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Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery.

Thomas AX, Cruz Del Angel Y, Gonzalez MI, Carrel AJ, Carlsen J, Lam PM, Hempstead BL, Russek SJ, Brooks-Kayal AR - eNeuro (2016)

Bottom Line: In contrast to previous reports (Unsain et al., 2008; Volosin et al., 2008; VonDran et al., 2014), our studies found that levels of proBDNF in the hippocampus are markedly elevated as early as 3 h after SE onset and remain elevated for 7 d.In vitro treatment of hippocampal slices from animals 24 h after SE with a PAI-1 inhibitor reduces proBDNF levels.These findings suggest that rapid proBDNF increases following SE are due in part to reduced cleavage, and that proBDNF may be part of the initial neurotrophin response driving intracellular signaling during the acute phase of epileptogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Neuroscience Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Medical Scientist Training Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.

ABSTRACT
Brain-derived neurotrophic factor (BDNF) levels are elevated after status epilepticus (SE), leading to activation of multiple signaling pathways, including the janus kinase/signal transducer and activator of transcription pathway that mediates a decrease in GABAA receptor α1 subunits in the hippocampus (Lund et al., 2008). While BDNF can signal via its pro or mature form, the relative contribution of these forms to signaling after SE is not fully known. In the current study, we investigate changes in proBDNF levels acutely after SE in C57BL/6J mice. In contrast to previous reports (Unsain et al., 2008; Volosin et al., 2008; VonDran et al., 2014), our studies found that levels of proBDNF in the hippocampus are markedly elevated as early as 3 h after SE onset and remain elevated for 7 d. Immunohistochemistry studies indicate that seizure-induced BDNF localizes to all hippocampal subfields, predominantly in principal neurons and also in astrocytes. Analysis of the proteolytic machinery that cleaves proBDNF to produce mature BDNF demonstrates that acutely after SE there is a decrease in tissue plasminogen activator and an increase in plasminogen activator inhibitor-1 (PAI-1), an inhibitor of extracellular and intracellular cleavage, which normalizes over the first week after SE. In vitro treatment of hippocampal slices from animals 24 h after SE with a PAI-1 inhibitor reduces proBDNF levels. These findings suggest that rapid proBDNF increases following SE are due in part to reduced cleavage, and that proBDNF may be part of the initial neurotrophin response driving intracellular signaling during the acute phase of epileptogenesis.

No MeSH data available.


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BDNF protein is expressed in neurons and astrocytes of hippocampus after pilocarpine-induced SE. A, Representative confocal images of hippocampal subfields from HA-tagged mice 3 h after SE and an age- and handling-matched control (20× magnification; scale bar, 100 µm) shows the presence of HA immunoreactivity in principal cells, glia, and mossy fiber layers. The first column shows anti-HA (green) immunoreactivity with DAPI (blue) in each condition. The second column demonstrates the colocalization of immunoreactivity for HA (green) and the neuronal marker MAP2 (red). The third column demonstrates colocalization of immunoreactivity for HA (green) and the glial marker GFAP (red). B, High-magnification confocal image of CA3 hippocampal subfield (63× magnification; scale bar, 20 µm). White arrowheads correspond to neuronal localization of HA immunoreactivity in pyramidal cells of CA3; blue arrowheads correspond to the localization of HA immunoreactivity in mossy fibers. SL, Stratum lucidum; SP, stratum pyramidale. C, High-magnification confocal image of CA3 hippocampal subfield (63× magnification; scale bar, 20 µm), demonstrating glial expression of BDNF.
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Figure 4: BDNF protein is expressed in neurons and astrocytes of hippocampus after pilocarpine-induced SE. A, Representative confocal images of hippocampal subfields from HA-tagged mice 3 h after SE and an age- and handling-matched control (20× magnification; scale bar, 100 µm) shows the presence of HA immunoreactivity in principal cells, glia, and mossy fiber layers. The first column shows anti-HA (green) immunoreactivity with DAPI (blue) in each condition. The second column demonstrates the colocalization of immunoreactivity for HA (green) and the neuronal marker MAP2 (red). The third column demonstrates colocalization of immunoreactivity for HA (green) and the glial marker GFAP (red). B, High-magnification confocal image of CA3 hippocampal subfield (63× magnification; scale bar, 20 µm). White arrowheads correspond to neuronal localization of HA immunoreactivity in pyramidal cells of CA3; blue arrowheads correspond to the localization of HA immunoreactivity in mossy fibers. SL, Stratum lucidum; SP, stratum pyramidale. C, High-magnification confocal image of CA3 hippocampal subfield (63× magnification; scale bar, 20 µm), demonstrating glial expression of BDNF.

