Cu-II(atsm) Attenuates Neuroinflammation

Background: Neuroinflammation and biometal dyshomeostasis are key pathological features of several neurodegenerative diseases, including Alzheimer's disease (AD). Inflammation and biometals are linked at the molecular level through regulation of metal buffering proteins such as the metallothioneins. Even though the molecular connections between metals and inflammation have been demonstrated, little information exists on the effect of copper modulation on brain inflammation. Methods: We demonstrate the immunomodulatory potential of the copper bis(thiosemicarbazone) complex Cu-II(atsm) in an neuroinflammatory model in vivo and describe its anti-inflammatory effects on microglia and astrocytes in vitro. Results: By using a sophisticated in vivo magnetic resonance imaging (MRI) approach, we report the efficacy of Cu-II(atsm) in reducing acute cerebrovascular inflammation caused by peripheral administration of bacterial lipopolysaccharide (LPS). Cu-II(atsm) also induced anti-inflammatory outcomes in primary microglia [significant reductions in nitric oxide (NO), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor (TNF)] and astrocytes [significantly reduced NO, MCP-1, and interleukin 6 (IL-6)] in vitro. These anti-inflammatory actions were associated with increased cellular copper levels and increased the neuroprotective protein metallothionein-1 (MT1) in microglia and astrocytes. Conclusion: The beneficial effects of Cu-II(atsm) on the neuroimmune system suggest copper complexes are potential therapeutics for the treatment of neuroinflammatory conditions.Peer reviewe

CuII(atsm) Attenuates Neuroinflammation

Background: Neuroinflammation and biometal dyshomeostasis are key pathological features of several neurodegenerative diseases, including Alzheimer’s disease (AD). Inflammation and biometals are linked at the molecular level through regulation of metal buffering proteins such as the metallothioneins. Even though the molecular connections between metals and inflammation have been demonstrated, little information exists on the effect of copper modulation on brain inflammation.Methods: We demonstrate the immunomodulatory potential of the copper bis(thiosemicarbazone) complex CuII(atsm) in an neuroinflammatory model in vivo and describe its anti-inflammatory effects on microglia and astrocytes in vitro.Results: By using a sophisticated in vivo magnetic resonance imaging (MRI) approach, we report the efficacy of CuII(atsm) in reducing acute cerebrovascular inflammation caused by peripheral administration of bacterial lipopolysaccharide (LPS). CuII(atsm) also induced anti-inflammatory outcomes in primary microglia [significant reductions in nitric oxide (NO), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor (TNF)] and astrocytes [significantly reduced NO, MCP-1, and interleukin 6 (IL-6)] in vitro. These anti-inflammatory actions were associated with increased cellular copper levels and increased the neuroprotective protein metallothionein-1 (MT1) in microglia and astrocytes.Conclusion: The beneficial effects of CuII(atsm) on the neuroimmune system suggest copper complexes are potential therapeutics for the treatment of neuroinflammatory conditions

Data defining markers of human neural stem cell lineage potential

Neural stem cells (NSCs) and neural progenitor cells (NPCs) are self-renewing and multipotent cells, however, NPCs are considered to be more lineage-restricted with a reduced self-renewing capacity. We present data comparing the expression of 21 markers encompassing pluripotency, self-renewal (NSC) as well as neuronal and glial (astrocyte and oligodendrocyte) lineage specification and 28 extracellular proteoglycan (PG) genes and their regulatory enzymes between embryonic stem cell (ESC)-derived human NSCs (hNSC H9 cells, Thermo Fisher) and human cortex-derived normal human NPCs (nhNPCs, Lonza). The data demonstrates expression differences of multiple lineage and proteoglycan-associated genes between hNSC H9 cells and nhNPCs. Data interpretation of markers and proteoglycans defining NSC and neural cell lineage characterisation can be found in “Cell surface heparan sulfate proteoglycans as novel markers of human neural stem cell fate determination” (Oikari et al. 2015) [1]. Keywords: Neural stem cells, Neural progenitor cells, Proteoglycans, Lineage, Characterisatio

