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Impaired ILK Function Is Associated with Deficits in Hippocampal Based Memory and Synaptic Plasticity in a FASD Rat Model.

Bhattacharya D, Dunaway EP, Bhattacharya S, Bloemer J, Buabeid M, Escobar M, Suppiramaniam V, Dhanasekaran M - PLoS ONE (2015)

Bottom Line: In FASD, a downstream effector of ILK, Glycogen Synthase Kinase 3β (GSK3β) remains highly active (reduced Ser9 phosphorylation).This reduced memory performance was consistent with decrease in LTP as compared to controls.These impairments appear to be mediated by reduced GSK3β regulation and increased synaptic stabilization of the calcium-impermeable GluR2 AMPA receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Drug, Discovery and Development, Auburn University, Auburn, Alabama, United States of America.

ABSTRACT
Fetal Alcohol Spectrum Disorder (FASD) is an umbrella term that encompasses a wide range of anatomical and behavioral problems in children who are exposed to alcohol during the prenatal period. There is no effective treatment for FASD, because of lack of complete characterization of the cellular and molecular mechanisms underlying this condition. Alcohol has been previously characterized to affect integrins and growth factor signaling receptors. Integrin Linked Kinase (ILK) is an effector of integrin and growth-factor signaling which regulates various signaling processes. In FASD, a downstream effector of ILK, Glycogen Synthase Kinase 3β (GSK3β) remains highly active (reduced Ser9 phosphorylation). GSK3β has been known to modulate glutamate receptor trafficking and channel properties. Therefore, we hypothesize that the cognitive deficits accompanying FASD are associated with impairments in the ILK signaling pathway. Pregnant Sprague Dawley rats consumed a "moderate" amount of alcohol throughout gestation, or a calorie-equivalent sucrose solution. Contextual fear conditioning was used to evaluate memory performance in 32-33-day-old pups. Synaptic plasticity was assessed in the Schaffer Collateral pathway, and hippocampal protein lysates were used to evaluate ILK signaling. Alcohol exposed pups showed impaired contextual fear conditioning, as compared to control pups. This reduced memory performance was consistent with decrease in LTP as compared to controls. Hippocampal ILK activity and GSK3β Ser21/9 phosphorylation were significantly lower in alcohol-exposed pups than controls. Increased synaptic expression of GluR2 AMPA receptors was observed with immunoprecipitation of post-synaptic density protein 95 (PSD95). Furthermore, immunoprecipitation of ILK revealed a decreased interaction with GluR2. The ILK pathway appears to play a significant role in memory and synaptic plasticity impairments in FASD rats. These impairments appear to be mediated by reduced GSK3β regulation and increased synaptic stabilization of the calcium-impermeable GluR2 AMPA receptors.

No MeSH data available.


Related in: MedlinePlus

Prenatal alcohol impairs ILK activity.(a) Western blot analysis of total GSK3β and its Ser9 phosphorylation state was performed in hippocampal lysates from rats prenatally exposed to alcohol and nonexposed controls (n = 4). There was a significant decrease in the pGSK3β/GSK3β ratio in exposed rats, suggesting increased GSK3β activity as a result of alcohol exposure (p < 0.05). (b) Western blot analysis of expression of ILK in brain hippocampal protein lysates from exposed and nonexposed rats (n = 4). There were no differences in densitometric evaluation shows in expression of ILK as a result of alcohol exposure. (c) ILK activity assay was performed with pooled hippocampal protein lysates from control and alcohol rats (n = 4) and Akt ser473 phosphorylation assessed with western blot analysis. The quantitation of band density analysis shows reduced ILK activity in alcohol-exposed animals (p < 0.05).
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pone.0135700.g003: Prenatal alcohol impairs ILK activity.(a) Western blot analysis of total GSK3β and its Ser9 phosphorylation state was performed in hippocampal lysates from rats prenatally exposed to alcohol and nonexposed controls (n = 4). There was a significant decrease in the pGSK3β/GSK3β ratio in exposed rats, suggesting increased GSK3β activity as a result of alcohol exposure (p < 0.05). (b) Western blot analysis of expression of ILK in brain hippocampal protein lysates from exposed and nonexposed rats (n = 4). There were no differences in densitometric evaluation shows in expression of ILK as a result of alcohol exposure. (c) ILK activity assay was performed with pooled hippocampal protein lysates from control and alcohol rats (n = 4) and Akt ser473 phosphorylation assessed with western blot analysis. The quantitation of band density analysis shows reduced ILK activity in alcohol-exposed animals (p < 0.05).

