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

Proteasome Inhibitors against Glioblastoma—Overview of Molecular Mechanisms of Cytotoxicity, Progress in Clinical Trials, and Perspective for Use in Personalized Medicine

Proteasome inhibitors are moieties targeting the proteolytic activity of a proteasome, with demonstrated efficacy in certain hematological malignancies and candidate drugs in other types of cancer, including glioblastoma (GBM). They disturb the levels of proteasome-regulated proteins and lead to the cell cycle inhibition and apoptosis of GBM cells. The accumulation of cell cycle inhibitors p21 and p27, and decreased levels of prosurvival molecules NFKB, survivin, and MGMT, underlie proteasome inhibitors’ cytotoxicity when used alone or in combination with the anti-GBM cytostatic drug temozolomide (TMZ). The evidence gathered in preclinical studies substantiated the design of clinical trials that employed the two most promising proteasome inhibitors, bortezomib and marizomib. The drug safety profile, maximum tolerated dose, and interaction with other drugs were initially evaluated, mainly in recurrent GBM patients. A phase III study on newly diagnosed GBM patients who received marizomib as an adjuvant to the Stupp protocol was designed and completed in 2021, with the Stupp protocol receiving patients as a parallel control arm. The data from this phase III study indicate that marizomib does not improve the PFS and OS of GBM patients; however, further analysis of the genetic and epigenetic background of each patient tumor may shed some light on the sensitivity of individual patients to proteasome inhibition. The mutational and epigenetic makeup of GBM cells, like genetic alterations to TP53 and PTEN, or MGMT promoter methylation levels may actually determine the response to proteasome inhibition

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

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

Antibodies used in the study.

<p>Antibodies used in the study.</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

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