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

Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5'-GGTAC^C-3' in the presence of Mg2+ as showngenerating 3' four base overhangs. Bioinformatics analysis reveals thatR.KpnI contains a \beta\beta\alpha-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonucleaseI-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequencenon-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148,H149 and Q175 correspond to the critical D, H and N or H residues ofthe HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD...D/EXK superfamily of nucleases, instead is a member of the HNH superfamily

Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily

The restriction endonuclease (REase) R.KpnI is an orthodox Type IIP enzyme, which binds to DNA in the absence of metal ions and cleaves the DNA sequence 5′-GGTAC^C-3′ in the presence of Mg(2+) as shown generating 3′ four base overhangs. Bioinformatics analysis reveals that R.KpnI contains a ββα-Me-finger fold, which is characteristic of many HNH-superfamily endonucleases, including homing endonuclease I-HmuI, structure-specific T4 endonuclease VII, colicin E9, sequence non-specific Serratia nuclease and sequence-specific homing endonuclease I-PpoI. According to our homology model of R.KpnI, D148, H149 and Q175 correspond to the critical D, H and N or H residues of the HNH nucleases. Substitutions of these three conserved residues lead to the loss of the DNA cleavage activity by R.KpnI, confirming their importance. The mutant Q175E fails to bind DNA at the standard conditions, although the DNA binding and cleavage can be rescued at pH 6.0, indicating a role for Q175 in DNA binding and cleavage. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD…D/EXK superfamily of nucleases, instead is a member of the HNH superfamily

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

Dextran Nanoparticle Synthesis and Properties.

Dextran is widely exploited in medical products and as a component of drug-delivering nanoparticles (NPs). Here, we tested whether dextran can serve as the main substrate of NPs and form a stable backbone. We tested dextrans with several molecular masses under several synthesis conditions to optimize NP stability. The analysis of the obtained nanoparticles showed that dextran NPs that were synthesized from 70 kDa dextran with a 5% degree of oxidation of the polysaccharide chain and 50% substitution with dodecylamine formed a NP backbone composed of modified dextran subunits, the mean diameter of which in an aqueous environment was around 100 nm. Dextran NPs could be stored in a dry state and reassembled in water. Moreover, we found that different chemical moieties (e.g., drugs such as doxorubicin) can be attached to the dextran NPs via a pH-dependent bond that allows release of the drug with lowering pH. We conclude that dextran NPs are a promising nano drug carrier

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