Heparan Sulfate Proteoglycans as Drivers of Neural Progenitors Derived From Human Mesenchymal Stem Cells

Background: Due to their relative ease of isolation and their high ex vivo and in vitro expansive potential, human mesenchymal stem cells (hMSCs) are an attractive candidate for therapeutic applications in the treatment of brain injury and neurological diseases. Heparan sulfate proteoglycans (HSPGs) are a family of ubiquitous proteins involved in a number of vital cellular processes including proliferation and stem cell lineage differentiation.Methods: Following the determination that hMSCs maintain neural potential throughout extended in vitro expansion, we examined the role of HSPGs in mediating the neural potential of hMSCs. hMSCs cultured in basal conditions (undifferentiated monolayer cultures) were found to co-express neural markers and HSPGs throughout expansion with modulation of the in vitro niche through the addition of exogenous HS influencing cellular HSPG and neural marker expression.Results: Conversion of hMSCs into hMSC Induced Neurospheres (hMSC IN) identified distinctly localized HSPG staining within the spheres along with altered gene expression of HSPG core protein and biosynthetic enzymes when compared to undifferentiated hMSCs.Conclusion: Comparison of markers of pluripotency, neural self-renewal and neural lineage specification between hMSC IN, hMSC and human neural stem cell (hNSC H9) cultures suggest that in vitro generated hMSC IN may represent an intermediary neurogenic cell type, similar to a common neural progenitor cell. In addition, this data demonstrates HSPGs and their biosynthesis machinery, are associated with hMSC IN formation. The identification of specific HSPGs driving hMSC lineage-specification will likely provide new markers to allow better use of hMSCs in therapeutic applications and improve our understanding of human neurogenesis

Cell surface heparan sulfate proteoglycans as novel markers of human neural stem cell fate determination

Multipotent neural stem cells (NSCs) provide a model to investigate neurogenesis and develop mechanisms of cell transplantation. In order to define improved markers of stemness and lineage specificity, we examined self-renewal and multi-lineage markers during long-term expansion and under neuronal and astrocyte differentiating conditions in human ESC-derived NSCs (hNSC H9 cells). In addition, with proteoglycans ubiquitous to the neural niche, we also examined heparan sulfate proteoglycans (HSPGs) and their regulatory enzymes. Our results demonstrate that hNSC H9 cells maintain self-renewal and multipotent capacity during extended culture and express HS biosynthesis enzymes and several HSPG core protein syndecans (SDCs) and glypicans (GPCs) at a high level. In addition, hNSC H9 cells exhibit high neuronal and a restricted glial differentiative potential with lineage differentiation significantly increasing expression of many HS biosynthesis enzymes. Furthermore, neuronal differentiation of the cells upregulated SDC4, GPC1, GPC2, GPC3 and GPC6 expression with increased GPC4 expression observed under astrocyte culture conditions. Finally, downregulation of selected HSPG core proteins altered hNSC H9 cell lineage potential. These findings demonstrate an involvement for HSPGs in mediating hNSC maintenance and lineage commitment and their potential use as novel markers of hNSC and neural cell lineage specification

HSPGs glypican‐1 and glypican‐4 are human neuronal proteins characteristic of different neural phenotypes

