CDKL5 ensures excitatory synapse stability by reinforcing NGL-1-PSD95 interaction in the postsynaptic compartment and is impaired in patient iPSC-derived neurons

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 (RTT), 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 while its phospho-mimetic form binds PSD95 more efficiently and partially rescues the CDKL5-specific spine defects. Interestingly, similar to rodent neurons, iPSC-derived neurons from patients with CDKL5 mutations display aberrant dendritic spines, thus suggesting a common function of CDKL5 in mice and humans.


Introduction
Rett syndrome (RTT) is a severe neurodevelopmental disorder and is the most common genetic cause of intellectual disability in females 1,2 . The disorder is mainly caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene 3,4 . In addition to classical RTT, several atypical forms exist, including the Hanefeld variant, which is caused by mutations in the X-linked CDKL5 gene. Patients present with early-onset intractable seizures starting before the age of 6 months and also show some typical symptoms of RTT although the clinical spectrum is heterogeneous [5][6][7][8][9][10][11] . This suggests that CDKL5 mutations cause an early-onset neurodevelopmental disorder 12 . In a small number of patients CDKL5 deletions have been reported [13][14][15] . These very likely caused loss-of CDKL5 function, similarly to what has been previously found in patients with a chromosome translocation that disrupted CDKL5 8,9 .CDKL5 encodes a serine-threonine kinase whose catalytic domain shares homology with members of the cyclin-dependent kinase family and mitogen-activated protein kinases 16,17 . Interestingly, we showed that CDKL5 and MeCP2 are both induced at high levels during neuronal maturation and synaptogenesis. In addition, CDKL5 binds and phosphorylates MeCP2 in vitro 18 . Despite this molecular interplay, it is now well assessed that CDKL5 exerts its own functions, regardless of MeCP2 [19][20][21][22] . However, the functional role of CDKL5 is presently unknown and thus, the question of how CDKL5 mutations affect neuronal development and function and contribute to the pathophysiology of RTT remains to be answered.
Herein, we describe that CDKL5 silencing leads to severe deficits in spine density and morphology. Notably, similar alterations were found in neurons established from patient fibroblast-derived pluripotent stem cells (iPSCs). Furthermore, we identified the netrin-G1 ligand (NGL-1, also known as LRRC4C) as a direct interactor and substrate of CDKL5. Importantly, NGL-1 phosphorylation strengthens the NGL-1-PSD95 interaction. Our findings demonstrate a novel role for CDKL5 in spine development and synapse morphogenesis.
number of CDKL5 puncta (61 ± 3%) co-localized with PSD95 in DIV15 neurons (Fig. 1df). Likewise, CDKL5 staining closely matched the pattern of other PSD markers (Fig. 1j). Consistent with a postsynaptic localization, CDKL5 immunolabeling was closely juxtaposed with presynaptic VGLUT1 (Fig. 1g). Triple staining for CDKL5, PSD95 and VGLUT1 confirmed CDKL5 localization at excitatory synapses (Fig. 1h, i). CDKL5 puncta coincided with the dot-like immunostaining of PSD95 and SHANK (Fig. 1k-m) and were apposed to VGLUT1 puncta also in brain (Fig. 1n-o'). To confirm the presence of CDKL5 at the PSD we performed a subcellular fractionation of mouse brain. CDKL5 was included in the synaptic fraction and in the entire PSD fraction (Fig. 1t). Further detergent solubilization of the synaptic plasma membrane fraction showed that CDKL5 is detectable in all PSD fractions, indicating its association with the PSD (Fig. 1t). Our studies also showed that CDKL5 co-localized marginally with inhibitory synaptic markers (Fig. 1p-s). Taken together, these findings indicate that CDKL5 is almost exclusively localized at excitatory synapses both in vitro and in vivo.

