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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 may control GluR2 protein at the synapse through ILK.(a). Immunoprecipitation (IP) with anti–PSD-95 from pooled hippocampal protein lysates of rats prenatally-exposed to alcohol and nonexposed controls (n = 5). In exposed rats, precipitate of GluR2 increased as compared to controls, while precipitates of GluR1and ILK did not change. The same blotting membrane was reprobed with anti–PSD-95 as a control for PSD95 pull down. The quantitation is shown adjacent to the blot image (p < 0.05). (b) Immunoprecipitation (IP) with anti–ILK coprecipitates GluR2 from hippocampal protein lysates of exposed and nonexposed animals (n = 5). There was reduced interaction in the prenatal exposed rats as compared to the nonexposed controls. The western blot analysis of immunoprecipitated ILK with anti–ILK antibody was used to confirm equal ILK immunoprecipitation. The quantitation is shown adjacent to the blot image (p < 0.05).
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pone.0135700.g004: Prenatal alcohol may control GluR2 protein at the synapse through ILK.(a). Immunoprecipitation (IP) with anti–PSD-95 from pooled hippocampal protein lysates of rats prenatally-exposed to alcohol and nonexposed controls (n = 5). In exposed rats, precipitate of GluR2 increased as compared to controls, while precipitates of GluR1and ILK did not change. The same blotting membrane was reprobed with anti–PSD-95 as a control for PSD95 pull down. The quantitation is shown adjacent to the blot image (p < 0.05). (b) Immunoprecipitation (IP) with anti–ILK coprecipitates GluR2 from hippocampal protein lysates of exposed and nonexposed animals (n = 5). There was reduced interaction in the prenatal exposed rats as compared to the nonexposed controls. The western blot analysis of immunoprecipitated ILK with anti–ILK antibody was used to confirm equal ILK immunoprecipitation. The quantitation is shown adjacent to the blot image (p < 0.05).

Mentions: ILK is known to interact with PSD95, an important scaffolding protein present at the synapse that interacts with several surface receptors. To understand the changes in AMPAR expression at the synaptic surface, we performed protein immunoprecipitation assays using anti-PSD95 antibody [37], and synaptic expression of GluR1, GluR2 and ILK was measured (Fig 4A). Synaptic expression of GluR2 was increased while synaptic expression of GluR1 was unchanged in animals exposed to alcohol prenatally. Increased GluR2 expression suggest an increase in calcium impermeable receptors at the synapse (GluR2 containing), which reduces the probability of action potential generation and, consequently, affect the NMDA-dependent LTP generation unlike calcium permeable (GluR2 lacking) receptors. Furthermore, calcium permeable receptors may also help in maintaining the availability of calcium ions necessary for LTP maintenance [38].


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 may control GluR2 protein at the synapse through ILK.(a). Immunoprecipitation (IP) with anti–PSD-95 from pooled hippocampal protein lysates of rats prenatally-exposed to alcohol and nonexposed controls (n = 5). In exposed rats, precipitate of GluR2 increased as compared to controls, while precipitates of GluR1and ILK did not change. The same blotting membrane was reprobed with anti–PSD-95 as a control for PSD95 pull down. The quantitation is shown adjacent to the blot image (p < 0.05). (b) Immunoprecipitation (IP) with anti–ILK coprecipitates GluR2 from hippocampal protein lysates of exposed and nonexposed animals (n = 5). There was reduced interaction in the prenatal exposed rats as compared to the nonexposed controls. The western blot analysis of immunoprecipitated ILK with anti–ILK antibody was used to confirm equal ILK immunoprecipitation. The quantitation is shown adjacent to the blot image (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135700.g004: Prenatal alcohol may control GluR2 protein at the synapse through ILK.(a). Immunoprecipitation (IP) with anti–PSD-95 from pooled hippocampal protein lysates of rats prenatally-exposed to alcohol and nonexposed controls (n = 5). In exposed rats, precipitate of GluR2 increased as compared to controls, while precipitates of GluR1and ILK did not change. The same blotting membrane was reprobed with anti–PSD-95 as a control for PSD95 pull down. The quantitation is shown adjacent to the blot image (p < 0.05). (b) Immunoprecipitation (IP) with anti–ILK coprecipitates GluR2 from hippocampal protein lysates of exposed and nonexposed animals (n = 5). There was reduced interaction in the prenatal exposed rats as compared to the nonexposed controls. The western blot analysis of immunoprecipitated ILK with anti–ILK antibody was used to confirm equal ILK immunoprecipitation. The quantitation is shown adjacent to the blot image (p < 0.05).
Mentions: ILK is known to interact with PSD95, an important scaffolding protein present at the synapse that interacts with several surface receptors. To understand the changes in AMPAR expression at the synaptic surface, we performed protein immunoprecipitation assays using anti-PSD95 antibody [37], and synaptic expression of GluR1, GluR2 and ILK was measured (Fig 4A). Synaptic expression of GluR2 was increased while synaptic expression of GluR1 was unchanged in animals exposed to alcohol prenatally. Increased GluR2 expression suggest an increase in calcium impermeable receptors at the synapse (GluR2 containing), which reduces the probability of action potential generation and, consequently, affect the NMDA-dependent LTP generation unlike calcium permeable (GluR2 lacking) receptors. Furthermore, calcium permeable receptors may also help in maintaining the availability of calcium ions necessary for LTP maintenance [38].

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