Reference:
Abstract:
Functional studies of CDKL5 implicated in an X-linked intellectual disability syndrome with early onset epileptic encelopathy. We have shown that mutations in the X-linked gene CDKL5/STK9 are a significant cause of a severe neurodevelopmental disorder (previously called atypical Rett syndrome (RTT) or variant of RTT), which affects predominantly girls (Kalscheuer et al, Am J Hum Genet 2003; Tao et al, Am J Hum Genet 2004; C�rdova-Fletes et al, Clin Genet 2010; Rademacher et al, Neurogenetics 2011). The main clinical features include early onset epileptic encephalopathy with seizures starting within the fifth month of life, severe developmental delay, deceleration of head growth, impaired communication and, often, hand stereotypies. CDKL5 encodes a serine-threonine kinase. Its N-terminal catalytic domain shares homology with members of the cyclin-dependent kinase (CDK) family and mitogen activated proteins (MAP) kinases. We have established that in primary neurons CDKL5 is localized at excitatory synapses and contributes to correct dendritic spine structure and synapse activity. This finding has prompted us to search for novel CDKL5 binding partners within dendritic spines. Having previously found that truncation of Netrin G1 (NTNG1) by a balanced chromosome translocation caused a clinical phenotype that largely overlapped with RTT (Borg et al, Eur J Hum Genet 2005), we hypothesized that Netrin G1 and CDKL5 could act in the same signaling pathway. To test our hypothesis, we have investigated if the specific Netrin G1 receptor NGL-1, which plays a crucial role in early synapse formation and maturation, interacts with CDKL5. Interestingly, our results indicated that NGL-1 and CDKL5 directly interact and in primary neurons both proteins co-localized at the dendritic spines. Next, we investigated if NGL-1 could be a phospho-substrate of CDKL5 and we indeed could show that CDKL5 phosphorylates NGL-1 on a serine residue (S631) close to the cytoplasmic C-terminal PDZ binding domain. This phosphorylation is necessary (i) for reinforcing the interaction between CDKL5 and NGL-1 and (ii) for promoting a stable association between NGL-1 and PSD95, a major protein of the postsynaptic density which plays an important role in synaptic plasticity. Accordingly, phospho-mutant NGL-1 was not any longer able to induce synaptic contacts while its phospho-mimetic form bound PSD95 more efficiently and partially rescued the CDKL5-specific spine defects. We also investigated the phosphorylation level of NGL-1 in humans using a fibroblast cell line derived from a girl who carried a balanced chromosome translocation that truncated CDKL5 and due to inactivation of the normal X-chromosome lacked functional CDKL5. Importantly, these cells when compared to normal control fibroblasts which express endogenous CDKL5 exhibited reduced levels of phosphorylated NGL1 with respect to the total amount of NGL1 protein and this level could be increased by overexpression of CDKL5. Together, our findings suggest a critical regulatory role for CDKL5 in the formation of excitatory synapses by coupling, through NGL-1 phosphorylation, the Netrin G1-NGL-1 adhesion with the recruitment of PSD95 and thereby provide important molecular insights into the pathophysiology of this disorder (Ricciardi et al, Nat Cell Biol 2012).
PROJECT DETAILS
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Country of research: Germany
Counry of funding source: Germany
Funding organization: Max Planck Institute for Molecular Genetics
Financing: NATIONAL FUNDINGS – 0 €