Mutations of the cyclin-dependent kinase-like 5 (CDKL5) and netrin-G1 (NTNG1) genes cause a severe neurodevelopmental disorder with clinical features that are closely related to Rett syndrome, including intellectual disability, early-onset intractable epilepsy and autism. We report here that CDKL5 is localized at excitatory synapses and contributes to correct dendritic spine structure and synapse activity. To exert this role, CDKL5 binds and phosphorylates the cell adhesion molecule NGL-1. This phosphorylation event ensures a stable association between NGL-1 and PSD95. Accordingly, phospho-mutant NGL-1 is unable to induce synaptic contacts whereas its phospho-mimetic form binds PSD95 more efficiently and partially rescues the CDKL5-specific spine defects. Interestingly, similarly to rodent neurons, iPSC-derived neurons from patients with CDKL5 mutations exhibit aberrant dendritic spines, thus suggesting a common function of CDKL5 in mice and humans.

SummaryElectrophysiological studies demonstrate that transient receptor potential vanilloid subtype 1 (TRPV1) is involved in neuronal transmission. Although it is expressed in the peripheral as well as the central nervous system, the questions remain whether TRPV1 is present in synaptic structures and whether it is involved in synaptic processes. In the present study we gathered evidence that TRPV1 can be detected in spines of cortical neurons, that it colocalizes with both pre-and postsynaptic proteins, and that it regulates spine morphology. Moreover, TRPV1 is also present in biochemically prepared synaptosomes endogenously. In F11 cells, a cell line derived from dorsal-root-ganglion neurons, TRPV1 is enriched in the tips of elongated filopodia and also at sites of cell-cell contact. In addition, we also detected TRPV1 in synaptic transport vesicles, and in transport packets within filopodia and neurites. Using FM4-64 dye, we demonstrate that recycling and/or fusion of these vesicles can be rapidly modulated by TRPV1 activation, leading to rapid reorganization of filopodial structure. These data suggest that TRPV1 is involved in processes such as neuronal network formation, synapse modulation and release of synaptic transmitters.Key words: Capsaicin receptor, Synapse, Active zone, FM4-64 dye, Synaptic-vesicle recycling Journal of Cell Sciencefrom rat DRG neurons and mouse neuroblastoma cells, results in increased neuritogenesis and the formation of extensive filopodial structures (Goswami and Hucho, 2007). Surprisingly, most of these filopodia contain elevated levels of TRPV1 at their tips and morphologically resemble elongated club-shaped spines. In the present study we demonstrate that TRPV1 is present in pre-and postsynaptic structures, and that it is transported to synaptic sites by synaptic transport packets. We also provide evidence that TRPV1 regulates filopodial dynamics and vesicle recycling, processes that are important for synaptogenesis. Results TRPV1 localizes to the spines of primary cortical neurons and is present in synaptic protein preparationsOn the basis of our previous work (Goswami and Hucho, 2007), we hypothesized the presence of TRPV1 in synapses. Primary cortical neurons were employed to test this hypothesis. Because the expression level of endogenous TRPV1 in these neurons was too low for immunocytochemical analysis, we expressed TRPV1 for 6 hours in cortical neurons and analyzed its localization. In spite of the short expression period, TRPV1 became enriched in distinct spots resembling synapses (Fig. 1A). To test whether these puncta represent dendritic spines carrying synapses, we co-stained TRPV1 with endogenous pre-and postsynaptic markers. TRPV1 colocalizes with endogenous pre-and postsynaptic marker proteins, including the active-zone protein Bassoon and postsynaptic elements such as GluR2-containing AMPA receptors, NMDA receptors and PSD95 (Fig. 1A,B). We also observed that TRPV1 colocalizes with ProSAP1 (also known as Shank2; a protein that is mainly present in the postsynapti...

The c-Jun N-terminal kinases (JNKs) are stress-activated serine-threonine kinases that have recently been linked to various neurological disorders. We previously described a patient with intellectual disability (ID) and seizures (Patient 1), carrying a de novo chromosome translocation affecting the CNS-expressed MAPK10/JNK3 gene. Here, we describe a second ID patient (Patient 2) with a similar translocation that likewise truncates MAPK10/JNK3, highlighting a role for JNK3 in human brain development. We have pinpointed the breakpoint in Patient 2, which is just distal to that in Patient 1. In both patients, the rearrangement resulted in a predicted protein interrupted towards the C-terminal end of the kinase domain. We demonstrate that these truncated proteins, although capable of weak interaction with various known JNK scaffolds, are not capable of phosphorylating the classical JNK target c-Jun in vitro, which suggests that the patient phenotype potentially arises from partial loss of JNK3 function. We next investigated JNK3-binding partners to further explore potential disease mechanisms. We identified PSD-95, SAP102 and SHANK3 as novel interaction partners for JNK3, and we demonstrate that JNK3 and PSD-95 exhibit partially overlapping expression at synaptic sites in cultured hippocampal neurons. Moreover, JNK3, like JNK1, is capable of phosphorylating PSD-95 in vitro, whereas disease-associated mutant JNK3 proteins do not. We conclude that reduced JNK3 activity has potentially deleterious effects on neuronal function via altered regulation of a set of post-synaptic proteins.

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