Mentions: To determine the cellular distribution of BDNF protein after SE, HA immunoreactivity was analyzed in coronal sections colabeled with a neuronal (MAP2) and an astrocytic (GFAP) marker (Fig. 4). It is important to note that the HA immunostaining is unable to distinguish between proBDNF and mBDNF; therefore, in these experiments the signal detected was considered as total BDNF expression. Another important limitation of these findings is that one cannot distinguish between HA immunoreactivity indicating the site of BDNF prerelease or internalization. Colocalization of HA and MAP2 immunoreactivity was assessed to identify BDNF expression in neurons and colocalization of HA and GFAP immunoreactivity was assessed to identify BDNF expression in astrocytes. The observed pattern of protein expression detected in control animals is consistent with the pattern of expression previously reported by others using the same HA-tagged BDNF knock-in mice (Yang et al., 2009; Dieni et al., 2012). The most prominent HA immunoreactivity signal is detected in the mossy fiber pathway, CA3 pyramidal neurons, and CA1 pyramidal neurons (Fig. 4). When compared with controls, mice at 3 h post-SE showed an increase in HA immunoreactivity as well as MAP2 immunoreactivity that can be detected in all hippocampal regions analyzed. In SE animals, the pattern of HA immunoreactivity is well colocalized with both MAP2 (Fig. 4A,B) and GFAP immunostaining (Fig. 4A,C), suggesting that within the hippocampus of animals acutely following SE, BDNF is localized in principal neurons and astrocytes.


Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery.

Thomas AX, Cruz Del Angel Y, Gonzalez MI, Carrel AJ, Carlsen J, Lam PM, Hempstead BL, Russek SJ, Brooks-Kayal AR - eNeuro (2016)

