Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling.

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.

Zfp423 Regulates Sonic Hedgehog Signaling via Primary Cilium Function.

Zfp423 encodes a 30-zinc finger transcription factor that intersects several canonical signaling pathways. Zfp423 mutations result in ciliopathy-related phenotypes, including agenesis of the cerebellar vermis in mice and Joubert syndrome (JBTS19) and nephronophthisis (NPHP14) in humans. Unlike most ciliopathy genes, Zfp423 encodes a nuclear protein and its developmental expression is complex, leading to alternative proposals for cellular mechanisms. Here we show that Zfp423 is expressed by cerebellar granule cell precursors, that loss of Zfp423 in these precursors leads to cell-intrinsic reduction in proliferation, loss of response to Shh, and primary cilia abnormalities that include diminished frequency of both Smoothened and IFT88 localization. Loss of Zfp423 alters expression of several genes encoding key cilium components, including increased expression of Tulp3. Tulp3 is a direct binding target of Zfp423 and reducing the overexpression of Tulp3 in Zfp423-deficient cells suppresses Smoothened translocation defects. These results define Zfp423 deficiency as a bona fide ciliopathy, acting upstream of Shh signaling, and indicate a mechanism intrinsic to granule cell precursors for the resulting cerebellar hypoplasia.

Syndecan-2 induces filopodia and dendritic spine formation via the neurofibromin-PKA-Ena/VASP pathway.

Syndecan-2 induced filopodia before spinogenesis; therefore, filopodia formation was used here as a model to study the early downstream signaling of syndecan-2 that leads to spinogenesis. Screening using kinase inhibitors indicated that protein kinase A (PKA) is required for syndecan-2-induced filopodia formation in both human embryonic kidney cells and hippocampal neurons. Because neurofibromin, a syndecan-2-binding partner, activates the cyclic adenosine monophosphate pathway, the role of neurofibromin in syndecan-2-induced filopodia formation was investigated by deletion mutant analysis, RNA interference, and dominant-negative mutant. The results showed that neurofibromin mediates the syndecan-2 signal to PKA. Among actin-associated proteins, Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) were predicted as PKA effectors downstream of syndecan-2, as Ena/VASP, which is activated by PKA, induces actin polymerization. Indeed, when the activities of Ena/VASP were blocked, syndecan-2 no longer induced filopodia formation. Finally, in addition to filopodia formation, neurofibromin and Ena/VASP contributed to spinogenesis. This study reveals a novel signaling pathway in which syndecan-2 activates PKA via neurofibromin and PKA consequently phosphorylates Ena/VASP, promoting filopodia and spine formation.

Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK.

Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain of function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, we investigated potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. We found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-alpha. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis.

X-linked mental retardation gene CASK interacts with Bcl11A/CTIP1 and regulates axon branching and outgrowth.

Calcium/calmodulin-dependent serine kinase (CASK), a causative gene in X-linked mental retardation, carries out multiple functions in neurons, including vesicle trafficking of ion channels, synapse formation, and gene transcription. From a yeast two-hybrid screen, Krüppel-like zinc finger protein B cell lymphoma/COUP-TF-interacting protein 1 (Bcl11A/CTIP1) was identified as a CASK binding protein. Through alternative splicing, a single Bcl11A gene encodes two major protein products in neurons, Bcl11A-S and Bcl11A-L. CASK interacted with both Bcl11A-S and Bcl11A-L in transfected COS cells and brain. Immunofluorescence staining further indicated the colocalization of CASK and Bcl11A in the nuclei of neurons. These studies supported an interaction between CASK and Bcl11A in vivo. Bcl11A-L has previously been shown to play a role in gene transcription as well as axon outgrowth and branching. Here, we further show that Bcl11A-L rearranges the distribution of nuclear actin, which may be related to the function of Bcl11A-L in gene expression. More importantly, using cultured hippocampal neurons as a model system, we show that CASK enhances the ability of Bcl11A-L to restrict axon outgrowth and branching. Interruption of the interaction between CASK and Bcl11A increased the outgrowth and branching of axons, suggesting that the interaction between CASK and Bcl11A controls axon arborization. In conclusion, our results suggest that, through the interaction with Bcl11A, CASK plays a role in axonogenesis, which may be related to brain anatomical characteristics in humans.

Bcl11A/CTIP1 regulates expression of DCC and MAP1b in control of axon branching and dendrite outgrowth.