Loss of CDKL5 impairs spine structure and synaptic activity
To investigate the role of CDKL5 in dendritic spines, we silenced CDKL5 in hippocampal neurons using two short-hairpin RNAs (sh-CDKL5#1, sh-CDKL5#2). In HEK293T cells both shRNAs down-regulated exogenous mouse CDKL5 levels by almost 80% (Supplementary Fig. S2a). Next, we infected DIV 7 hippocampal neurons with sh-CDKL5#1 and 7 days later observed a consistent reduction of CDKL5 ( Supplementary Fig.  S2b-h). Knock-down neurons showed a significant increase in protrusion density ( Fig. 2a-i), the dendritic protrusions were significantly thinner compared to controls and showed a filopodia-like morphology ( Fig. 2a-i). Notably, some of these thin filopodia-like spines were particularly branched and displayed an aberrant morphology ( Fig. 2b'', b'''). Furthermore, the percentage of filopodia-like spines and thin headed spines increased, while the percentage of stubby and mushroom-shaped spines decreased (Fig. 2h, i). We obtained similar results for neurons infected at DIV 10 and analyzed at DIV 15 (data not shown). Conversely, we did not score any significant alteration in dendritic arborization (data not shown). These results indicate that CDKL5 is required for ensuring a correct number of well-shaped spines. Specificity was confirmed by the ability of a CDKL5 knock-down resistant form to rescue both, normal spine morphology and density (Supplementary Fig.  S2i-o). The morphological alterations reported in CDKL5 silenced neurons were associated with a reduction in excitatory synapses and synaptophysin puncta in these neurons ( Fig. 2js). To determine whether these alterations were accompanied with a reduction in overall synaptic activity, we examined spontaneous miniature excitatory post-synaptic currents (mEPSCs) in sh-CDKL5#1 transfected neurons. Consistent with the reduced excitatory synapse density, CDKL5 knock-down caused a significant decrease of mEPSCs ( Fig. 2t-y) but had no significant effect on inhibitory synapse density and accordingly we did not detect any significant changes in miniature inhibitory postsynaptic currents (mIPSCs) (Supplementary Fig. S3). These results indicate that CDKL5 is necessary for correct dendritic spine structure and synapse activity.
Considering that CDKL5 is a kinase, we hypothesized that its phosphorylation activity is necessary for such function. To address this issue we used the CDKL5 K42R kinase-dead form, which has a dominant-negative effect on wild-type CDKL5 16,19 . To analyze the effects of CDKL5K42R on spine density and morphology, we transfected DIV 7 hippocampal neurons with DsRed alone or combined with GFP-CDKL5K42R. Interestingly, GFP-CDKL5K42R immunolabeling was very similar to that of endogenous CDKL5 and there was a clear enrichment of the immunostaining in dendritic spines ( Supplementary Fig. S2pq'). Consistent with its dominant-negative action, neurons overexpressing GFP-CDKL5K42R displayed a significant higher number of thinner and longer dendritic protrusions and a corresponding loss of stubby and mushroom-shaped spines with respect to controls ( Supplementary Fig. S2r-w). Since the phenotype discovered for the CDKL5K42R mutant was similar to the phenotype of CDKL5 knock-down neurons, it is conceivable that CDKL5 promotes spine formation through a mechanism that is dependent on its kinase activity.
Next, we sought to confirm the significance of our findings in vivo by electroporating E13.5 embryos in utero with a plasmid containing cassettes for independent expression of sh-CDKL5#1 and EGFP. Interestingly, at E18.5 CDKL5 down-regulated neurons showed a delay in radial migration. On the contrary, at P11 their relative location within the cerebral cortex was comparable to that of controls, thus indicating a transient delay in the definitive neuronal positioning (Fig. 3a, b and data not shown). Imaging of the GFP-labeled dendritic protrusions at P11, a crucial period of synaptogenesis, showed numerous protrusions with a large fraction having a well-defined head structure, characteristic of mature spines. Similar to the in vitro studies, sh-CDKL5#1 knock-down cortical pyramidal neurons showed a robust increase in protrusion density with dendritic protrusions significantly longer and dysmorphic ( Fig. 3c-h). Next, we asked whether CDKL5 deficit might affect the number of functional excitatory synapses also in vivo. The density of VGLUT1 puncta identifying excitatory presynaptic buttons was significantly lower in sh-CDKL5#1 than in sh-Control pyramidal neurons (Fig. 3e, f, i), confirming that CDKL5 is crucial for dendritic spine morphogenesis and synaptic contact maintenance in vivo.

NGL cell adhesion molecules interact with CDKL5 at the PSD
Next, we set out to identify new CDKL5 interacting partners within dendritic spines. On the basis of our previous results showing that truncation of NTNG1 caused atypical RTT with early-onset seizures 23 , we considered the netrin-G1 receptor NGL-1 and the other members of the NGL family (NGL-2, -3, also known as LRRC4 and LRRC4B), which play a crucial role in early synapse formation and subsequent maturation 24,25 as a good starting point. Induction of NGL-netrin-G interaction promotes synapse formation 26,27 . In cells transfected with CDKL5-V5 and the intracellular domain of each of the NGLs, all three members were immunoprecipitated by the V5 antibody (Fig. 4a). In particular, CDKL5 binds specifically to a stretch of 5 amino acids located at the N-terminal side of the intracellular domain ( Fig. 4bd). Additionally, we screened other PSD enriched proteins that were directly or indirectly implicated in RTT-related disorders for CDKL5 interaction, but the results were negative (data not shown).
This prompted us to particularly characterize the association of CDKL5 with NGL-1. A coclustering assay performed in cells co-transfected with GFP-CDKL5 and Myc-NGL-1, indicated that these proteins form intracellular and surface co-clusters ( Fig. 4e-i). In fact, while CDKL5 localized in the cytoplasm when transfected alone, co-expression with NGL-1 resulted in CDKL5 translocation to the cell membrane and exact overlapping with NGL-1 ( Fig. 4g-h). Further tests in a more physiological context showed that in hippocampal neurons both proteins co-localized precisely at dendritic spines ( Fig. 4j-j'''). Similarly, Myc-NGL-1 also highly co-localized with endogenous CDKL5 (Fig. 4k-m). Next, the interaction of the proteins was assessed using P4 synaptosome brain extracts. Indeed, NGL-1 was coimmunoprecipitated by the CDKL5 antibody (Fig. 4n), indicating that NGL-1 assembles in the CDKL5 complex in vivo. Notably, both proteins even directly interact as ascertained by a pull-down assay (Fig. 4o).
These data prompted us to speculate that CDKL5 might phosphorylate NGL-1. At first, we analyzed whether NGL-1 is phosphorylated in mouse brain by performing 2D-SDS-PAGE from P10 extracts (Fig. 5a). NGL-1 specific staining was resolved in two protein spots. Since addition of the phosphatase CIP robustly reduced the intensity of only spot 2, while spot 1 increased its relative signal, we concluded that spot 2 did represent the phosphorylated form of NGL-1 (Fig. 5a).
Subsequently, we subjected the entire or parts of the NGL-1 intracellular domain to Phos-Tag staining. Interestingly, only the entire and the C2 N-terminal part produced an additional upper band that presents a putatively phosphorylated protein as confirmed by its loss after CIP treatment (Fig. 5b). These results indicate that NGL-1 is present with a monophosphorylated form both, in vitro and in vivo.