Mentions: Prenatal alcohol increases GSK3β activation [35], and GSK3β activity impairs learning and memory by modulating expression of surface receptor proteins (as well as affecting other physiological neuronal functions [36]). ILK phosphorylates both GSK3β and Akt (which is, in turn, the primary regulator of GSK3β phosphorylation) as downstream signaling molecules. Phosphorylation of GSK3β inhibits its activity [13]; thus, deficits in ILK should result in enhanced GSK3β and, consequently, impairments in learning and memory. However, the role of ILK in learning and memory after prenatal alcohol exposure has not yet been investigated. In the present study, phosphorylation of GSK3β in the hippocampus of rats prenatally exposed to alcohol was reduced by approx. 20% as compared to nonexposed controls (Fig 3A, p < 0.05). Interestingly, expression of ILK in the hippocampus was equivalent in both exposed and nonexposed animals (Fig 3B). Nonetheless, despite equivalent expression, changes in ILK activity may impair downstream phosphorylation. An ILK kinase assay revealed that ILK activity was indeed reduced by approximately 60% in the hippocampus of exposed animals, as compared to nonexposed controls (Fig 3C). These observations appear to confirm our hypothesis that decreased ILK activity reduces GSK3β phosphorylation, and that this reduced phosphorylation may be responsible for the memory and LTP impairments observed in the exposed animals.


Impaired ILK Function Is Associated with Deficits in Hippocampal Based Memory and Synaptic Plasticity in a FASD Rat Model.

Bhattacharya D, Dunaway EP, Bhattacharya S, Bloemer J, Buabeid M, Escobar M, Suppiramaniam V, Dhanasekaran M - PLoS ONE (2015)

Prenatal alcohol impairs ILK activity.(a) Western blot analysis of total GSK3β and its Ser9 phosphorylation state was performed in hippocampal lysates from rats prenatally exposed to alcohol and nonexposed controls (n = 4). There was a significant decrease in the pGSK3β/GSK3β ratio in exposed rats, suggesting increased GSK3β activity as a result of alcohol exposure (p < 0.05). (b) Western blot analysis of expression of ILK in brain hippocampal protein lysates from exposed and nonexposed rats (n = 4). There were no differences in densitometric evaluation shows in expression of ILK as a result of alcohol exposure. (c) ILK activity assay was performed with pooled hippocampal protein lysates from control and alcohol rats (n = 4) and Akt ser473 phosphorylation assessed with western blot analysis. The quantitation of band density analysis shows reduced ILK activity in alcohol-exposed animals (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549293&req=5