Generating neurons from human stem cells has potential for brain damage therapy and neurogenesis modeling, but current efficacy is limited by culture heterogeneity and the lack of markers. We have previously reported the heparan sulfate proteoglycans (HSPGs) glypican-1 (GPC1) and -4 (GPC4) as the markers of lineage-specific human neural stem cells (hNSCs) and mediators of hNSC lineage potential. Here, we further examined phenotypical characteristics and GPC1 and GPC4 during neural differentiation of hNSCs in the presence of two neurogenic growth factors reported to bind to heparan sulfate: brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor-B (PDGF-B). In hNSC neural cultures, GPC1 and GPC4 were expressed along neurites and cell bodies in long-term (40–60 days) neural differentiation cultures demonstrating the areas of differential localization—suggesting potentially different functions. Neural differentiation cultures in the presence of BDNF or PDGF-B generated phenotypically different neural cells with BDNF treatment associated with higher GPC4 versus GPC1 expression, increased heterogeneity, and differential neuron subtype marker expression to PDGF-B cultures. PDGF-B cultures exhibited higher levels of spontaneous activity and reduced heterogeneity over long-term culture associated with decreased GPC4. Untreated neural cultures were highly variable, supporting the use of neuroregulatory growth factors for guided differentiation. Targeted siRNA downregulation of GPC1/4 reduced neural differentiation markers and altered response to exogenous BDNF and PDGF-B. This work confirms GPC1 and GPC4 as regulators of human neural differentiation and supports their use as novel markers of neural cell characterization.</p

Table_1_Heparan Sulfate Proteoglycans as Drivers of Neural Progenitors Derived From Human Mesenchymal Stem Cells.DOCX

<p>Background: Due to their relative ease of isolation and their high ex vivo and in vitro expansive potential, human mesenchymal stem cells (hMSCs) are an attractive candidate for therapeutic applications in the treatment of brain injury and neurological diseases. Heparan sulfate proteoglycans (HSPGs) are a family of ubiquitous proteins involved in a number of vital cellular processes including proliferation and stem cell lineage differentiation.</p><p>Methods: Following the determination that hMSCs maintain neural potential throughout extended in vitro expansion, we examined the role of HSPGs in mediating the neural potential of hMSCs. hMSCs cultured in basal conditions (undifferentiated monolayer cultures) were found to co-express neural markers and HSPGs throughout expansion with modulation of the in vitro niche through the addition of exogenous HS influencing cellular HSPG and neural marker expression.</p><p>Results: Conversion of hMSCs into hMSC Induced Neurospheres (hMSC IN) identified distinctly localized HSPG staining within the spheres along with altered gene expression of HSPG core protein and biosynthetic enzymes when compared to undifferentiated hMSCs.</p><p>Conclusion: Comparison of markers of pluripotency, neural self-renewal and neural lineage specification between hMSC IN, hMSC and human neural stem cell (hNSC H9) cultures suggest that in vitro generated hMSC IN may represent an intermediary neurogenic cell type, similar to a common neural progenitor cell. In addition, this data demonstrates HSPGs and their biosynthesis machinery, are associated with hMSC IN formation. The identification of specific HSPGs driving hMSC lineage-specification will likely provide new markers to allow better use of hMSCs in therapeutic applications and improve our understanding of human neurogenesis.</p

HSPGs glypican‐1 and glypican‐4 are human neuronal proteins characteristic of different neural phenotypes

Generating neurons from human stem cells has potential for brain damage therapy and neurogenesis modeling, but current efficacy is limited by culture heterogeneity and the lack of markers. We have previously reported the heparan sulfate proteoglycans (HSPGs) glypican-1 (GPC1) and -4 (GPC4) as the markers of lineage-specific human neural stem cells (hNSCs) and mediators of hNSC lineage potential. Here, we further examined phenotypical characteristics and GPC1 and GPC4 during neural differentiation of hNSCs in the presence of two neurogenic growth factors reported to bind to heparan sulfate: brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor-B (PDGF-B). In hNSC neural cultures, GPC1 and GPC4 were expressed along neurites and cell bodies in long-term (40–60 days) neural differentiation cultures demonstrating the areas of differential localization—suggesting potentially different functions. Neural differentiation cultures in the presence of BDNF or PDGF-B generated phenotypically different neural cells with BDNF treatment associated with higher GPC4 versus GPC1 expression, increased heterogeneity, and differential neuron subtype marker expression to PDGF-B cultures. PDGF-B cultures exhibited higher levels of spontaneous activity and reduced heterogeneity over long-term culture associated with decreased GPC4. Untreated neural cultures were highly variable, supporting the use of neuroregulatory growth factors for guided differentiation. Targeted siRNA downregulation of GPC1/4 reduced neural differentiation markers and altered response to exogenous BDNF and PDGF-B. This work confirms GPC1 and GPC4 as regulators of human neural differentiation and supports their use as novel markers of neural cell characterization.</p