CDKL5 phosphorylates NGL-1 at S631
NGLs' C2 N-terminal domain has several amino acids conserved between the family members, including S600, S620 and S631. We mutated these serines into nonphosphorylable alanines and repeated the Phos-Tag assays. Importantly, our results identified NGL-1 S631, but not S600 or S620, to be phosphorylated (Fig. 5c). To obtain direct evidence of the CDKL5 ability to induce NGL-1 protein phosphorylation, the Cterminal half of NGL-1 (aa 550-640) was purified and used for in vitro kinase assays.
Interestingly, NGL-1 is readily phosphorylated in the presence of CDKL5. In contrast, mutant S631A was not phosphorylated by CDKL5 (Fig. 5d). The catalytic activity of CDKL5 has been reported to be involved in its auto-phosphorylation in vitro 16,18 . Consistent with this, we observed auto-phosphorylation when CDKL5 was added (Fig. 5d).
To address if the NGL-1 S631A mutant could be co-immunoprecipitated by CDKL5, we analyzed their interaction. Noteworthy, GFP-CDKL5 failed to co-immunoprecipitate NGL-1 S631A, thus suggesting the relevance of the phoshorylation for reinforcing the interaction (Fig. 5e). To gain further support, we analyzed the NGL-1 binding ability of various CDKL5 deletion mutants. Notably, none of the mutants lacking the kinase domain or its activity were able to immunoprecipitate NGL-1 ( Supplementary Fig. S4) suggesting that the kinase domain and CDKL5 auto-phosphorylation are necessary for the stable interaction of CDKL5 with NGL-1. We next investigated the phosphorylation level of NGL-1 in a fibroblast cell line derived from a female patient who carried a X;7 chromosome translocation that trunctated CDKL5 and due to inactivation of the normal X-chromosome lacked functional CDKL5 8 . Importantly, the patient cells when compared to normal control fibroblasts which express endogenous CDKL5 exhibited reduced levels of the NGL-1 phosphorylated form with respect to the total amount of the protein (Fig. 5f). Thus, these data suggest that the CDKL5/ NGL-1 protein interaction is conserved in human cells and that also in fibroblasts NGL-1 is phosphorylated by CDKL5. Along the same line, an increment of the NGL-1 phosphorylated form was detected when CDKL5 was overexpressed in the patient fibroblasts. This effect was not seen in normal control fibroblasts, suggesting that in these cells NGL-1 S631 phosphorylation was already saturated by endogenous CDKL5 (Fig. 5g).