BDNF protein is expressed in neurons and astrocytes of hippocampus after pilocarpine-induced SE. A, Representative confocal images of hippocampal subfields from HA-tagged mice 3 h after SE and an age- and handling-matched control (20× magnification; scale bar, 100 µm) shows the presence of HA immunoreactivity in principal cells, glia, and mossy fiber layers. The first column shows anti-HA (green) immunoreactivity with DAPI (blue) in each condition. The second column demonstrates the colocalization of immunoreactivity for HA (green) and the neuronal marker MAP2 (red). The third column demonstrates colocalization of immunoreactivity for HA (green) and the glial marker GFAP (red). B, High-magnification confocal image of CA3 hippocampal subfield (63× magnification; scale bar, 20 µm). White arrowheads correspond to neuronal localization of HA immunoreactivity in pyramidal cells of CA3; blue arrowheads correspond to the localization of HA immunoreactivity in mossy fibers. SL, Stratum lucidum; SP, stratum pyramidale. C, High-magnification confocal image of CA3 hippocampal subfield (63× magnification; scale bar, 20 µm), demonstrating glial expression of BDNF.
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Figure 4: BDNF protein is expressed in neurons and astrocytes of hippocampus after pilocarpine-induced SE. A, Representative confocal images of hippocampal subfields from HA-tagged mice 3 h after SE and an age- and handling-matched control (20× magnification; scale bar, 100 µm) shows the presence of HA immunoreactivity in principal cells, glia, and mossy fiber layers. The first column shows anti-HA (green) immunoreactivity with DAPI (blue) in each condition. The second column demonstrates the colocalization of immunoreactivity for HA (green) and the neuronal marker MAP2 (red). The third column demonstrates colocalization of immunoreactivity for HA (green) and the glial marker GFAP (red). B, High-magnification confocal image of CA3 hippocampal subfield (63× magnification; scale bar, 20 µm). White arrowheads correspond to neuronal localization of HA immunoreactivity in pyramidal cells of CA3; blue arrowheads correspond to the localization of HA immunoreactivity in mossy fibers. SL, Stratum lucidum; SP, stratum pyramidale. C, High-magnification confocal image of CA3 hippocampal subfield (63× magnification; scale bar, 20 µm), demonstrating glial expression of BDNF.
Mentions: To determine the cellular distribution of BDNF protein after SE, HA immunoreactivity was analyzed in coronal sections colabeled with a neuronal (MAP2) and an astrocytic (GFAP) marker (Fig. 4). It is important to note that the HA immunostaining is unable to distinguish between proBDNF and mBDNF; therefore, in these experiments the signal detected was considered as total BDNF expression. Another important limitation of these findings is that one cannot distinguish between HA immunoreactivity indicating the site of BDNF prerelease or internalization. Colocalization of HA and MAP2 immunoreactivity was assessed to identify BDNF expression in neurons and colocalization of HA and GFAP immunoreactivity was assessed to identify BDNF expression in astrocytes. The observed pattern of protein expression detected in control animals is consistent with the pattern of expression previously reported by others using the same HA-tagged BDNF knock-in mice (Yang et al., 2009; Dieni et al., 2012). The most prominent HA immunoreactivity signal is detected in the mossy fiber pathway, CA3 pyramidal neurons, and CA1 pyramidal neurons (Fig. 4). When compared with controls, mice at 3 h post-SE showed an increase in HA immunoreactivity as well as MAP2 immunoreactivity that can be detected in all hippocampal regions analyzed. In SE animals, the pattern of HA immunoreactivity is well colocalized with both MAP2 (Fig. 4A,B) and GFAP immunostaining (Fig. 4A,C), suggesting that within the hippocampus of animals acutely following SE, BDNF is localized in principal neurons and astrocytes.

Bottom Line: In contrast to previous reports (Unsain et al., 2008; Volosin et al., 2008; VonDran et al., 2014), our studies found that levels of proBDNF in the hippocampus are markedly elevated as early as 3 h after SE onset and remain elevated for 7 d.In vitro treatment of hippocampal slices from animals 24 h after SE with a PAI-1 inhibitor reduces proBDNF levels.These findings suggest that rapid proBDNF increases following SE are due in part to reduced cleavage, and that proBDNF may be part of the initial neurotrophin response driving intracellular signaling during the acute phase of epileptogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Neuroscience Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045; Medical Scientist Training Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.

ABSTRACT
Brain-derived neurotrophic factor (BDNF) levels are elevated after status epilepticus (SE), leading to activation of multiple signaling pathways, including the janus kinase/signal transducer and activator of transcription pathway that mediates a decrease in GABAA receptor α1 subunits in the hippocampus (Lund et al., 2008). While BDNF can signal via its pro or mature form, the relative contribution of these forms to signaling after SE is not fully known. In the current study, we investigate changes in proBDNF levels acutely after SE in C57BL/6J mice. In contrast to previous reports (Unsain et al., 2008; Volosin et al., 2008; VonDran et al., 2014), our studies found that levels of proBDNF in the hippocampus are markedly elevated as early as 3 h after SE onset and remain elevated for 7 d. Immunohistochemistry studies indicate that seizure-induced BDNF localizes to all hippocampal subfields, predominantly in principal neurons and also in astrocytes. Analysis of the proteolytic machinery that cleaves proBDNF to produce mature BDNF demonstrates that acutely after SE there is a decrease in tissue plasminogen activator and an increase in plasminogen activator inhibitor-1 (PAI-1), an inhibitor of extracellular and intracellular cleavage, which normalizes over the first week after SE. In vitro treatment of hippocampal slices from animals 24 h after SE with a PAI-1 inhibitor reduces proBDNF levels. These findings suggest that rapid proBDNF increases following SE are due in part to reduced cleavage, and that proBDNF may be part of the initial neurotrophin response driving intracellular signaling during the acute phase of epileptogenesis.

No MeSH data available.


Related in: MedlinePlus