCre;Nf1fl/fl mice. These tumors contained EGFP+/Sca-1+ stromal cells among E...
Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the... more Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the NF1 tumor suppressor gene in the Schwann cell lineage. To understand whether neurofibroma formation is possible after birth, we induced Nf1 loss of function with an inducible proteolipid protein Cre allele. Perinatal loss of Nf1 resulted in the development of small plexiform neurofibromas late in life, whereas loss in adulthood caused large plexiform neurofibromas and morbidity beginning 4 months after onset of Nf1 loss. A conditional EGFP reporter allele identified cells showing recombination, including peripheral ganglia satellite cells, peripheral nerve S100bþ myelinating Schwann cells, and peripheral nerve p75þ cells. Neurofibromas contained cells with Remak bundle disruption but no recombination within GFAPþ nonmyelinating Schwann cells. Extramedullary lymphohematopoietic expansion was also observed in PlpCre;Nf1fl/fl mice. These tumors contained EGFPþ/Sca-1þ stromal cells among EGFP-negative lympho-hematopoietic cells indicating a noncell autonomous effect and unveiling a role of Nf1-deleted microenvironment on lympho-hematopoietic proliferation in vivo. Together these findings define a tumor suppressor role for Nf1 in the adult and narrow the range of potential neurofibromainitiating cell populations. Cancer Res; 71(13); 4675-85. Ó2011 AACR.
There are pluripotent stem cells in the adult brain that might not be very different from those f... more There are pluripotent stem cells in the adult brain that might not be very different from those found in bone marrow. Recent and profound advances in the field of neuropoiesis, which often rely on insights from studies of hematopoiesis and in some instances use cross-paradigms with this field, have already revealed that bone marrow has much in common with so-called &#39;brain marrow&#39;. Proliferative primogenitors and developmentally regulated molecules are hallmarks of both neuropoiesis and hematopoiesis. This article will focus on recent advances in neuropoiesis within a central core of the mature brain that is referred to as brain marrow, discussing its pluripotency and proliferative capacity, in vitro and molecular assays used in its study, and markers of neuropoietic stem/progenitor cells. As hematopoiesis research has led to the discovery of numerous morphogenetic factors, it is anticipated that studies of neuropoiesis should also uncover many new factors and genes that affect the growth and differentiation of neural cells. Recent breakthroughs in the stem-cell field prompt an inclusion of rationale for the persistence of normal stem/progenitor cells even in the aged brain. By analogy with hematopoiesis research, a thorough investigation of brain marrow should provide basic insights into developmental and persistent neurogenesis while anticipating cell-transplant and gene therapies for debilitating neurological diseases.
Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes 30-50 % of affected i... more Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes 30-50 % of affected individuals to develop plexiform neurofibromas. We found that macrophage infiltration of both mouse and human neurofibromas correlates with disease progression. Macrophages accounted for almost half of neurofibroma cells, leading us to hypothesize that nerve macrophages are inflammatory effectors in neurofibroma development and/or growth. We tested the effects of PLX3397, a dual kit/fms kinase inhibitor that blocks macrophage infiltration, in the Dhh-Cre; Nf1 flox/flox mouse model of GEM grade I neurofibroma. In mice aged 1-4 months, prior to development of nerve pathology and neurofibroma formation, PLX3397 did not impair tumor initiation and increased tumor volume compared to controls. However, in mice aged 7-9 months, after tumor establishment, a subset of mice demonstrating the largest reductions in macrophages after PLX3397 exhibited cell death and tumor volume regression. Macrophages are likely to provide an initial line of defense against developing tumors. Once tumors are established, they become tumor permissive. Macrophage depletion may result in impaired tumor maintenance and represent a therapeutic strategy for neurofibroma therapy.