NGL-1 binding to PSD95 is sustained by CDKL5 phosphorylation on S631
NGLs show a conserved C-terminal PDZ binding domain (VQETQI), which specifically binds to PSD95 27 that plays a significant role in learning and memory 29 . NGL-1 spine inducing capability is promoted by targeting PSD95 to new forming dendritic protrusions 27 . Given that CDKL5 interacts directly with NGL-1, we wondered whether CDKL5 might influence the interaction of these proteins. To test this, we performed a coimmunoprecipitation assay in cortical neurons infected at DIV 7 with sh-CDKL5#1. Seven days later, NGL-1 was immunoprecipitated and the amount of PSD95 in the complex was determined. Remarkably, we observed a robust reduction of the NGL-1-PSD95 interaction in CDKL5 knock-down neurons (Fig. 6a). These results indicate that CDKL5 functions in promoting and/or maintaining the NGL-1-PSD95 protein association.
NGL-1 phosphorylated residue S631 is very close to the terminal PDZ binding domain and therefore located at a convenient site for regulating NGL-1-PSD95 interaction. To test this, we transfected cells with either NGL-1 or its S631A phospho-mutant in combination with GFP-PSD95, immunoprecipitated the lysates for Myc-NGL-1 and measured the amount of associated PSD95. Indeed, the phospho-mutant showed reduced association with PSD95 ( Fig. 6a). Next, we tested the ability of the mutant to promote synaptogenesis in hippocampal neurons. Notably, dendritic protrusions were reduced in numbers and showed a significantly elongated morphology (Fig. 6b, c). These alterations are both qualitatively and quantitatively similar to the phenotype observed in CDKL5 down-regulated neurons. Therefore, it is plausible that the NGL-1 S631A mutant acts through a dominant-negative effect on endogenous NGL-1 and its ability to associate with PSD95.
To further strengthen this assumption, the phospho-mimetic NGL-1 mutant S631E was challenged in similar experiments. In particular, this mutant associated better with PSD95 although it was not able to significantly increase spine formation upon overexpression (Fig.  6b, c). Despite that, when expressed in CDKL5 knock-down neurons, the mutant was capable to restore nearly correct length and morphology of dendritic spines, but not their numbers (Fig. 6f, g). Conversely, both phospho-mutants retained the ability to induce presynaptic terminals similar to the native NGL-1 when expressed by heterologous cells cocultured with primary neurons (Supplementary Fig. S5) 27 . These findings are consistent with the NGL-1 extracellular domain being the main molecular player of this activity.
In overall, these data indicate that CDKL5 is of critical relevance for the maintenance of synaptic contacts, which is mainly achieved by regulating the NGL-1 phosphorylated state and, thereby, its ability to bind PSD95 and stabilize their association.

Patient-specific alterations in iPSC-derived neurons
iPSC technology offers the possibility to model in vitro neurodevelopmental disorders by providing access to human neurons. Therefore, we set out to establish iPSC lines from two female patients diagnosed with atypical RTT caused by CDKL5 pathogenic mutations (R59X, L220P) and whose primary fibroblasts were already characterized 20,30 . Fibroblasts were infected with OCT4, SOX2 and KLF4-expressing retroviruses and cultured in the presence of SR and FGF2. After 6-8 weeks, the iPSC-like clones expressed pluripotency markers and showed silencing of the retroviral exogenous genes ( Fig. 7a and Supplementary  Fig. S6a,b, Fig. S7a,b). Pluripotent properties were also ascertained by testing the capability to differentiate into cells of the endoderm (SOX17 + ), mesoderm (SMA + ) and neuroectoderm (TuJ1 + ) germ layers in vitro and the potential to generate teratomas ( Supplementary Fig.   S6c,d). Further, correct chromosomal content was ascertained by karyotyping ( Supplementary Fig. S7c).
Increasing evidence shows that female iPSCs may or may not retain the inactivated Xchromosome status of the original fibroblasts [31][32][33] . Indeed, only iPSCs exclusively expressing the mutant CDKL5 allele will represent a coherent model of disease-affected cells. Thus, we only investigated those clones in which inherent X-chromosome inactivation was revealed by H3K27me3 nuclear staining (Fig. 7a) and selected only cell lines, which showed expression of either the mutant or the normal CDKL5 allele for further analysis.
This approach allowed the generation of both, pathogenic and healthy iPSCs from the same individual providing stringent control cells for comparative analyses. A list of the cell lines used for each experiment is detailed in Supplementary Table 1.
Undifferentiated mutant and control iPSC clones did not show any evident difference (data not shown). We then induced their differentiation into neurons through generation of neuralrosette intermediate neural progenitors and their subsequent differentiation for 55-60 days 34 . In these conditions, neuronal cultures developed a highly intricate neuronal network highly enriched in forebrain committed glutamatergic neurons ( Fig. 7b-h). Interestingly, CDKL5 expression was found to strongly increase following neuronal differentiation (Fig. 8a). In particular, CDKL5 was found enriched in dendritic spines in well-matured neurons (Fig. 8b). These findings establish iPSC-derived neurons as an adequate in vitro model to study CDKL5-dependent biological mechanisms. Absence of CDKL5 did not impair the rate of neuronal differentiation or morphology ( Fig. 8c-g). Interestingly, a significantly reduced number of synaptic contacts, indicated by the number of VGLUT1 and PSD95 puncta was detected in the mutants (Fig. 8h-m). To highlight spine morphology, neurons were infected with a constitutively expressing GFP lentivirus at low multiplicity. This staining permitted to visualize frequent abnormalities in the overall spine structure of mutant neurons (Fig. 8n-r). In particular, many of these altered spines did not present an evident pre-synaptic terminal (Fig. 8p, q). These results are well in line with those obtained for CDKL5 silenced mouse neurons and suggest a conserved role of this protein in neuronal structure homeostasis and activity.

