GSK‐3α/β‐mediated phosphorylation of CRMP‐2 regulates activity‐dependent dendritic growth

Abstract

Neuronal activity shapes the dendritic arbour; however, most of the molecular players in this process remain to be identified. We observed that depolarization-induced neuronal activity causes an increase in the phosphorylation of glycogen synthase kinase-3 (GSK-3)a/b on Ser21/9 in cerebellar granule neurons. Using several approaches, including gene knockdown and GSK-3a/b S21A/S21A/S9A/S9A double knockin mice, we demonstrated that both GSK-3b and GSK-3a mediate activity-dependent dendritic growth and that Ser21/9 phosphorylation of GSK-3a/b plays an important role in this process. Collapsin response mediator protein-2 (CRMP-2), which is crucial for axon development, is phosphorylated at Thr514 and inactivated by GSK-3. We found CRMP-2 was located mainly in the dendrites of cerebellar granule neurons, in contrast to the axonal distribution in hippocampal neurons. Over-expression of CRMP-2 promoted and knockdown of CRMP-2 impaired dendritic growth, suggesting that CRMP-2 is necessary and sufficient for activity-dependent dendritic development. Furthermore, silencing CRMP-2 completely blocked the dendritic growth-promoting effects of GSK-3 knockdown, and expression of Thr514 nonphosphorylated form of CRMP-2 counteracted the inhibitory effect of constitutively active GSK-3. This data indicate that CRMP-2 functions downstream of GSK-3. Together, these findings identify a novel GSK-3/CRMP-2 pathway that connects neuronal activity to dendritic growth.

The functionalized amino acid (S)-Lacosamide subverts CRMP2-mediated tubulin polymerization to prevent constitutive and activity-dependent increase in neurite outgrowth

“…To determine the involvement of CRMP2 in activity-driven neurite outgrowth, cortical neurons overexpressing EGFP were exposed to 25 mM KCl and maintained for 96 h to ascertain the extent of activity dependent neurite outgrowth (Figure 8A). Consistent with previous findings (Tan et al, 2013), chronic depolarization with 25 mM KCl led to a ~43% increase in total neurite outgrowth (143.1 ± 11.5) compared to control (100 ± 6.6) (Figure 8B–G). Notably, blockade of CRMP2-mediated neurite outgrowth by ( S )-LCM was sufficient to prevent activity dependent growth induced by KCl (68.4 ± 3.8 vs. 61.7 ± 3.5) ( p > 0.05).…”

“…EGFP-expressing cells were then imaged at 6 DIV. The use of 25 mM KCl is consistent with previous work reporting the involvement of CRMP2 in activity-dependent outgrowth (Tan et al, 2013). Analysis of neurite outgrowth was completed using a neurite outgrowth analysis protocol with the MetaXpress software (Molecular Devices, Sunnyvale, CA).…”

“…CRMP2 has recently been suggested as a potential mediator of activity-dependent neurite outgrowth in cerebellar granule neurons (Tan et al, 2013). Unlike other central neurons, cerebellar granule cells require slightly depolarizing conditions for survival in vitro .…”

“…Further studies also suggest the importance of kinase cascades (Solem et al, 1995; Wayman et al, 2006). Recently, activity-induced outgrowth in cerebellar granule cells was attributed to changes in the kinase GSK3β and its substrate, collapsin response mediator protein 2 (CRMP2) (Tan et al, 2013). CRMP2 is an intracellular phosphoprotein originally identified for coordinating axon guidance and growth cone collapse (Goshima et al, 1995).…”

Molecular and Cellular Mechanisms of Axonal Regeneration After Spinal Cord Injury

“…In hippocampal neurons, inactivation of GSK3␤ is crucial for axon specification and growth through the phosphorylation of CRMP-2 (93)(94)(95). In cerebellar granule neurons, GRMP-2 is localized to dendritic branches, where its phosphorylation by GSK3␤ inhibits dendritic growth (96).…”