pone.0135700.g003: Prenatal alcohol impairs ILK activity.(a) Western blot analysis of total GSK3β and its Ser9 phosphorylation state was performed in hippocampal lysates from rats prenatally exposed to alcohol and nonexposed controls (n = 4). There was a significant decrease in the pGSK3β/GSK3β ratio in exposed rats, suggesting increased GSK3β activity as a result of alcohol exposure (p < 0.05). (b) Western blot analysis of expression of ILK in brain hippocampal protein lysates from exposed and nonexposed rats (n = 4). There were no differences in densitometric evaluation shows in expression of ILK as a result of alcohol exposure. (c) ILK activity assay was performed with pooled hippocampal protein lysates from control and alcohol rats (n = 4) and Akt ser473 phosphorylation assessed with western blot analysis. The quantitation of band density analysis shows reduced ILK activity in alcohol-exposed animals (p < 0.05).
Mentions: Prenatal alcohol increases GSK3β activation [35], and GSK3β activity impairs learning and memory by modulating expression of surface receptor proteins (as well as affecting other physiological neuronal functions [36]). ILK phosphorylates both GSK3β and Akt (which is, in turn, the primary regulator of GSK3β phosphorylation) as downstream signaling molecules. Phosphorylation of GSK3β inhibits its activity [13]; thus, deficits in ILK should result in enhanced GSK3β and, consequently, impairments in learning and memory. However, the role of ILK in learning and memory after prenatal alcohol exposure has not yet been investigated. In the present study, phosphorylation of GSK3β in the hippocampus of rats prenatally exposed to alcohol was reduced by approx. 20% as compared to nonexposed controls (Fig 3A, p < 0.05). Interestingly, expression of ILK in the hippocampus was equivalent in both exposed and nonexposed animals (Fig 3B). Nonetheless, despite equivalent expression, changes in ILK activity may impair downstream phosphorylation. An ILK kinase assay revealed that ILK activity was indeed reduced by approximately 60% in the hippocampus of exposed animals, as compared to nonexposed controls (Fig 3C). These observations appear to confirm our hypothesis that decreased ILK activity reduces GSK3β phosphorylation, and that this reduced phosphorylation may be responsible for the memory and LTP impairments observed in the exposed animals.

Bottom Line: In FASD, a downstream effector of ILK, Glycogen Synthase Kinase 3β (GSK3β) remains highly active (reduced Ser9 phosphorylation).This reduced memory performance was consistent with decrease in LTP as compared to controls.These impairments appear to be mediated by reduced GSK3β regulation and increased synaptic stabilization of the calcium-impermeable GluR2 AMPA receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Drug, Discovery and Development, Auburn University, Auburn, Alabama, United States of America.

ABSTRACT
Fetal Alcohol Spectrum Disorder (FASD) is an umbrella term that encompasses a wide range of anatomical and behavioral problems in children who are exposed to alcohol during the prenatal period. There is no effective treatment for FASD, because of lack of complete characterization of the cellular and molecular mechanisms underlying this condition. Alcohol has been previously characterized to affect integrins and growth factor signaling receptors. Integrin Linked Kinase (ILK) is an effector of integrin and growth-factor signaling which regulates various signaling processes. In FASD, a downstream effector of ILK, Glycogen Synthase Kinase 3β (GSK3β) remains highly active (reduced Ser9 phosphorylation). GSK3β has been known to modulate glutamate receptor trafficking and channel properties. Therefore, we hypothesize that the cognitive deficits accompanying FASD are associated with impairments in the ILK signaling pathway. Pregnant Sprague Dawley rats consumed a "moderate" amount of alcohol throughout gestation, or a calorie-equivalent sucrose solution. Contextual fear conditioning was used to evaluate memory performance in 32-33-day-old pups. Synaptic plasticity was assessed in the Schaffer Collateral pathway, and hippocampal protein lysates were used to evaluate ILK signaling. Alcohol exposed pups showed impaired contextual fear conditioning, as compared to control pups. This reduced memory performance was consistent with decrease in LTP as compared to controls. Hippocampal ILK activity and GSK3β Ser21/9 phosphorylation were significantly lower in alcohol-exposed pups than controls. Increased synaptic expression of GluR2 AMPA receptors was observed with immunoprecipitation of post-synaptic density protein 95 (PSD95). Furthermore, immunoprecipitation of ILK revealed a decreased interaction with GluR2. The ILK pathway appears to play a significant role in memory and synaptic plasticity impairments in FASD rats. These impairments appear to be mediated by reduced GSK3β regulation and increased synaptic stabilization of the calcium-impermeable GluR2 AMPA receptors.

No MeSH data available.


Related in: MedlinePlus