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Activation of GSK-3β and caspase-3 occurs in Nigral dopamine neurons during the development of apoptosis activated by a striatal injection of 6-hydroxydopamine.

Hernandez-Baltazar D, Mendoza-Garrido ME, Martinez-Fong D - PLoS ONE (2013)

Bottom Line: The disappearance of TH(+) cells was associated with a decrease in NeuN and β-III tubulin immunoreactivity and an increase in Apostain, cleaved caspase-3, and GSK-3β pY216 in the SNc.Increased levels of caspase-3 immunoreactivity in TH(+) cells were detected from days 1 to 15, and the levels then decreased to day 30 postlesion.Our results suggest that caspase-3 and GSK-3β pY216 activation might participate in the DA cell death and that the active caspase-3 might also participate in the neuroinflammation caused by the striatal 6-OHDA injection.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, DF, México.

ABSTRACT
The 6-Hydroxydopamine (6-OHDA) rat model of Parkinson's disease is essential for a better understanding of the pathological processes underlying the human disease and for the evaluation of promising therapeutic interventions. This work evaluated whether a single striatal injection of 6-OHDA causes progressive apoptosis of dopamine (DA) neurons and activation of glycogen synthase kinase 3β (GSK-3β) and caspase-3 in the substantia nigra compacta (SNc). The loss of DA neurons was shown by three neuron markers; tyrosine hydroxylase (TH), NeuN, and β-III tubulin. Apoptosis activation was determined using Apostain and immunostaining against cleaved caspase-3 and GSK-3β pY216. We also explored the possibility that cleaved caspase-3 is produced by microglia and astrocytes. Our results showed that the 6-OHDA caused loss of nigral TH(+) cells, progressing mainly in rostrocaudal and lateromedial directions. In the neostriatum, a severe loss of TH(+) terminals occurred from day 3 after lesion. The disappearance of TH(+) cells was associated with a decrease in NeuN and β-III tubulin immunoreactivity and an increase in Apostain, cleaved caspase-3, and GSK-3β pY216 in the SNc. Apostain immunoreactivity was observed from days 3 to 21 postlesion. Increased levels of caspase-3 immunoreactivity in TH(+) cells were detected from days 1 to 15, and the levels then decreased to day 30 postlesion. The cleaved caspase-3 also collocated with microglia and astrocytes indicating its participation in glial activation. Our results suggest that caspase-3 and GSK-3β pY216 activation might participate in the DA cell death and that the active caspase-3 might also participate in the neuroinflammation caused by the striatal 6-OHDA injection.

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The injection of 6-OHDA into the neostriatum leads to the activation of caspase-3 in the SNc.Representative confocal micrographs of mesencephalon immunostained against TH and cleaved caspase-3. The numbers at the left margin of the figures are the days after the 6-OHDA lesion. The primary antibodies were a mouse monoclonal anti-TH and a rabbit polyclonal anti-cleaved-caspase-3. The secondary antibodies were FITC goat anti-mouse IgG (H+L) conjugated and Texas red goat anti-rabbit H+L IgG. The scale bars = 50 µm in the first row are common for all the micrographs.
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pone-0070951-g005: The injection of 6-OHDA into the neostriatum leads to the activation of caspase-3 in the SNc.Representative confocal micrographs of mesencephalon immunostained against TH and cleaved caspase-3. The numbers at the left margin of the figures are the days after the 6-OHDA lesion. The primary antibodies were a mouse monoclonal anti-TH and a rabbit polyclonal anti-cleaved-caspase-3. The secondary antibodies were FITC goat anti-mouse IgG (H+L) conjugated and Texas red goat anti-rabbit H+L IgG. The scale bars = 50 µm in the first row are common for all the micrographs.

Mentions: The confocal microscopy analysis showed that cleaved caspase-3 immunostaining was absent in the SNc without a lesion and was present as early as day 1 postlesion (Fig. 5). The cleaved caspase-3-immunostaining reached the maximum intensity at day 7 postlesion and decreased thereafter but was present until the end of the study (Fig. 5). The cleaved caspase-3 immunoreactivity was collocated with most of TH(+) cells, showing a nuclear (day 3) and perinuclear (day 7) presence (Fig. 5 and Fig. 6A). A great number of small cells with only cleaved caspase-3 immunostaining were also seen scattered in the neuropil of the SNc after day 7 postlesion (Fig. 5 and Fig. 6A). To identify to what cell population would correspond the non-TH cleaved caspase-3 expression, we explored two glial populations, namely the OX42-positive microglia and GFAP-positive astrocytes at day 7 after the 6-OHDA lesion (Fig. 6B,C). An increase in the number of those glial cell populations was measured in the lesion side suggesting the presence of neuroinflammation (data not shown). The presence of cleaved caspase-3 immunostaining was observed in the cytoplasm and nucleus of both OX42-positive microglia (6B) and GFAP-positive astrocyte cells (6C).


