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Neuroinflammation increases GABAergic tone and impairs cognitive and motor function in hyperammonemia by increasing GAT-3 membrane expression. Reversal by sulforaphane by promoting M2 polarization of microglia.

Hernandez-Rabaza V, Cabrera-Pastor A, Taoro-Gonzalez L, Gonzalez-Usano A, Agusti A, Balzano T, Llansola M, Felipo V - J Neuroinflammation (2016)

Bottom Line: This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze.Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum.This is associated with astrocytes deactivation and normalization of GAT-3 membrane expression, extracellular GABA, glutamate-nitric oxide-cGMP pathway, and learning and motor coordination.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.

ABSTRACT

Background: Hyperammonemia induces neuroinflammation and increases GABAergic tone in the cerebellum which contributes to cognitive and motor impairment in hepatic encephalopathy (HE). The link between neuroinflammation and GABAergic tone remains unknown. New treatments reducing neuroinflammation and GABAergic tone could improve neurological impairment. The aims were, in hyperammonemic rats, to assess whether: (a) Enhancing endogenous anti-inflammatory mechanisms by sulforaphane treatment reduces neuroinflammation and restores learning and motor coordination. (b) Reduction of neuroinflammation by sulforaphane normalizes extracellular GABA and glutamate-NO-cGMP pathway and identify underlying mechanisms. (c) Identify steps by which hyperammonemia-induced microglial activation impairs cognitive and motor function and how sulforaphane restores them.

Methods: We analyzed in control and hyperammonemic rats, treated or not with sulforaphane, (a) learning in the Y maze; (b) motor coordination in the beam walking; (c) glutamate-NO-cGMP pathway and extracellular GABA by microdialysis; (d) microglial activation, by analyzing by immunohistochemistry or Western blot markers of pro-inflammatory (M1) (IL-1b, Iba-1) and anti-inflammatory (M2) microglia (Iba1, IL-4, IL-10, Arg1, YM-1); and (e) membrane expression of the GABA transporter GAT-3.

Results: Hyperammonemia induces activation of astrocytes and microglia in the cerebellum as assessed by immunohistochemistry. Hyperammonemia-induced neuroinflammation is associated with increased membrane expression of the GABA transporter GAT-3, mainly in activated astrocytes. This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze. Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum. This is associated with astrocytes deactivation and normalization of GAT-3 membrane expression, extracellular GABA, glutamate-nitric oxide-cGMP pathway, and learning and motor coordination.

Conclusions: Neuroinflammation increases GABAergic tone in the cerebellum by increasing GAT-3 membrane expression. This impairs motor coordination and learning in the Y maze. Sulforaphane could be a new therapeutic approach to improve cognitive and motor function in hyperammonemia, hepatic encephalopathy, and other pathologies associated with neuroinflammation by promoting microglia differentiation from M1 to M2.

No MeSH data available.


Related in: MedlinePlus

Hyperammonemia increases and treatment with sulforaphane normalizes membrane expression of GAT-3 in the cerebellum. a Membrane expression of GAT-3 was analyzed by Western blot after cross-linking with BS3. A typical image of the blots with and without BS3 is shown. Membrane expression was quantified as described in methods. Values are the mean ± SEM of six rats per group. Values significantly different from control rats are indicated by asterisks. Values significantly different from hyperammonemic rats are indicated by “a”. **p < 0.01; “aa” p < 0.01. b Double immunofluorescence staining of GFAP (green) and GAT-3 (red). Nuclei are stained with DAPI (blue). In the merged image, co-localization of GAT-3 and GFAP appears in yellow. It can be seen that GAT-3 is expressed in astrocytes surrounding Purkinje neurons (P). c Double immunofluorescence staining of Neun (red) and GAT-3 (green). Nuclei are stained with DAPI (blue). In the merged image, no co-localization of GAT-3 and Neun is observed
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Fig4: Hyperammonemia increases and treatment with sulforaphane normalizes membrane expression of GAT-3 in the cerebellum. a Membrane expression of GAT-3 was analyzed by Western blot after cross-linking with BS3. A typical image of the blots with and without BS3 is shown. Membrane expression was quantified as described in methods. Values are the mean ± SEM of six rats per group. Values significantly different from control rats are indicated by asterisks. Values significantly different from hyperammonemic rats are indicated by “a”. **p < 0.01; “aa” p < 0.01. b Double immunofluorescence staining of GFAP (green) and GAT-3 (red). Nuclei are stained with DAPI (blue). In the merged image, co-localization of GAT-3 and GFAP appears in yellow. It can be seen that GAT-3 is expressed in astrocytes surrounding Purkinje neurons (P). c Double immunofluorescence staining of Neun (red) and GAT-3 (green). Nuclei are stained with DAPI (blue). In the merged image, no co-localization of GAT-3 and Neun is observed

Mentions: Results are expressed as mean ± SEM. In Figs. 2a, b, 3a, 4a, and 5a–f, data were analyzed by analysis of variance (one-way ANOVA) with Newman-Keuls multiple post hoc test. Two-way ANOVA was used in Fig. 3b, with fraction number and experimental group as variables. Significance levels were set at p < 0.05.Fig. 2


Neuroinflammation increases GABAergic tone and impairs cognitive and motor function in hyperammonemia by increasing GAT-3 membrane expression. Reversal by sulforaphane by promoting M2 polarization of microglia.