The extension of axon branches is important for target innervation but how axon branching is regulated is currently not well understood. Here, we report that Bcl11A/CTIP1/Evi9, a zinc finger transcription factor, downregulates axon branching. Knockdown of Bcl11A induced axon branching and multi-axon formation, as well as dendrite outgrowth. Due to alternative splicing, a single Bcl11A gene encodes two protein products, Bcl11A-L and -S. Bcl11A-L was found to be the main Bcl11A player in regulation of neurite arborization; Bcl11A-S is an antagonist of Bcl11A-L. Time-lapse study further suggests that Bcl11A-L knockdown enhances axon dynamics and increases the duration of axon outgrowth. Finally, the expression of DCC and MAP1b, two molecules involved in direction and branching of axon outgrowth, is controlled by Bcl11A-L. DCC overexpression rescues the phenotype induced by Bcl11A-L knockdown. In conclusion, this report provides the first evidence that Bcl11A is important for neurite arborization.

Neuroprotection in the Acute Stage Enables Functional Recovery Following Repair of Chronic Cervical Root Transection After a 3-Week Delay.

BACKGROUND: Preganglionic cervical root transection (PCRT) is the most severe type of brachial plexus injury. In some cases, surgical procedures must be postponed for ≥3 wk until electromyographic confirmation. However, research works have previously shown that treating PCRT after a 3-wk delay fails to result in functional recovery. OBJECTIVE: To assess whether the immunosuppressive drug sirolimus, by promoting neuroprotection in the acute phase of PCRT, could enable functional recovery in cases of delayed repair. METHODS: First, rats received a left 6th to 8th cervical root transection, after which half were administered sirolimus for 1 wk. Markers of microglia, astrocytes, neurons, and autophagy were assessed at days 7 and 21. Second, animals with the same injury received nerve grafts, along with acidic fibroblast growth factor and fibrin glue, 3 wk postinjury. Sirolimus was administered to half of them for the first week. Mechanical sensation, grasping power, spinal cord morphology, functional neuron survival, nerve fiber regeneration, and somatosensory-evoked potentials (SSEPs) were assessed 1 and 23 wk postinjury. RESULTS: Sirolimus was shown to attenuate microglial and astrocytic proliferation and enhance neuronal autophagy and survival; only rats treated with sirolimus underwent significant sensory and motor function recovery. In addition, rats who achieved functional recovery were shown to have abundant nerve fibers and neurons in the dorsal root entry zone, dorsal root ganglion, and ventral horn, as well as to have SSEPs reappearance. CONCLUSION: Sirolimus-induced neuroprotection in the acute stage of PCRT enables functional recovery, even if surgical repair is performed after a 3-wk delay.

Transcriptional modification by a CASK-interacting nucleosome assembly protein.

CASK acts as a coactivator for Tbr-1, an essential transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK coactivation effect is unclear. Here, we report that CASK binds to another nuclear protein, CINAP, which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b and reelin. A knockdown of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1/CASK/CINAP protein complex and thus modifies expression of the NR2b gene.

Cytoplasmic distribution of T-box transcription factor Tbr-1 in adult rodent brain.

T-brain-1 (Tbr-1), a brain-specific T-box transcription factor, plays a critical role in cerebral cortex and olfactory bulb development. The expression levels of Tbr-1 are highest at the embryonic stage and are gradually reduced during the developmental process. In adult brain, Tbr-1 is expressed at a lower, but still significant level. Tbr-1 transcriptional activity is enhanced via interaction with CASK, a membrane-associated guanylate kinase, but it is not clear whether any other mechanism regulates Tbr-1 activity. We examined the subcellular distribution of Tbr-1 in adult and postnatal rat brains using DAB stain and confocal imaging analysis. In contrast to the embryonic stage, Tbr-1 was distributed in P3 and adult rat brain in the nucleus as well as the cytoplasm of neurons in the cerebral cortex and hippocampus. Confocal analysis clearly showed dendritic distribution of Tbr-1 in pyramidal neurons. In the cerebellum of P15, P22, and adult rats, Tbr-1 was specifically expressed in Purkinje cells, where Tbr-1 was localised in the cytoplasm, including the dendritic tree. In addition, biochemical fractionation of adult cerebral cortex and hippocampus showed that cytoplasmic Tbr-1 is highly enriched in the lysed synaptosomal fraction, further indicating a synaptic distribution of cytoplasmic Tbr-1 in adult brain. Our study suggests that translocation from synapse to the nucleus is involved in regulation of Tbr-1 function in postnatal and adult brains.

Leptin is essential for microglial activation and neuropathic pain after preganglionic cervical root avulsion.