Image_1_Heparan Sulfate Proteoglycans as Drivers of Neural Progenitors Derived From Human Mesenchymal Stem Cells.tif

<p>Background: Due to their relative ease of isolation and their high ex vivo and in vitro expansive potential, human mesenchymal stem cells (hMSCs) are an attractive candidate for therapeutic applications in the treatment of brain injury and neurological diseases. Heparan sulfate proteoglycans (HSPGs) are a family of ubiquitous proteins involved in a number of vital cellular processes including proliferation and stem cell lineage differentiation.</p><p>Methods: Following the determination that hMSCs maintain neural potential throughout extended in vitro expansion, we examined the role of HSPGs in mediating the neural potential of hMSCs. hMSCs cultured in basal conditions (undifferentiated monolayer cultures) were found to co-express neural markers and HSPGs throughout expansion with modulation of the in vitro niche through the addition of exogenous HS influencing cellular HSPG and neural marker expression.</p><p>Results: Conversion of hMSCs into hMSC Induced Neurospheres (hMSC IN) identified distinctly localized HSPG staining within the spheres along with altered gene expression of HSPG core protein and biosynthetic enzymes when compared to undifferentiated hMSCs.</p><p>Conclusion: Comparison of markers of pluripotency, neural self-renewal and neural lineage specification between hMSC IN, hMSC and human neural stem cell (hNSC H9) cultures suggest that in vitro generated hMSC IN may represent an intermediary neurogenic cell type, similar to a common neural progenitor cell. In addition, this data demonstrates HSPGs and their biosynthesis machinery, are associated with hMSC IN formation. The identification of specific HSPGs driving hMSC lineage-specification will likely provide new markers to allow better use of hMSCs in therapeutic applications and improve our understanding of human neurogenesis.</p

Image_2_Heparan Sulfate Proteoglycans as Drivers of Neural Progenitors Derived From Human Mesenchymal Stem Cells.TIF

<p>Background: Due to their relative ease of isolation and their high ex vivo and in vitro expansive potential, human mesenchymal stem cells (hMSCs) are an attractive candidate for therapeutic applications in the treatment of brain injury and neurological diseases. Heparan sulfate proteoglycans (HSPGs) are a family of ubiquitous proteins involved in a number of vital cellular processes including proliferation and stem cell lineage differentiation.</p><p>Methods: Following the determination that hMSCs maintain neural potential throughout extended in vitro expansion, we examined the role of HSPGs in mediating the neural potential of hMSCs. hMSCs cultured in basal conditions (undifferentiated monolayer cultures) were found to co-express neural markers and HSPGs throughout expansion with modulation of the in vitro niche through the addition of exogenous HS influencing cellular HSPG and neural marker expression.</p><p>Results: Conversion of hMSCs into hMSC Induced Neurospheres (hMSC IN) identified distinctly localized HSPG staining within the spheres along with altered gene expression of HSPG core protein and biosynthetic enzymes when compared to undifferentiated hMSCs.</p><p>Conclusion: Comparison of markers of pluripotency, neural self-renewal and neural lineage specification between hMSC IN, hMSC and human neural stem cell (hNSC H9) cultures suggest that in vitro generated hMSC IN may represent an intermediary neurogenic cell type, similar to a common neural progenitor cell. In addition, this data demonstrates HSPGs and their biosynthesis machinery, are associated with hMSC IN formation. The identification of specific HSPGs driving hMSC lineage-specification will likely provide new markers to allow better use of hMSCs in therapeutic applications and improve our understanding of human neurogenesis.</p