Hernandez-Rabaza V, Cabrera-Pastor A, Taoro-Gonzalez L, Gonzalez-Usano A, Agusti A, Balzano T, Llansola M, Felipo V - J Neuroinflammation (2016)

Hyperammonemia increases and treatment with sulforaphane normalizes membrane expression of GAT-3 in the cerebellum. a Membrane expression of GAT-3 was analyzed by Western blot after cross-linking with BS3. A typical image of the blots with and without BS3 is shown. Membrane expression was quantified as described in methods. Values are the mean ± SEM of six rats per group. Values significantly different from control rats are indicated by asterisks. Values significantly different from hyperammonemic rats are indicated by “a”. **p < 0.01; “aa” p < 0.01. b Double immunofluorescence staining of GFAP (green) and GAT-3 (red). Nuclei are stained with DAPI (blue). In the merged image, co-localization of GAT-3 and GFAP appears in yellow. It can be seen that GAT-3 is expressed in astrocytes surrounding Purkinje neurons (P). c Double immunofluorescence staining of Neun (red) and GAT-3 (green). Nuclei are stained with DAPI (blue). In the merged image, no co-localization of GAT-3 and Neun is observed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4835883&req=5

Fig4: Hyperammonemia increases and treatment with sulforaphane normalizes membrane expression of GAT-3 in the cerebellum. a Membrane expression of GAT-3 was analyzed by Western blot after cross-linking with BS3. A typical image of the blots with and without BS3 is shown. Membrane expression was quantified as described in methods. Values are the mean ± SEM of six rats per group. Values significantly different from control rats are indicated by asterisks. Values significantly different from hyperammonemic rats are indicated by “a”. **p < 0.01; “aa” p < 0.01. b Double immunofluorescence staining of GFAP (green) and GAT-3 (red). Nuclei are stained with DAPI (blue). In the merged image, co-localization of GAT-3 and GFAP appears in yellow. It can be seen that GAT-3 is expressed in astrocytes surrounding Purkinje neurons (P). c Double immunofluorescence staining of Neun (red) and GAT-3 (green). Nuclei are stained with DAPI (blue). In the merged image, no co-localization of GAT-3 and Neun is observed
Mentions: Results are expressed as mean ± SEM. In Figs. 2a, b, 3a, 4a, and 5a–f, data were analyzed by analysis of variance (one-way ANOVA) with Newman-Keuls multiple post hoc test. Two-way ANOVA was used in Fig. 3b, with fraction number and experimental group as variables. Significance levels were set at p < 0.05.Fig. 2

Bottom Line: This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze.Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum.This is associated with astrocytes deactivation and normalization of GAT-3 membrane expression, extracellular GABA, glutamate-nitric oxide-cGMP pathway, and learning and motor coordination.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.

ABSTRACT

Background: Hyperammonemia induces neuroinflammation and increases GABAergic tone in the cerebellum which contributes to cognitive and motor impairment in hepatic encephalopathy (HE). The link between neuroinflammation and GABAergic tone remains unknown. New treatments reducing neuroinflammation and GABAergic tone could improve neurological impairment. The aims were, in hyperammonemic rats, to assess whether: (a) Enhancing endogenous anti-inflammatory mechanisms by sulforaphane treatment reduces neuroinflammation and restores learning and motor coordination. (b) Reduction of neuroinflammation by sulforaphane normalizes extracellular GABA and glutamate-NO-cGMP pathway and identify underlying mechanisms. (c) Identify steps by which hyperammonemia-induced microglial activation impairs cognitive and motor function and how sulforaphane restores them.

Methods: We analyzed in control and hyperammonemic rats, treated or not with sulforaphane, (a) learning in the Y maze; (b) motor coordination in the beam walking; (c) glutamate-NO-cGMP pathway and extracellular GABA by microdialysis; (d) microglial activation, by analyzing by immunohistochemistry or Western blot markers of pro-inflammatory (M1) (IL-1b, Iba-1) and anti-inflammatory (M2) microglia (Iba1, IL-4, IL-10, Arg1, YM-1); and (e) membrane expression of the GABA transporter GAT-3.

Results: Hyperammonemia induces activation of astrocytes and microglia in the cerebellum as assessed by immunohistochemistry. Hyperammonemia-induced neuroinflammation is associated with increased membrane expression of the GABA transporter GAT-3, mainly in activated astrocytes. This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze. Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum. This is associated with astrocytes deactivation and normalization of GAT-3 membrane expression, extracellular GABA, glutamate-nitric oxide-cGMP pathway, and learning and motor coordination.

Conclusions: Neuroinflammation increases GABAergic tone in the cerebellum by increasing GAT-3 membrane expression. This impairs motor coordination and learning in the Y maze. Sulforaphane could be a new therapeutic approach to improve cognitive and motor function in hyperammonemia, hepatic encephalopathy, and other pathologies associated with neuroinflammation by promoting microglia differentiation from M1 to M2.

No MeSH data available.


Related in: MedlinePlus