GSK3 alpha and GSK3 beta phosphorylate arc and regulate its degradation

The selective and neuronal activity-dependent degradation of synaptic proteins appears to be crucial for long-term synaptic plasticity. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which regulates the synaptic content of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), excitatory synapse strength and dendritic spine morphology. The levels of Arc protein are tightly regulated, and its removal occurs via proteasome-mediated degradation that requires prior ubiquitination. Glycogen synthase kinases α and β (GSK3α, GSKβ; collectively named GSK3α/β) are serine-threonine kinases with abundant expression in the central nervous system. Both GSK3 isozymes are tonically active under basal conditions, but their activity is regulated by intra- and extracellular factors, intimately involved in neuronal activity. Similar to Arc, GSK3α and GSK3β contribute to synaptic plasticity and the structural plasticity of dendritic spines. The present study identified Arc as a GSK3α/β substrate and showed that GSKβ promotes Arc degradation under conditions that induce de novo Arc synthesis. We also found that GSK3α/β inhibition potentiated spine head thinning that was caused by the prolonged stimulation of N-methyl-D-aspartate receptors (NMDAR). Furthermore, overexpression of Arc mutants that were resistant to GSK3β-mediated phosphorylation or ubiquitination resulted in a stronger reduction of dendritic spine width than wildtype Arc overexpression. Thus, GSK3β terminates Arc expression and limits its effect on dendritic spine morphology. Taken together, the results identify GSK3α/β-catalyzed Arc phosphorylation and degradation as a novel mechanism for controlling the duration of Arc expression and function

GSK3α and GSK3β Phosphorylate Arc and Regulate its Degradation

The selective and neuronal activity-dependent degradation of synaptic proteins appears to be crucial for long-term synaptic plasticity. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which regulates the synaptic content of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), excitatory synapse strength and dendritic spine morphology. The levels of Arc protein are tightly regulated, and its removal occurs via proteasome-mediated degradation that requires prior ubiquitination. Glycogen synthase kinases α and β (GSK3α, GSKβ; collectively named GSK3α/β) are serine-threonine kinases with abundant expression in the central nervous system. Both GSK3 isozymes are tonically active under basal conditions, but their activity is regulated by intra- and extracellular factors, intimately involved in neuronal activity. Similar to Arc, GSK3α and GSK3β contribute to synaptic plasticity and the structural plasticity of dendritic spines. The present study identified Arc as a GSK3α/β substrate and showed that GSKβ promotes Arc degradation under conditions that induce de novo Arc synthesis. We also found that GSK3α/β inhibition potentiated spine head thinning that was caused by the prolonged stimulation of N-methyl-D-aspartate receptors (NMDAR). Furthermore, overexpression of Arc mutants that were resistant to GSK3β-mediated phosphorylation or ubiquitination resulted in a stronger reduction of dendritic spine width than wildtype Arc overexpression. Thus, GSK3β terminates Arc expression and limits its effect on dendritic spine morphology. Taken together, the results identify GSK3α/β-catalyzed Arc phosphorylation and degradation as a novel mechanism for controlling the duration of Arc expression and function

Structural Plasticity of Dendritic Spines Requires GSK3α and GSK3β

<div><p>Although memories appear to be elusive phenomena, they are stored in the network of physical connections between neurons. Dendritic spines, which are actin-rich dendritic protrusions, serve as the contact points between networked neurons. The spines’ shape contributes to the strength of signal transmission. To acquire and store information, dendritic spines must remain plastic, i.e., able to respond to signals, by changing their shape. We asked whether glycogen synthase kinase (GSK) 3α and GSK3β, which are implicated in diseases with neuropsychiatric symptoms, such as Alzheimer's disease, bipolar disease and schizophrenia, play a role in a spine structural plasticity. We used Latrunculin B, an actin polymerization inhibitor, and chemically induced Long-Term Depression to trigger fast spine shape remodeling in cultured hippocampal neurons. Spine shrinkage induced by either stimulus required GSK3α activity. GSK3β activity was only important for spine structural changes after treatment with Latrunculin B. Our results indicate that GSK3α is an essential component for short-term spine structural plasticity. This specific function should be considered in future studies of neurodegenerative diseases and neuropsychiatric conditions that originate from suboptimal levels of GSK3α/β activity.</p></div

Antibodies used in the study.