Discussion
In this study, we revealed how loss of CDKL5, the molecule responsible for the Hanefeld variant of RTT and early-onset intractable encephalopathy, impacts neuronal function. Our first observations indicate that CDKL5 mainly localizes to dendritic spines where it is juxtaposed to the PSD. Interestingly, CDKL5 was found in the human PSD 35 (www.genes2cognition.org/HUMAN-PSD), suggesting that its localization at the synaptic PSD is evolutionary conserved. Our data provide evidence that CDKL5 is necessary for correct genesis and maintenance of dendritic spines and for synapse formation. Indeed, knocking down CDKL5 significantly decreases the density and length of dendritic spines both, in vitro and in vivo.
In line with the compromised development of spines, CDKL5 down-regulated neurons exhibit abnormal mEPSCs. Thus, our results reveal that CDKL5 is a key-limiting factor in regulating glutamatergic synapse formation and suggest that changes in excitatory synaptic strength might be responsible, at least in part, for the neurodevelopmental symptoms associated with its deficiency.
We identified CDKL5 as an interactor of NGLs, which are synaptic cell adhesion molecules (CAMs) that exert a pervasive role in synapse homeostasis 24,27,[36][37][38] . Very intriguingly, we havepreviously found that NTNG1, which encodes the specific NGL-1 partner netrin-G1, was disrupted by a chromosome translocation in a patient with atypical RTT 23 . It is then highly plausible that CDKL5 and NGL-1 might work, at least in part, in a common pathway thus providing a molecular framework explaining the similar neuropathological outcome of NTNG1 and CDKL5 mutations.
Our findings prompted us to search for pathogenic mutations in NGL-1, -2 in patients with atypical RTT. No likely pathogenic changes were detected in these genes, suggesting that they are not a major contributor (data not shown). Despite we did not detect NGL-1, -2 mutations, other groups reported deletions including NTNG1 in patients presented with intellectual disability and seizures 39 , NTNG1 missense mutations in autism 40 , and the association of NTNG1 non-coding single nucleotide polymorphisms with schizophrenia 41 .
We provide evidence that CDKL5 phosphorylates NGL-1 on a unique serine, which is very close to the PDZ binding domain that interacts with PSD95. This phosphorylation can also be extracted from a phospho-proteomic survey of mouse tissues 28 . To date, little evidence exists showing that the activity of synaptic CAMs is controlled by post-translational mechanisms. The discovery of a regulated phosphorylation process in NGL-1 opens the exciting possibility that the activity of other CAMs can be controlled by their phosphorylation state. We demonstrated that CDKL5 is able to phosphorylate NGL-1 at S631 both, in vitro and in mouse brain. Importantly, the same results were obtained for human cells. In fact, CDKL5 negative patient fibroblasts exhibited reduced NGL-1 phosphorylation when compared to control cells. Since NGL-1 phosphorylation was not completely absent, it is plausible to assume that other kinases substitute for CDKL5 in these cells. To overcome the limitations of using skin fibroblasts, we generated iPSC-derived neurons from two female patients with atypical RTT. Interestingly, in these neurons we detected a loss of synaptic contacts together with an increased number of aberrant dendritic spines indicating that they represent a reliable system for studying CDKL5 function.
We showed that CDKL5-mediated phosphorylation of NGL-1 is necessary to ensure stable binding to PSD95. This mechanism resembles the phosphorylation dynamics of ion channels that mediate changes in the interaction with PSD95 42,43 . Furthermore, phosphorylation of serine at position -9 outside the PDZ-binding domain in ephrin B ligands is required for the stable interaction with GRIP at the PSD and ensures correct synaptic activity 44 . These results mirror our findings and highlight a new aspect of fine-tuning protein-protein interaction by serine phosphorylation.
Interestingly, overexpression of a NGL-1 non-phosphorylable mutant leads to significant elongation of dendritic protrusions. This phenotype closely resembles the defects observed in CDKL5 silenced neurons, suggesting that the mutant could act in a dominant-negative manner leading to disruption of the NGL-1-PSD95 interaction. Thus, these data indicate that synaptic strength and stability can be modified by altering the ratio between phosphorylated and unphosphorylated NGL-1 and, possibly, its homologs.
Our results are consistent with a model in which loss of CDKL5 elicits spine length and synaptic strength by regulating NGL-1-PSD95 interaction. On the other hand, CDKL5 interacts with Rac1 21 , which participates in stabilizing the actin cytoskeleton. Therefore, it is possible that Rac1 activity is altered by CDKL5 deletion. This is particularly relevant given that actin filament dynamics within the spines control their morphology and stability 45 . Thus, we cannot exclude that some aspects of the alterations described in this study can be ascribed to defects in such mechanisms. In agreement with this assumption, rescue of CDKL5-dependent spine deficits by a phospho-mimetic NGL-1 mutant is incomplete suggesting that other mechanisms are dysregulated by CDKL5 impairment.
In conclusion, we demonstrate that loss of CDKL5 severely affects spine morphology and reduces functional synaptic contacts impairing neuronal activity.
We propose a model in which CDKL5-dependent phosphorylation on S631 controls the association of NGL-1 with the postsynaptic molecular hub PSD95. In light of these findings, we provide novel mechanistic insights into how CDKL5 mutations can impact on neuronal function in atypical forms of RTT and other CDKL5-related neurodevelopmental disorders.
The lentiviral knock-down constructs were generated by cloning oligoduplexes into pLentiLox 3.7 (pLL 3.7) according to the protocol published online by Science gateway (http://www.sciencegateway.org/protocols/lentivirus/cloning.htm). The sh-CDKL5#1 construct targets the region AGGAGCCTATGGAGTTGTA, the sh-CDKL5#2 construct is directed against the target region TGATAGCAGTTCTGGTACA and the sh-control construct is directed against Renilla luciferase and targets the region GGCCTTTCACTACTCCTAC. pMyc-hNGL1 was a gift from E. Kim (Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea). pEGFP-PSD95 was a gift from L. Muzio (San Raffaele Scientific Institute, Milan, Italy). pMXOCT4, pMXSOX2, pMXKLF4 were a gift by S.Yamanaka (Gladstone Institute, University of California). The S631A, S631E and S600A derivatives were generated by site-directed mutagenesis using the Quick change II site-directed Mutagenesis Kit (Stratagene). pCAGGS-hNGL1-iresEGFP and pCAGGS-NGL1-S631A-iresEGFP were generated by PCR and cloned into the EcoRI site of pCAGGS-iresEGFP. Recombinant hNGL1 (550-640AA) and hNGL1 (550-640AA) S631A were produced from the pTYB1-NGL1 and pTYB1-NGL1-S631A constructs cloned by PCR into the NdeI and XhoI sites of pTYB1 (New England Biolabs) generating a chitinbinding fusion protein. All PCR-generated constructs were verified by sequencing.

