Sbp2l contributes to oligodendrocyte maturation through translational control in Tcf7l2 signaling.

Oligodendrocytes (OLs) are the myelin-forming cells in the CNS that support neurons through the insulating sheath of axons. This unique feature and developmental processes are achieved by extrinsic and intrinsic gene expression programs, where RNA-binding proteins can contribute to dynamic and fine-tuned post-transcriptional regulation. Here, we identified SECIS-binding protein 2-like (Sbp2l), which is specifically expressed in OLs by integrated transcriptomics. Histological analysis revealed that Sbp2l is a molecular marker of OL maturation. Sbp2l knockdown (KD) led to suppression of matured OL markers, but not a typical selenoprotein, Gpx4. Transcriptome analysis demonstrated that Sbp2l KD decreased cholesterol-biosynthesis-related genes regulated by Tcf7l2 transcription factor. Indeed, we confirmed the downregulation of Tcf7l2 protein without changing its mRNA in Sbp2l KD OPCs. Furthermore, Sbp2l KO mice showed the decrease of Tcf7l2 protein and deficiency of OL maturation. These results suggest that Sbp2l contributes to OL maturation by translational control of Tcf7l2.

DNA damage stress-induced translocation of mutant FUS proteins into cytosolic granules and screening for translocation inhibitors.

Fused in sarcoma/translated in liposarcoma (FUS) is an RNA-binding protein, and its mutations are associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), through the DNA damage stress response, aberrant stress granule (SG) formation, etc. We previously reported that translocation of endogenous FUS into SGs was achieved by cotreatment with a DNA double-strand break inducer and an inhibitor of DNA-PK activity. In the present study, we investigated cytoplasmic SG formation using various fluorescent protein-tagged mutant FUS proteins in a human astrocytoma cell (U251) model. While the synergistic enhancement of the migration of fluorescent protein-tagged wild-type FUS to cytoplasmic SGs upon DNA damage induction was observed when DNA-PK activity was suppressed, the fluorescent protein-tagged FUSP525L mutant showed cytoplasmic localization. It migrated to cytoplasmic SGs upon DNA damage induction alone, and DNA-PK inhibition also showed a synergistic effect. Furthermore, analysis of 12 sites of DNA-PK-regulated phosphorylation in the N-terminal LC region of FUS revealed that hyperphosphorylation of FUS mitigated the mislocalization of FUS into cytoplasmic SGs. By using this cell model, we performed screening of a compound library to identify compounds that inhibit the migration of FUS to cytoplasmic SGs but do not affect the localization of the SG marker molecule G3BP1 to cytoplasmic SGs. Finally, we successfully identified 23 compounds that inhibit FUS-containing SG formation without changing normal SG formation. Highlights Characterization of DNA-PK-dependent FUS stress granule localization.A compound library was screened to identify compounds that inhibit the formation of FUS-containing stress granules.

A new l-arginine oxidase engineered from l-glutamate oxidase.

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 µM. The linear relationship is maintained around 1 µM of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.

DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2.

Microprocessor complex, including DiGeorge syndrome critical region gene 8 (DGCR8) and DROSHA, recognizes and cleaves primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical miRNAs. The study of DGCR8 haploinsufficiency reveals that the efficiency of this activity varies for different miRNA species. It is thought that this variation might be associated with the risk of schizophrenia with 22q11 deletion syndrome caused by disruption of the DGCR8 gene. However, the underlying mechanism for varying action of DGCR8 with each miRNA remains largely unknown. Here, we used in vivo monitoring to measure the efficiency of DGCR8-dependent microprocessor activity in cultured cells. We confirmed that this system recapitulates the microprocessor activity of endogenous pri-miRNA with expression of a ratiometric fluorescence reporter. Using this system, we detected mir-9-2 as one of the most efficient targets. We also identified a novel DGCR8-responsive RNA element, which is highly conserved among mammalian species and could be regulated at the epi-transcriptome (RNA modification) level. This unique feature between DGCR8 and pri-miR-9-2 processing may suggest a link to the risk of schizophrenia.

An RNA Switch of a Large Exon of Ninein Is Regulated by the Neural Stem Cell Specific-RNA Binding Protein, Qki5.