AIMS: Preganglionic cervical root avulsion (PCRA) affects both the peripheral and central nervous systems and is often associated with neuropathic pain. Unlike peripheral nerve injuries (PNI), central lesions caused by disruption of cervical roots from the spinal cord following PCRA contribute to the generation of neuropathic pain. Leptin is involved in the development of neuropathic pain after PNI by affecting neurons. However, whether leptin is involved in microglial activation leading to neuropathic pain after PCRA is unknown. MAIN METHODS: Preganglionic avulsion of the left 6th-8th cervical roots was performed in C57B/6J mice and leptin-deficient mice. A leptin antagonist or leptin was administered to C57B/6J mice and leptin-deficient mice after injury, respectively. The expression pattern of spinal and supraspinal microglia was examined by immunofluorescent staining. Von Frey filaments were used to test pain sensitivity. KEY FINDINGS: Leptin is essential for the development of neuropathic pain after PCRA. Allodynia was absent in the leptin-deficient mice and the mice administered the leptin antagonist. We also found that leptin deficiency or the administration of its antagonist inhibited the development of microgliosis in the dorsal horn and brainstem. Furthermore, increase in the expression of CD86 and iNOS, and Wallerian degeneration were noted in the spinal cord. The administration of exogenous leptin to leptin-deficient mice reversed these effects. SIGNIFICANCE: We concluded that leptin is involved in the proliferation and activation of microglia, which in turn enhances the development of neuropathic pain. Blocking the effects of leptin might be a target for the treatment of neuropathic pain after PCRA.

Zfp423 binds autoregulatory sites in p19 cell culture model.

Zfp423 is a 30 zinc finger transcription factor that forms regulatory complexes with EBF family members and factors targeted by canonical signaling pathways. Zfp423 mutations produce a range of developmental abnormalities in mice and humans related to the ciliopathies. Surprisingly, computational analysis of clustered Zfp423 and partner motifs in conserved genomic sequences predicts enrichment in Zfp423 and Ebf genes. In cell culture models selected for Zfp423 and EBF expression, we identify strong and reproducible occupancy of two Zfp423 intronic sites using chromatin immunoprecipitation with multiple independent antibodies. Both sites are significantly enriched in either quantitative PCR or massively parallel sequencing assays. A site in intron 5 acts as a classical enhancer in transient assays, but does not require the consensus motif for activity, suggesting a redundant or modulatory role for Zfp423 binding in this context. We speculate that Zfp423 may repress this enhancer as part of a developmental ratchet.

SUMOylation of the MAGUK protein CASK regulates dendritic spinogenesis.

Membrane-associated guanylate kinase (MAGUK) proteins interact with several synaptogenesis-triggering adhesion molecules. However, direct evidence for the involvement of MAGUK proteins in synapse formation is lacking. In this study, we investigate the function of calcium/calmodulin-dependent serine protein kinase (CASK), a MAGUK protein, in dendritic spine formation by RNA interference. Knockdown of CASK in cultured hippocampal neurons reduces spine density and shrinks dendritic spines. Our analysis of the time course of RNA interference and CASK overexpression experiments further suggests that CASK stabilizes or maintains spine morphology. Experiments using only the CASK PDZ domain or a mutant lacking the protein 4.1-binding site indicate an involvement of CASK in linking transmembrane adhesion molecules and the actin cytoskeleton. We also find that CASK is SUMOylated. Conjugation of small ubiquitin-like modifier 1 (SUMO1) to CASK reduces the interaction between CASK and protein 4.1. Overexpression of a CASK-SUMO1 fusion construct, which mimicks CASK SUMOylation, impairs spine formation. Our study suggests that CASK contributes to spinogenesis and that this is controlled by SUMOylation.

CASK associates with glutamate receptor interacting protein and signaling molecules.

CASK has been implicated in synaptic protein targeting, synaptic organization, and transcriptional regulation. Here, three more CASK associated proteins, GRIP1, PKCepsilon, and RGS4, were initially identified by immunoprecipitation and mass analysis, and confirmed by immunoprecipitation-immunoblotting assay using rat brain extract. Via the interaction with GRIP1, GluR2/3 was also co-immunoprecipitated by CASK antibody from rat brain. The PDZ and SH3-GK domains of CASK were demonstrated as the associated domains for GRIP1 and PKCepsilon, respectively. The associations between CASK, PKCepsilon, and RGS4 were up-regulated in the adult brain compared with postnatal day 11 rat brain. In contrast, the associations of CASK with Mint1, GRIP1, and GluR2/3 were down-regulated in the adult brain. These results suggest that CASK protein complex is developmentally regulated by unknown signals. In conclusion, our study suggests that the CASK protein complex not only functions as a scaffold but also recruits signaling molecules and may contribute to signal transduction.