Primary hippocampal and cortical cultures
Primary neuronal cultures were prepared as described previously 20 .

Mixed-culture assay
Mixed-culture assay was carried as described previously 40 . Ricciardi

Retrovirus infection, iPSCs derivation and differentiation
Female CDKL5 fibroblasts derived from two patients' specific dermal biopsies explants were kindly provided by Thierry Bienvenu's group (Cochin Institute, Paris, France). They were maintained in vitro for two passages before infection with three retrovirus cocktails (OCT4, SOX2 and KLF4). 2.5 × 10 5 fibroblasts were infected with the retroviral cocktail for each reprogramming experiment; two days after infection, fibroblasts were plated on mitotically inactivated murine embryonic fibroblasts (MEF) layer on Matrigel™ (BD) coated dishes. After 6-8 weeks in culture in hES medium (DMEM F12, 1 mM Sodium Pyruvate, 1X NEA, 20 μM β-Mercaptoethanol, 20% Knockout Serum Replacements, 10 ng/ml FGF2; Invitrogen), the first hiPSC clones appeared and were manually picked. Thus, they were cultured and expanded in hESCs conditions on MEF feeders layer on Matrigel™ (BD) coated dishes. For differentiation into the three germ layers, hiPSC colonies were incubated in 1 mg/ml collagenase IV (Invitrogen) for 1 hour at 37°C, detached and collected. They were cultured in suspension for 5 days in EB medium (hES without FGF2) and plated on 1 mg/ml gelatine (Sigma) coated dishes for up to 20 days in 20% Fetal Bovine Serum (Invitrogen). For neuronal differentiation, EBs were cultured for 5 days in suspension in EB medium, then plated and cultured on Matrigel™ coated dishes in N2/FGF medium (DMEM F12, 1X N2 supplement, 20 ng/ml FGF2; Invitrogen) for 3-5 days. After 3-5 days, appearing rosette-like structures were picked and manually disaggregated; then, they were plated on a layer of glial cells derived from E14.5 mouse cortices in N2/FGF medium for 1 day and then cultured in B27/AA medium (Neurobasal medium, 1X B27 supplement; Invitrogen; 20 μM Ascorbic Acid; Fluka; 20 ng/ml BDNF; Peprotech) for 55-60 days. For spine length analysis, manually disaggregated rosette structures were plated on Matrigel™ for 1 day, then infected by lentivirus carrying GFP reporter (pLL3.7-GFP) and maintained in culture for 3 days. Infected cells were manually detached and plated again on mouse astrocyte layers. They were cultured on astrocytes for 55-60 days in B27/AA medium and then fixed for further analysis.

Immunofluorescence
Hippocampal neurons were fixed for 10 min in methanol (-20°C) or 4% paraformaldehyde for 20 min at room temperature. Immunofluorescence was performed as described previously 20 . The following primary antibodies were used: immunopurified rabbit polyclonal anti-CDKL5 (1:5) 19  Bright field images were detected by a upright Leica microscope.