PLPCre;Nf1fl/fl mice. Gross micrographs (left panels) show representative malignant Grade III GEM... more PLPCre;Nf1fl/fl mice. Gross micrographs (left panels) show representative malignant Grade III GEM peripheral nerve sheath tumors (PNST). Arrows in the lower left panel point to two hindlimb tumors. Rulers show 1mm markings. In panels at right, H&E staining of paraffin sections of these tumors are shown. A representative anti-S100β stain (DAB, brown reaction product) is shown in the inset on the top right. The star in the bottom right panel designates a prominent nerve bundle. Supplemental Figure 2: EGFP+ non-neural cells in heart, lung, thyroid, and spleen. EGFP immunohistochemistry was performed on a variety of paraffin embedded organs. Tissuespecific EGFP+ cells (green) within the heart (top left), lung (top right), thyroid (bottom left), and spleen (bottom right) 15 months post adult Tamoxifen exposure in PlpCre;Nf1fl/fl animals. Tissue sections were photographed at 20X magnification using 300ms exposure of FITC fluorochrome.
2809 Glioblastoma multiform (GBM) is the most common and most malignant primary brain tumor. We h... more 2809 Glioblastoma multiform (GBM) is the most common and most malignant primary brain tumor. We have reported that the expression of PAX6 is decreased in most GBM patient samples when compared to adjacent normal tissue and anaplastic astrocytomas (AAs). Lower PAX6 expression in tumor tissue is an indication of an unfavorable prognosis for patients with malignant astrocytic gliomas (Zhou et. al. Clin. Can. Res. 9:3369-75). PAX6 encodes a transcription factor critical in the development of the eye and central nervous system. In contrast to the decreased expression of PAX6 in GBMs, we observed an increase in the expression of PAX6 in the GBM cell line U251 after introducing a normal chromosome 10. Loss of heterozygosity (LOH) of chromosome 10 occurs in up to 80% of human GBM cases. Therefore, the reduced expression of PAX6 in GBMs may result from the loss of unidentified tumor suppression gene(s) in chromosome 10; however, this is not a simple side effect of LOH of chromosome 10. Overexpression of PAX6 via stable transfection of a PAX6/pRC-CMV construct in the GBM cell line U251HF suppressed anchorage-independent growth in vivo and tumor formation in vitro. This data is consistent with the hypothesis that PAX6 may have a tumor-suppressor function in GBM (Zhou et. al. J. Neuro-Oncology, in print). Microarray hybridization using Affymetrix chips revealed alterations of matrix metalloproteinase-2 (MMP2) gene expression in the suppressed U251HF PAX6-transfected cells. This data was confirmed by real-time quantitative reverse transcription PCR from cells grown in vitro and in the brain of nude mice. Moreover, there is a reverse correlation between the expression of PAX6 and MMP2 in GBMs, but not in AAs. MMP2 is a well-known protease involved in cell invasion and the malignant progression of gliomas. MMP2 was also shown to be suppressed at a functional level in gelatin zymography assays in PAX6 transfectant cells. This suppression correlates with the near abolition of the ability of all PAX6 transfectants to invade in matrigel invasion assays; however, cells expressing a dominant negative mutant form of PAX6 (PAX6-344) displayed a significant enhancement in their ability to invade in this assay. Luciferase MMP2 promoter assays have indicated that the lower expression level of MMP2 in PAX6-transfected cells is due to lower MMP2 promoter activity. In conclusion, our data indicate that PAX6 plays an important role in suppressing the formation of GBM by suppressing the expression of MMP2. Identification and functional characterization of the cis-element responsible for the suppression of the MMP2 promoter activity by PAX6 is being examined.
