A schizophrenia risk gene, NRGN, bidirectionally modulates synaptic plasticity via regulating the neuronal phosphoproteome

Background:

Dendritic spines are actin-rich structures that receive synaptic transmission. As synaptic activity increases, signaling pathways and cytoskeletal remodeling are initiated in dendritic spines to strengthen the synapse. Conversely, decreases in synaptic activity lead to synaptic weakening. This activity-based regulation of synaptic strength is known as synaptic plasticity.

Numerous signaling pathways required for synaptic plasticity are regulated by intracellular Ca2+ levels and the calcium-binding protein calmodulin (CaM). Both CaM and the CaM-Ca²⁺ complex have a wide variety of binding partners that can induce either synaptic strengthening or weakening. The relatively low concentration of CaM in the spine creates competition for both free CaM and CaM-Ca²⁺ complexes.

CaM-binding protein neurogranin (Ng) is required for long-term potentiation and memory formation. As intracellular Ca2+ increases, Ng releases CaM. In turn, CaM-Ca²⁺ complexes can then induce numerous signaling cascades in the post-synapse. CaM-Ca²⁺ binds and activates protein phosphatase 2B (PP2B/calcineurin) with a high affinity. However, as CaM-Ca²⁺ levels increase, CaM-Ca²⁺ protein kinase II (CaMKII) is also activated. Although CaMKII has a weaker affinity for CaM-Ca²⁺, the concentration of CaMKII is much higher than PP2B in the post-synapse. Therefore, CaMKII activity dominates at high CaM-Ca²⁺ levels. The authors hypothesized that Ng regulates CaM levels and subsequently control phosphorylation events and downstream signaling in the post-synapse. As changes in Ng expression levels have been linked to several diseases, understanding Ng function is critical.

Key Findings:

To probe the role of Ng in synaptic plasticity, the authors overexpressed the protein in mouse hippocampal CA1 neurons. Overexpression of Ng induced LTP during spike-timing-dependent plasticity (STDP) with longer (20 s) pairing intervals in a CaM-dependent manner. Furthermore, Ng knockdown neurons lost the ability to induce LTP during shorter (10 s) pairing intervals. The authors predicted that loss of Ng would increase the amount of CaM-Ca²⁺ complexes in dendritic spines, thereby altering CaM-Ca²⁺ dependent kinase and phosphatase activation. To investigate the impact of Ng knockdown on the phosphoproteome, the authors used mass spectrometry. They showed 16% of the ~30,000 observed phosphorylation sites were significantly altered in Ng knockdown, including reduced phosphorylation on four residues of the NMDAR subunit Grin2A. As Grin2A dysfunction is associated with both schizophrenia and autism-spectrum disorder, the authors examined how hypo-phosphorylation of four Grin2A residues impacts synaptic function. Both Grin2A phospho-deficient mutants and Ng knockdown neurons accelerated the decay of NMDAR-currents, suggesting Grin2A phosphorylation influences synaptic Ca²⁺ influx.

To determine the downstream effects of Ng knockdown, the authors hypothesized that Ng loss increases the concentration of CaM-Ca²⁺, subsequently increasing phosphatase PP2B activation.  They found that FK506, a PP2B antagonist, abolished the accelerated NMDAR current decay in Ng knockdown neurons. Furthermore, FK506 treatment rescued the long-term potentiation (LTP) defects observed in Ng knockdown neurons. Similarly, blocking PP2B with FK506 treatment mimicked Ng overexpression, facilitating LTP induction. Collectively, these data show that Ng regulation of CaM-Ca²⁺ levels is critical for regulating synaptic plasticity.

Why I Selected & Importance

Regulation of synaptic calcium signaling is critical for regulating synaptic strength and defects in these pathways have been linked to numerous neurological disorders. Here, the authors have shown that Ng is critical in regulating CaM-Ca²⁺ levels in the postsynaptic compartment, influencing downstream kinase/phosphatase activity and ultimately NMDAR-mediated current kinetics and LTP induction.  Dysregulation of Ng has been linked to Alzheimer’s, Jacobsen syndrome and schizophrenia. Directly linking Ng function to critical downstream CaM-Ca²⁺ signaling pathways may ultimately reveal new targets for treating these disorders.

Future Directions & Questions for the Authors:

• Phosphorylation of Ng by PKC has been well studied and this modification reduces Ng-CaM binding. In vitro, Ng phosphorylation can be reversed by PP2B. If Ng dephosphorylation is mediated by PP2B in the post-synaptic compartment, could this serve as some sort of feedback mechanism in regulating Ng-CaM binding and therefore PP2B activity?
• Does Ng play a role in regulating CaM-Ca²⁺ during dendritic spine formation/maturation in addition to synaptic plasticity?
• What is the consequence of the hyperphosphorylation of S882/S890 in Grin2A observed in the Ng KD mass spectrometry data?

Further Reading:

1. Petersen, A., & Gerges, N. Z. (2015). Neurogranin regulates CaM dynamics at dendritic spines. Scientific Reports, 5, 11135. doi:10.1038/srep11135
2. Díez‐Guerra, F. J. (2010), Neurogranin, a link between calcium/calmodulin and protein kinase C signaling in synaptic plasticity. IUBMB Life, 62: 597-606. doi:1002/iub.357
3. Jones, K.J, Templet, S., Zemoura, K., Kuzniewska, B., Pena, F.X., Hwang, H. et al. (2018). Rapid, experience-dependent translation of neurogranin enables memory encoding. Proc Natl Acad Sci U S A, 115(25): E5805-E5814.

 

Tags: calcium, dendritic spine, phosphorylation, plasticity

doi: https://doi.org/10.1242/prelights.7546

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