RNA Extraction and qRT-PCR
All RNA samples were extracted with RNeasy Micro Kit (Quiagen) from 1 to 5×10 6 cells, according to the manufacturer's instruction. RNA strands were retro-transcribed using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) and cDNAs were amplified by Amplibiotherm DNA polymerase (Fisher Molecular Biology). For qRT-PCR, SsoFast EvaGreen Supermix™ (BioRad) was used according to the manufacturer's instruction.

Immunoprecipitation and Western blot
Immunoprecipitation on cell transiently co-transfected using the calcium phosphate precipitation method or Lipofectamine 2000 (Invitrogen) and Western blot were performed as described previously 20

Phosphate-affinity polyacrylamide gel electrophoresis
The dinuclear metal complex Mn2+-Phos-Tag™ acts as a phosphate-binding tag molecule and allows separation of phosphorylated and non-phosphorylated proteins in SDS-PAGE by mobility shift detection.Here we used 10% SDS-gels with 50μM Phos-Tag™ acrylamide (Nard Institute, Ltd) and 100 μM MnCl 2 (Sigma) according to the manufacturer's recommendations.

2D-PAGE
Mouse brains were manually broken up using a dounce homogenizer and then resuspended in 500 μl of lysis buffer (5 M urea, 2 M thiourea, 2% CHAPS w/v, 2% Zwittergent v/v, protease and phosphatase inhibitors). The lysates were centrifuged at 16,000 g for 30 minutes at 15°C and stored at -80°C. The recovered supernatant was analyzed to determine total protein concentration using BioRad protein assay and BSA as standard. For 2D electrophoresis, 300 μg total protein were loaded on each gel and each sample was run in triplicates. Prior to the first dimension of isoelectrofocusing, each sample was added with

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Europe PMC Funders Author Manuscripts Destreak (final concentration: 100 mM) and IPG buffer (final concentration: 0.5%) and then run at 50,000Vhr on 7 cm strips. The second dimension was SDS-PAGE 6%, upon reduction with DTE and alkylation with iodoacetamide of the strips. Resolved proteins were electrotransferred onto Hybond-ECL nitrocellulose membrane with a miniVE Blot Module (GE Healthcare) for 2 h at 300 mA in transfer buffer (25mM trizma base, 40mM glycine, 20% methanol, 0.05% SDS). Membranes were probed for NGL-1. Detection was by enhanced chemiluminescence (ECL) reaction (ECL detection kit, GE Healthcare) and proteins were visualized on autoradiography films (Hyperfilm ECL, GE Healthcare). The lysate (300 μg total protein), without phosphatase inhibitors, was subjected to dephosphorylation reaction using CIP. Membrane images were acquired with ProXPRESS 2D Proteomic Imaging System and quantitative comparisons of spot intensities were carried out using Progenesis SameSpots (Nonlinear Dynamics, Newcastle upon Tyne, UK).

In uterus electroporation
In uterus electroporation was performed as described previously 41 .

Immunocytochemistry for light microscopy
Immunocytochemistry on floating 50 μm brain sections, made with a CM 1850 UV cryostat (Leica), was performed as described previously 41 . Sections were incubated with mouse anti-GFP (clone 3E6; 1:2,000, Invitrogen) and guinea-pig anti-VGLUT1 (1:500, Synaptic System). For teratoma analysis, up to 2×10 6 iPSCs were injected subcutaneously into the dorsal flanks of nude mice. After 3 months, teratoma were explanted, fixed and dissected as described above. Sections were stained with hematoxylin and eosin for further analysis.

Dendritic protrusion analysis
Fluorescent images were acquired using a TCS SP5 laser scanning confocal (Leica) 63× oilimmersion lens as z series of 15-25 images taken at 0.3-μm intervals at 1.024 × 1.024 pixels resolution with 1x zoom. The confocal microscope acquisition parameters were kept the same for all scans for each experiment. Each experiment was repeated at least two, mostly three times and images analyzed in blind using ImageJ (NIH) software. Dendritic protrusion density was measured by counting the number of dendritic protrusions on both, primary and secondary dendrites and expressed as the number of dendritic protrusions per 10 μm dendritic length. Dendritic protrusion length was measured by manually drawing a vertical line from the protrusion's tip to the point where it met the dendritic shaft while head width was measured by drawing a perpendicular line to the length. The proportion of spines in each morphology category was determined for each dendrite. For each experiment, about 300-600 spines from 25-30 dendrites derived from 10-20 neurons were analyzed per condition. In figure legends, n refers to the number of neurons quantified. Statistical significance was determined by Student's t-test.