International journal of molecular biology, Nov 16, 2018
One common symptom of most neurodegenerative diseases related to aging is the presence of blood-b... more One common symptom of most neurodegenerative diseases related to aging is the presence of blood-brain barrier disruption and mitochondrial disfunction. N-acetylcysteine (NAC) is an FDA approved drug commonly utilized for a variety of neuropsychiatric and neurodegenerative diseases; however, while studies have shown that NAC can improve mitochondrial function in mouse models of some of these diseases, the mechanism(s) of action have yet to be fully defined. Our previous studies have shown that NAC treatment can alter blood-brain barrier (BBB) permeability in mice via changes in reactive oxygen species (ROS). To further examine the effects of NAC on both cell-specificity and the mitochondrial respiration of brain vascular endothelial cells and astrocytes that make up the blood-brain barrier, we utilized brain microvascular endothelial and cerebrum astrocytic cells and an in vitro mouse BBB model. We show that NAC caused time-and dose-dependent changes in BBB permeability that were correlated to significant changes in tight and gap junction proteins, change in endothelial nNOS, and downregulation of mitochondrial complex proteins in endothelial cells. Extracellular flux analysis revealed a significant reduction in endothelial cell mitochondrial maximal respiration after 48hrs. In contrast, astrocyte respiration showed no change after 48hours but was significantly increased after 24hours of NAC treatment corresponding to the decreased permeability noted at this time. These changes paralleled alterations in NOS in endothelial cells but not astrocytes. We conclude that murine astrocytes and endothelial cells respond to NAC treatment in a cell-specific fashion, altering their perspective metabolic profiles and ultimately the tight and gap junctions that determine the permeability of the BBB. Given the current theory that mitochondrial dysfunction and BBB leakiness are predisposing, possibly causative symptoms, for most neurodegenerative diseases associated with aging, our findings suggest that NAC's cell-specific effects upon both metabolism and BBB permeability may be concurrent mechanisms by which this drug is effective.
Background: Ras is a small G-protein switch, activated when GTP-bound; in the GTP-bound state Ras... more Background: Ras is a small G-protein switch, activated when GTP-bound; in the GTP-bound state Ras signals to downstream effector proteins. GTPase inactivating proteins (GAPs) including neurofibromain (Nf1) accelerate hydrolysis of Ras-GTP to inactive Ras-GDP. We showed that activation of Ras or loss of Nf1 in brain oligodendrocytes correlates with myelin decompaction, down-regulation of claudins, and down-regulation and mis-localization of connexins (Mayes et al., Cell Reports, 2013). Non-cell autonomous defects in perivascular astrocytes and blood-brain barrier (BBB) are also observed. The bloodbrain barrier becomes leaky, implicating a soluble mediator. NO and nitric oxide synthases (NOS1-3) are up-regulated in mutant white matter. Treating mice with the NOS inhibitor L-NAME or the antioxidant NAC corrected cellular phenotypes. Consistent with recent data implicating white matter changes in learning, CNP-HRasG12V mice displayed locomotor hyperactivity which could be rescued by antioxidant treatment. Results: Using flow cytometry to define reactive species in cells in sorted brain non-neuronal cells we have now identified alterations in reactive species (NO, SO and/or peroxynitrites) in oligodendrocyte precursors, oligodendrocytes, astrocytes, pericytes and endothelial cells when Ras-GTP is elevated in oligodendrocytes. We also found that many changes are absent in Nf1+/− mice, when cells in the brain in addition to oligodendrocytes are hemizygous for Nf1 loss. Mating mice in which oligodendrocyte only have activated Ras with Nf1+/− mice confirmed that effects on reactive species are mediated by hemizygosity in Nf1+/− astrocytes, pericytes and/or endothelial cells. FACs analysis showed that NO is significantly diminished in oligodendrocytes in these mutants, and shuttled to surrounding cells of the vasculature. Conclusion: We conclude that signaling between oligodendrocytes and cells of white matter blood vessels contributes to homeostasis of the blood-brain barrier. NR is supported by the NIH, the DOD Program in Neurofibromatosis and the Children's Tumor Foundation.
1057 Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. High expression of ... more 1057 Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. High expression of vascular endothelial growth factor ( VEGF ) has been a hallmark of GBM. However, the regulation of VEGF expression in malignant gliomas is not well understood. We have previously reported that the transcription factor PAX6 suppresses the tumorigenecity of glioblastoma cells. In addition, there is a reverse correlation between the expression level of PAX6 and VEGF when human glioma tumors and the surrounding normal tissues were compared, suggesting that PAX6 might regulate VEGF expression. In this study, several techniques such as real-time quantitative reverse transcription PCR, enzyme-linked immunosorbent assay and immunohistochemistry were used to investigate if PAX6 regulates VEGF expression in glioma cell lines. Our results showed a significant reduction of VEGF expression in the PAX6 stable-transfected glioma cell line U251HF grown both in vitro and in subcutaneous xenografts in nude mice. We further demonstrated in two glioma cell lines (U251HF and U87) that VEGF expression was markedly suppressed by adenoviral-mediated overexpression of PAX6, as well as PTEN. Interestingly, in cells when both genes were overexpressed together, VEGF expression was further suppressed. Co-immunoprecipitation identified the existence of physical association between PAX6 and PTEN in the situation where the enhanced suppression of VEGF expression occurred. This discovery revealed a novel mechanism underlying the tumor-suppression functions of both PAX6 and PTEN in malignant gliomas. Data from this study are consistent with our previous finding in which patients with malignant gliomas have significantly more favorable outcomes when their tumors have high PAX6 and PTEN expression levels compared to patients with low expression values for either or both genes.
