Syu-ichi Hirai

De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy

Early infantile epileptic encephalopathy with suppression-burst (EIEE), also known as Ohtahara syndrome, is one of the most severe and earliest forms of epilepsy. Using array-based comparative genomic hybridization, we found a de novo 2.0-Mb microdeletion at 9q33.3–q34.11 in a girl with EIEE. Mutation analysis of candidate genes mapped to the deletion revealed that four unrelated individuals with EIEE had heterozygous missense mutations in the gene encoding syntaxin binding protein 1 (STXBP1). STXBP1 (also known as MUNC18-1) is an evolutionally conserved neuronal Sec1/Munc-18 (SM) protein that is essential in synaptic vesicle release in several species. Circular dichroism melting experiments revealed that a mutant form of the protein was significantly thermolabile compared to wild type. Furthermore, binding of the mutant protein to syntaxin was impaired. These findings suggest that haploinsufficiency of STXBP1 causes EIEE.

Inactivation of aPKCλ results in the loss of adherens junctions in neuroepithelial cells without affecting neurogenesis in mouse neocortex

In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKCλ, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKCλ knockout mice, neuroepithelial cells of the neocortical region lost aPKCλ protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKCλ is indispensable for the maintenance of adherens junctions and may function in the regulation of adherens junction integrity upon differentiation of neuroepithelial cells into neurons. In spite of the loss of adherens junctions in the neuroepithelium of conditional aPKCλ knockout mice, neurons were produced at a normal rate. Therefore, we concluded that, at least in the later stages of neurogenesis, regulation of cell cycle exit is independent of adherens junctions.

Degradation of transcription factors, c-Jun and c-Fos, by calpain

c‐Jun protein, and AP1/PEA1 transcription factor component, is a typical short‐lived protein, and like other short‐lived proteins such as c‐Fos, contains PEST regions. Calcium‐dependent neutral protease (calpain), a candidate for the degradation of PEST‐containing proteins, digests c‐Jun and c‐Fos efficiently in vitro. This is the first demonstration that transcription factors are substrates for calpain. The C‐terminal portion of c‐Jun is relatively resistant to calpain such that an 18kDa fragment, which includes the DNA binding domain, accumulates under moderate digestion conditions. The activity of c‐Jun in cultured cells can be modified by changing the level of calpastatin, an endogenous calpain inhibitor, indicating that c‐Jun is also a substrate for calpain in vivo.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

The c-Jun N-Terminal Kinase Activator Dual Leucine Zipper Kinase Regulates Axon Growth and Neuronal Migration in the Developing Cerebral Cortex

Mammalian corticogenesis substantially depends on migration and axon projection of newborn neurons that are coordinated by a yet unidentified molecular mechanism. Dual leucine zipper kinase (DLK) induces activation of c-Jun N-terminal kinase (JNK), a molecule that regulates morphogenesis in various organisms. We show here, using gene targeting in mice, that DLK is indispensable for establishing axon tracts, especially those originating from neocortical pyramidal neurons of the cerebrum. Direct and quantitative analysis of radial migration of pyramidal neurons using slice culture and a time-lapse imaging system revealed that acceleration around the subplate was affected by DLK gene disruption and by administration of a JNK inhibitor. Phosphorylation of JNK substrates, including c-Jun and doublecortin, and of JNK itself at the activation loop were partially affected in brains of DLK-deficient mouse embryos. These data suggest that DLK plays a significant role in the coordinated regulation of radial migration and axon projection by modulating JNK activity.

Dominant-Negative Mutations in α-II Spectrin Cause West Syndrome with Severe Cerebral Hypomyelination, Spastic Quadriplegia, and Developmental Delay

A de novo 9q33.3-q34.11 microdeletion involving STXBP1 has been found in one of four individuals (group A) with early-onset West syndrome, severe hypomyelination, poor visual attention, and developmental delay. Although haploinsufficiency of STXBP1 was involved in early infantile epileptic encephalopathy in a previous different cohort study (group B), no mutations of STXBP1 were found in two of the remaining three subjects of group A (one was unavailable). We assumed that another gene within the deletion might contribute to the phenotype of group A. SPTAN1 encoding alpha-II spectrin, which is essential for proper myelination in zebrafish, turned out to be deleted. In two subjects, an in-frame 3 bp deletion and a 6 bp duplication in SPTAN1 were found at the initial nucleation site of the alpha/beta spectrin heterodimer. SPTAN1 was further screened in six unrelated individuals with WS and hypomyelination, but no mutations were found. Recombinant mutant (mut) and wild-type (WT) alpha-II spectrin could assemble heterodimers with beta-II spectrin, but alpha-II (mut)/beta-II spectrin heterodimers were thermolabile compared with the alpha-II (WT)/beta-II heterodimers. Transient expression in mouse cortical neurons revealed aggregation of alpha-II (mut)/beta-II and alpha-II (mut)/beta-III spectrin heterodimers, which was also observed in lymphoblastoid cells from two subjects with in-frame mutations. Clustering of ankyrinG and voltage-gated sodium channels at axon initial segment (AIS) was disturbed in relation to the aggregates, together with an elevated action potential threshold. These findings suggest that pathological aggregation of alpha/beta spectrin heterodimers and abnormal AIS integrity resulting from SPTAN1 mutations were involved in pathogenesis of infantile epilepsy.