Electrophysiology
Standard patch-clamp whole cell recordings were taken from primary hippocampal neurons transfected at DIV 7 with sh-control or sh-CDKL5#1 and labeled for GFP at DIV 14 as described above. The neurons were continuously perfused with artificial cerebrospinal fluid . Miniature excitatory post-synaptic currents (mEPSCs) were observed in the presence of aCSF containing 1 mM tetrodotoxin and 10 mM (-)-bicuculline methiodide, using continuous 60 second epochs of mEPSCs recorded at − 60 mV membrane potential with an Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) with a low pass filter of 2 KHz. Data were digitized at 2-5 KHz with a Digidata 1322A (Molecular Devices) controlled by Clampex 9 software (Molecular Devices) and analyzed off-line with Clampfit 9 software (Molecular Devices), after processing with a band pass of ~2 Hz to 1 KHz using a single pole digital filter. Event selection was visually checked for all data. The statistical analysis was performed using the paired T-test expressing population mean as the mean ± SE.

Recombinant protein purification
hNGL-1 and hNGL-1-S631A were cloned in pTYB1 expression construct, expressed in E.coli ER2566 strain and induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 30°C for 5 h. Following induction, extracts were prepared by resuspending the bacteria in 750 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1% Triton X-100 with protease inhibitors (Sigma) and sonicated. Following centrifugation at 40,000 rpm for 10 min, the lysate was added to chitin-agarose beads (New England Biolabs Inc.). Fusion proteins were cleaved on the column overnight by incubation with lysis buffer containing 50 mM dithiothreitol (DTT). Eluted fractions containing the bulk of NGL-1 were pooled. Recombinant purified NGL-1 and NGL-1-S631A were dialyzed for 16 h at 4°C against the kinase buffer used in the phosphorylation assay.

In vitro phosphorylation assays
HEK293T cells were transfected with 6-Myc-CDKL5 and 6-Myc plasmids. 48 hours after transfection, transfected cells were lyzed in 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% NP-40, 1 mM DTT, phenylmethylsulfonyl fluoride and protease and phosphatase inhibitors (Sigma). Equal amounts of protein extracts were pre-cleared for 1 h with 40 μl of 100% mouse IgG-agarose beads at 4°C; 40 μl of 100% agarose conjugate anti-c-Myc (Sigma) was added to the extracts and the mixture incubated for 2 h at 4°C. Immunocomplexes were collected by centrifugation, washed five times and incubated with 1.5 μg of recombinant protein in a kinase buffer in the presence of 7 μCi of [γ-33 P] ATP and 25 μM of unlabeled ATP and incubated for 30 min at 30°C. The reaction was stopped by the addition of Laemmli buffer and directly loaded onto a 15% SDS-PAGE; 33 P-labeled proteins were detected by autoradiography.

Pull-down
Myc-CDKL5 was first translated in vitro using the TNT Quick Coupled Transcription/ Translation System (Promega) and then incubated with Intein-immobilized-chitin-agarose beads or with hNGL-1-Intein-immobilized-chitin agarose beads for 4 h at 4°C.
Immunocomplexes were collected by centrifugation, washed three times with 750 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1% Triton X-100 with protease and phosphatase inhibitors (Sigma), separated on SDS-PAGE and subjected to western blot with anti-Myc and anti-Chitin-Binding-Domain (CBD) antibodies.

Lentivirus and retrovirus production
VSVG-coated particles were packaged in HEK293T cells.

Synaptosome and PSD preparation
The synaptosome fraction was prepared from rat brains as described previously 42 . PSD fractions were prepared from the synaptosome fraction as described previously 43 .

Mutation search of NGL-1 and NGL-2
For mutation search the single coding exons of NGL-1 and NGL-2 together with their immediately flanking non-coding regions were screened for mutations in a cohort of 51 patients who had been diagnosed with RTT, infantile spasms or a variant of RTT, in whom no MECP2 or CDKL5 mutation had been identified. All blood samples were obtained after provision of informed consent. DNAs were amplified by PCR using several primer pairs. Primer sequences are available upon request from V.M.K. PCR products were subjected to denaturing high-performance liquid chromatography or directly sequenced using conventional Sanger sequencing.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. is detected in the synaptosomal fraction (Syn) and is enriched in the postsynaptic density fraction I (PSDI). Note that CDKL5 is also detected in postsynaptic density fractions II and III (PSDII and PSDIII). PSD95 and Synaptophysin (Syn) were used as a control. Scale bars: 10 μm (a, b, d, h, k, p,    Antitubulin antibody staining served as loading control. The relative NGL-1 phosphorylation is presented as means +/− SD of four independent experiments compared to the total amount of NGL-1 (***p<0.001, t test). (g) Total cell lysates of both, control and CDKL5 negative patient fibroblast cells were transfected with the indicated plasmids and resolved in a normal SDS gel or in a Phos-Tag SDS gel. GFP protein levels were used to monitor transfection efficiency as indicated in NGL-1/GFP ratios (given in arbitrary units). The relative NGL-1 phosphorylation is presented as means +/− SD of three independent experiments compared to the total amount of NGL-1 (**p<0.01, t test).