Activation of GSK-3β and caspase-3 occurs in Nigral dopamine neurons during the development of apoptosis activated by a striatal injection of 6-hydroxydopamine.

Hernandez-Baltazar D, Mendoza-Garrido ME, Martinez-Fong D - PLoS ONE (2013)

The injection of 6-OHDA into the neostriatum leads to the activation of caspase-3 in the SNc.Representative confocal micrographs of mesencephalon immunostained against TH and cleaved caspase-3. The numbers at the left margin of the figures are the days after the 6-OHDA lesion. The primary antibodies were a mouse monoclonal anti-TH and a rabbit polyclonal anti-cleaved-caspase-3. The secondary antibodies were FITC goat anti-mouse IgG (H+L) conjugated and Texas red goat anti-rabbit H+L IgG. The scale bars = 50 µm in the first row are common for all the micrographs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3733721&req=5

pone-0070951-g005: The injection of 6-OHDA into the neostriatum leads to the activation of caspase-3 in the SNc.Representative confocal micrographs of mesencephalon immunostained against TH and cleaved caspase-3. The numbers at the left margin of the figures are the days after the 6-OHDA lesion. The primary antibodies were a mouse monoclonal anti-TH and a rabbit polyclonal anti-cleaved-caspase-3. The secondary antibodies were FITC goat anti-mouse IgG (H+L) conjugated and Texas red goat anti-rabbit H+L IgG. The scale bars = 50 µm in the first row are common for all the micrographs.
Mentions: The confocal microscopy analysis showed that cleaved caspase-3 immunostaining was absent in the SNc without a lesion and was present as early as day 1 postlesion (Fig. 5). The cleaved caspase-3-immunostaining reached the maximum intensity at day 7 postlesion and decreased thereafter but was present until the end of the study (Fig. 5). The cleaved caspase-3 immunoreactivity was collocated with most of TH(+) cells, showing a nuclear (day 3) and perinuclear (day 7) presence (Fig. 5 and Fig. 6A). A great number of small cells with only cleaved caspase-3 immunostaining were also seen scattered in the neuropil of the SNc after day 7 postlesion (Fig. 5 and Fig. 6A). To identify to what cell population would correspond the non-TH cleaved caspase-3 expression, we explored two glial populations, namely the OX42-positive microglia and GFAP-positive astrocytes at day 7 after the 6-OHDA lesion (Fig. 6B,C). An increase in the number of those glial cell populations was measured in the lesion side suggesting the presence of neuroinflammation (data not shown). The presence of cleaved caspase-3 immunostaining was observed in the cytoplasm and nucleus of both OX42-positive microglia (6B) and GFAP-positive astrocyte cells (6C).

Bottom Line: The disappearance of TH(+) cells was associated with a decrease in NeuN and β-III tubulin immunoreactivity and an increase in Apostain, cleaved caspase-3, and GSK-3β pY216 in the SNc.Increased levels of caspase-3 immunoreactivity in TH(+) cells were detected from days 1 to 15, and the levels then decreased to day 30 postlesion.Our results suggest that caspase-3 and GSK-3β pY216 activation might participate in the DA cell death and that the active caspase-3 might also participate in the neuroinflammation caused by the striatal 6-OHDA injection.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, DF, México.

ABSTRACT
The 6-Hydroxydopamine (6-OHDA) rat model of Parkinson's disease is essential for a better understanding of the pathological processes underlying the human disease and for the evaluation of promising therapeutic interventions. This work evaluated whether a single striatal injection of 6-OHDA causes progressive apoptosis of dopamine (DA) neurons and activation of glycogen synthase kinase 3β (GSK-3β) and caspase-3 in the substantia nigra compacta (SNc). The loss of DA neurons was shown by three neuron markers; tyrosine hydroxylase (TH), NeuN, and β-III tubulin. Apoptosis activation was determined using Apostain and immunostaining against cleaved caspase-3 and GSK-3β pY216. We also explored the possibility that cleaved caspase-3 is produced by microglia and astrocytes. Our results showed that the 6-OHDA caused loss of nigral TH(+) cells, progressing mainly in rostrocaudal and lateromedial directions. In the neostriatum, a severe loss of TH(+) terminals occurred from day 3 after lesion. The disappearance of TH(+) cells was associated with a decrease in NeuN and β-III tubulin immunoreactivity and an increase in Apostain, cleaved caspase-3, and GSK-3β pY216 in the SNc. Apostain immunoreactivity was observed from days 3 to 21 postlesion. Increased levels of caspase-3 immunoreactivity in TH(+) cells were detected from days 1 to 15, and the levels then decreased to day 30 postlesion. The cleaved caspase-3 also collocated with microglia and astrocytes indicating its participation in glial activation. Our results suggest that caspase-3 and GSK-3β pY216 activation might participate in the DA cell death and that the active caspase-3 might also participate in the neuroinflammation caused by the striatal 6-OHDA injection.

Show MeSH
Related in: MedlinePlus