<p>Antibodies used in the study.</p

Knockdown of GSK3α but not GSK3β affects chLTD-induced changes to dendritic spine morphology

<p>A. Experimental outline with 3 time points for microscopy: baseline, chLTD induction, end of recovery period. Representative micrographs of cultured DIV18 murine hippocampal neurons transfected with shRNA constructs as indicated. Scale bar = 2.5 μm. B. Quantitative analysis of spine shape changes; * = <i>p</i> < 0.05, ** = <i>p</i> < 0.01 and x = <i>p</i> <0.05 and xxx = <i>p</i> < 0.001 for measurements of spines after GSK3α and GSK3α/β silencing compared to the control at the corresponding time points. ‡ = <i>p</i> < 0.05 difference between GSK3α and GSK3α/β silencing. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as the mean spine width per cell ± s.e.m. The curve between time points is extrapolated. C. Spine l/w ratio changes are presented as cumulative histograms of the l/w ratio at 3 time points.</p

GSK3α and GSK3β knockdown alter LatrB-induced changes to dendritic spine morphology.

<p>A. GSK3 α/β knockdown does not affect basal fluctuations of dendritic spine morphology. Experimental outline with 4 time points for microscopy and quantitative analysis of spine shape; # indicates <i>p</i><0.05 for measurements of spines after GSK3β silencing compared to control values at the corresponding time points. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as the mean spine width per cell ± s.e.m. The curve between time points is extrapolated. B. Experimental outline with 3 time points for microscopy: baseline, LatrB treatment, end of recovery period. Representative micrographs of cultured DIV18 murine hippocampal neurons transfected with shRNA constructs as indicated. Scale bar = 2.5 μm. C. Quantitative analysis of spine shape changes; *** = <i>p</i> < 0.001 and xx = <i>p</i> < 0.01, xxx = <i>p</i> < 0.001 and ### = <i>p</i> < 0.001 for spine measurements of shRNA silenced GSK3α, GSK3β and GSK3α/β compared to the control at the corresponding time points. ‡ = <i>p</i> < 0.05 difference between shRNAGSK3α and shGSK3β. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as mean spine width per cell ± s.e.m. The curve between time points is extrapolated. D. Spine l/w ratio changes presented as cumulative histograms of length/width ratio at 3 time points.</p

Inhibition of GSK3α/β activity in cultured neurons treated with LatrB hinders fast spine structural changes.

<p>A. Efficiency of GSK3α/β chemical inhibition. The level of phosphorylation for glycogen synthase Ser 641 at time points after GSK3α/β inhibition with Ch98 and BIO. Lysates from hippocampal neurons on DIV18. Tubulin was the loading control. B. Pharmacological inhibition of GSK3α/β does not affect basal fluctuations of dendritic spine morphology. Experimental outline with 4 time points for microscopy and quantitative analysis of spine width; ## indicates <i>p</i><0.01 for measurements of spines after GSK3α/β inhibition with BIO compared with control values at the corresponding time point. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as the mean spine width per cell ± s.e.m. The curve between time points is extrapolated. C. Experimental outline with 3 time points for microscopy: baseline, LatrB treatment, end of recovery period. Representative micrographs of DIV18 cultured murine hippocampal neurons. Scale bar = 2.5 μm. D. Quantitative analysis of spine shape changes; *** and ### indicates <i>p</i><0.001 for measurements of spines after GSK3α/β inhibition by Ch98 and BIO when compared to control values at the corresponding time points. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as mean spine width per cell ± s.e.m. The curve between time points is extrapolated. E. Spine l/w ratio changes are presented as cumulative histograms of the l/w ratio at 3 time points.</p