Association of ASIP/mPAR-3 with adherens junctions of mouse neuroepithelial cells

Polarity proteins play fundamental roles in asymmetric cell division, which is essential for the production of different types of cells in multicellular organisms. Here, we explore the localization of atypical PKC isotype‐specific interacting protein (ASIP), a mammalian homologue of the Caenorhabditis elegans polarity protein PAR‐3, in embryonic neural tissues. Although ASIP is localized on tight junctions in cultured epithelial cells, it localizes on adherens junctions outlined by β‐catenin and afadin at the luminal surface, an apical end of the neuroepithelium in developing mouse central nervous systems. Mammalian homologues of other C. elegans polarity proteins, mPAR‐6 and aPKC, also localize in the adherens junctions. In dorsal root ganglia of the peripheral nervous system, ASIP is found predominantly in the cytoplasm of ganglion cells. In dividing preneural cells at the ventricular (luminal) surface of the embryonic telencephalon, ASIP localize in adherence junctions of luminal surface regardless of the axis of cell division. Therefore, only the daughter cell facing the lumen (apical daughter) may inherit ASIP when the division plate is oriented parallel to the surface. Given the roles of Bazooka, a Drosophila homologue of ASIP/PAR‐3, in the asymmetric division of the Drosophila neuroblast, these observations suggest that ASIP, along with other polarity proteins and adherens junction proteins, plays an important role in neural cell differentiation by means of asymmetric cell division.

UCN-01, an anti-tumor drug, is a selective inhibitor of the conventional PKC subfamily.

A selective PKC inhibitor, UCN‐01, was shown to exhibit anti‐tumor activity in vitro and in vivo. We investigated UCN‐01 with respect to isozyme‐specific PKC inhibition using purified recombinant or rabbit brain PKC isozymes, cPKCα, β and γ, nPKCδ, ϵ and ν, and aPKCζ. Of the PKC isozymes examined, cPKCα was inhibited by UCN‐01 most effectively (K i = 0.44 nM), suggesting cPKCα is the prime candidate for the physiological target of UCN‐01. The K i values of UCN‐01 estimated from Dixon plots for cPKC isozymes are approximately 1 nM, whereas the K i values for nPKC isozymes are about 20 nM. Moreover, the K i value for aPKCζ is 3.8 μM. Thus, UCN‐01 discriminates between PKC subfamilies. In addition, the inhibitory effects of staurosporine, H7, and calphostin C on aPKCζ were examined and compared with those for cPKCα.

A PROTEIN KINASE CDELTA -BINDING PROTEIN SRBC WHOSE EXPRESSION IS INDUCED BY SERUM STARVATION

West-Western screening of a cDNA expression library using 32P-labeled, autophosphorylated protein kinase Cδ (PKCδ) as a probe, led us to identify cDNA clones encoding a PKCδ-binding protein that contains a leucine zipper-like motif in its N-terminal region and two PEST sequences in its C-terminal region. This protein shows overall sequence similarity (43.3%) to the serum deprivation response (sdr) gene product, and we named it SRBC (dr-elated gene product that inds to -kinase). PKCδ binds to the C-terminal half of SRBC through the regulatory domain and phosphorylates it in vitro. In COS1 cells, the phosphorylation of over-expressed SRBC is stimulated by 12-O-tetradecanoylphorbol-13-acetate and further enhanced by the over-expression of PKCδ. The mRNA for SRBC is detected in a wide variety of cultured cell lines and tissues and is strongly induced by serum starvation. Furthermore, SRBC mRNA is induced during retinoic acid-induced differentiation of P19 cells. These results suggest that SRBC serves as a substrate and/or receptor for PKC and might be involved in the control of cell growth mediated by PKC.

Collapsin response mediator protein 1 mediates reelin signaling in cortical neuronal migration

Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axon guidance molecules. Here, we show evidence that CRMP1 is involved in Reelin (Reln) signaling to regulate neuronal migration in the cerebral cortex. In crmp1−/− mice, radial migration of cortical neurons was retarded. This phenotype was not observed in the sema3A−/− and crmp1+/−;sema3A+/− cortices. However, CRMP1 was colocalized with disabled-1 (Dab1), an adaptor protein in Reln signaling. In the Relnrl/rl cortex, CRMP1 and Dab1 were expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss of crmp1 in a dab1 heterozygous background led to the disruption of hippocampal lamination, a Reeler-like phenotype. In addition to axon guidance, CRMP1 regulates neuronal migration by mediating Reln signaling.