Glioblastoma multiforme (GBM) is the most invasive brain tumor. We have previously reported that ... more Glioblastoma multiforme (GBM) is the most invasive brain tumor. We have previously reported that the transcription factor PAX6 suppresses the tumorigenecity of GBM cells. By an in vitro Matrigel invasion assay on two GBM cell lines stably transfected with wild-type and/or two mutant forms of PAX6, this study displays the first evidence that PAX6 inhibits the invasiveness of GBM cells and that the DNA-binding domain of PAX6 is required for this function. Using real-time quantitative reverse transcription-PCR (RT-PCR), gelatin zymography, and immunohistochemistry assays, the expression of the gene encoding matrix metalloproteinase-2 (MMP2) in GBM cell lines grown in vitro or in intracranial xenografts in nude mice was shown to be repressed by either stable or adenoviralmediated overexpression of PAX6. Luciferase promoter assays revealed PAX6-mediated suppression of MMP2 promoter activity. Electrophoretic mobility shift assays showed direct binding of PAX6 to the MMP2 promoter. A significant reverse correlation (P < 0.05) occurred between PAX6 and MMP2 expression quantified by real-time quantitative RT-PCR in 41 GBMs, 43 anaplastic astrocytomas, and 7 adjacent normal tissues. Interestingly, the degree and significance of the reverse correlation increased after excluding astrocytomas, whereas it became insignificant after excluding GBMs. In GBM cells stably transfected with a dominant negative mutant PAX6 showing increased MMP2 expression and invasiveness, knockdown of MMP2 revealed that MMP2 is one of the PAX6 target genes mediating its suppression of invasion. Overall data delineated a mechanism for the suppressive function of PAX6 in GBM: suppression of cell invasion by repressing the expression of proinvasive genes such as MMP2.
Dr. Mayes explains how studying an animal model can carry over to human patients in a short inter... more Dr. Mayes explains how studying an animal model can carry over to human patients in a short interview
Supplementary Figure 2 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre ... more Supplementary Figure 2 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre Each Cause Neurofibroma Formation
Supplementary Table 1 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre E... more Supplementary Table 1 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre Each Cause Neurofibroma Formation
Supplementary Figure 1 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre ... more Supplementary Figure 1 from Perinatal or Adult Nf1 Inactivation Using Tamoxifen-Inducible PlpCre Each Cause Neurofibroma Formation
Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the... more Plexiform neurofibromas are peripheral nerve sheath tumors initiated by biallelic mutation of the NF1 tumor suppressor gene in the Schwann cell lineage. To understand whether neurofibroma formation is possible after birth, we induced Nf1 loss of function with an inducible proteolipid protein Cre allele. Perinatal loss of Nf1 resulted in the development of small plexiform neurofibromas late in life, whereas loss in adulthood caused large plexiform neurofibromas and morbidity beginning 4 months after onset of Nf1 loss. A conditional EGFP reporter allele identified cells showing recombination, including peripheral ganglia satellite cells, peripheral nerve S100β+ myelinating Schwann cells, and peripheral nerve p75+ cells. Neurofibromas contained cells with Remak bundle disruption but no recombination within GFAP+ nonmyelinating Schwann cells. Extramedullary lympho-hematopoietic expansion was also observed in PlpCre;Nf1fl/fl mice. These tumors contained EGFP+/Sca-1+ stromal cells among E...
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