Inhibition of GSK3α/β activity in cultured neurons hinders spine structural plasticity upon chLTD

<p>A. chLTD activates GSK3α/β in synaptoneurosomes isolated from murine hippocampi. Representative immunoblots, scanned with a Li-Cor Odyssey imager, for phospho-GSK3α (Ser21)/β (Ser9), total GSK3α/β, phospho-GluA1 (Ser845; LTD control) and GAPDH (loading control). B. Quantitative analysis of phospho-GSK3α/β to the total-GSK3α/β ratio at the time points indicated, expressed as % values of an untreated control. Data (n = 3 experiments) are presented as means ± s.e.m. * indicates a <i>p</i><0.05 vs control. C. Pharmacological inhibition of GSK3α/β affects chemical LTD-induced changes of dendritic spine width. Experimental outline with 3 time points for microscopy: baseline, chLTD induction, end of recovery period. Representative micrographs of DIV18 cultured murine hippocampal neurons. Scale bar = 2.5 μm. D. Quantitative analysis of spine shape changes; *** = <i>p</i> < 0.001, # = <i>p</i> < 0.05, ## = <i>p</i> <0.01 for spine measurements after GSK3α/β inhibition with Ch98 and BIO compared to control values at the corresponding time points. For number of counted spines refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134018#pone.0134018.t003" target="_blank">Table 3</a>. Data are presented as mean spine width per cell ± s.e.m. The curve between time points is extrapolated. E. Spine l/w ratio changes are presented as cumulative histograms of the length/width ratio at 3 time points.</p

Validation of GSK3 shRNAs and their scrambled counterparts.

<p>A. Representative micrographs of neurons transfected with shRNA constructs (as indicated) and immunofluorescently labeled for GSK3α or GSK3β. Scale bar = 50 μm. B. Quantitative analysis of GSK3 silencing; * = <i>p</i> < 0.05, ** = <i>p</i> <0.01 and *** = <i>p</i> < 0.001 for intensity measurements compared to neurons expressing pSuper^GFP. ### = <i>p</i> < 0.001 for experimental variants indicated by brackets. Data are presented as the mean ± s.e.m.</p

Associations between food and beverage consumption and different types of sedentary behaviours in European preschoolers: the ToyBox-study

OBJECTIVE: To examine the association between food and beverage consumption and time spent in different sedentary behaviours such as watching TV and DVDs, playing computer/video games and quiet play/activities in preschoolers. METHODS: A sample of 6431 (51.8 % males) European preschoolers aged 3.5-5.5 years from six survey centres was included in the data analyses. Data on dietary habits and sedentary behaviours [watching TV, playing computer and quiet play (both during weekdays and weekend days)] were collected via standardized proxy-administered questionnaires. One-way analysis of covariance and general linear model (adjusted for sex, maternal education, body mass index and centre) were conducted. RESULTS: The results of the generalized linear model showed that the more strong associations in both males and females who were watching TV for > 1 h/day during weekdays were positively associated with increased consumption of fizzy drinks (β = 0.136 for males and β = 0.156 for females), fresh and packed juices (β = 0.069, β = 0.089), sweetened milk (β = 0.119, β = 0.078), cakes and biscuits (β = 0.116, β = 0.145), chocolate (β = 0.052, β = 0.090), sugar-based desserts and pastries (β = 0.234, β = 0.250), salty snacks (β = 0.067, β = 0.056), meat/poultry/processed meat (β = 0.067, β = 0.090) and potatoes (β = 0.071, β = 0.067), and negative associations were observed for the consumption of fruits (β = -0.057, β = -0.099), vegetables (β = -0.056, β = -0.082) and fish (β = -0.013, β = -0.013). During weekend days, results were comparable. CONCLUSIONS: In European preschoolers, sedentary behaviours were associated with consumption of energy-dense foods and fizzy drinks. The present findings will contribute to improve the strategies to prevent overweight, obesity and nutrition-related chronic diseases from early childhood