A set of tissue-specific splicing factors are thought to govern alternative splicing events during neural progenitor cell (NPC)-to-neuron transition by regulating neuron-specific exons. Here, we propose one such factor, RNA-binding protein Quaking 5 (Qki5), which is specifically expressed in the early embryonic neural stem cells. We performed mRNA-SEQ (Sequence) analysis using mRNAs obtained by developing cerebral cortices in Qk (Quaking) conditional knockout (cKO) mice. As expected, we found a large number of alternative splicing changes between control and conditional knockouts relative to changes in transcript levels. DAVID (The Database for Annotation, Visualization and Integrated Discovery) and Metascape analyses suggested that the affected spliced genes are involved in axon development and microtubule-based processes. Among these, the mRNA coding for the Ninein protein is listed as one of Qki protein-dependent alternative splicing targets. Interestingly, this exon encodes a very long polypeptide (2121 nt), and has been previously defined as a dynamic RNA switch during the NPC-to-neuron transition. Additionally, we validated that the regulation of this large exon is consistent with the Qki5-dependent alternative exon inclusion mode suggested by our previous Qki5 HITS-CLIP (high throughput sequencing-cross linking immunoprecipitation) analysis. Taken together, these data suggest that Qki5 is an important factor for alternative splicing in the NPC-to-neuron transition.

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.

RNA regulation went wrong in neurodevelopmental disorders: The example of Msi/Elavl RNA binding proteins.

RNA regulation participates in many aspects of brain development. There is substantial evidence that RNA dysregulation is critical in the pathogenesis of neurodevelopmental disorders, neurological diseases, and cancer. Several gene families encode RNA-binding proteins (RNABPs) that bind directly to RNA and orchestrate the post-transcriptional regulation of gene expression, including pre-mRNA splicing, stability, and poly(A) site usage. Among neural RNABPs, the Elavl and Msi families are the focus of neuronal development research owing to their hierarchical expression pattern: Msi1 is expressed in neural progenitor/stem cells, Elavl2 is expressed in early neuronal progenitors to mature neurons, and Elavl3/4 expression begins slightly later, during cortical neuron development. Traditional biochemical analyses provide mechanistic insight into RNA regulation by these RNABPs, and Drosophila and mouse genetic studies support a relationship between these RNABPs and several neurodevelopmental disorders. In addition, a recent cohort analysis of the human genome shows that genetic mutations and SNPs in these RNABPs are associated with various neurological disorders. Newly emerged technologies assess transcriptome-wide RNA-protein interactions in vivo. These technologies, combined with classical genetics methods, provide new insight into Elavl and Msi, not only with respect to their neurodevelopmental functions, but also their roles in several diseases. We review recent discoveries related to the two RNABP families in brain development and disease.

Polyglutamine expansion disturbs the endoplasmic reticulum formation, leading to caspase-7 activation through Bax.

The endoplasmic reticulum (ER) plays a pivotal role in cellular functions such as the ER stress response. However, the effect of the ER membrane on caspase activation remains unclear. This study reveals that polyglutamine oligomers augmented at ER induce insertion of Bax into the ER membrane, thereby activating caspase-7. In line with the role of ER in cell death induced by polyglutamine expansion, the ER membrane was found to be disrupted and dilated in the brain of a murine model of Huntington's disease. We can conclude that polyglutamine expansion may drive caspase-7 activation by disrupting the ER membrane.

Olfaxin as a novel Prune2 isoform predominantly expressed in olfactory system.

Prune homolog 2 (Drosophila) (PRUNE2) encodes a BCH motif-containing protein that shares homology with the Cayman ataxia-related protein Caytaxin. Caytaxin is a substrate of caspase-3 and is specifically expressed at the presynapse of vesicular-type glutamate transporter (VGLUT)-positive neurons, where it plays a role in glutamate neurotransmission primarily in the cerebellum and hippocampus. Here, we showed that a novel Prune2 isoform contains a BCH motif and localizes predominantly to the synaptic cytosol, similar to Caytaxin. However, the isoform is expressed predominantly in the olfactory bulb and layer Ia of the piriform cortex, where Caytaxin is scarcely expressed. The isoform expression is upregulated during development, similar to that in the presynaptic-localizing proteins Synapsin I and Bassoon. Prune2 and its previously identified isoforms have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein, and an antagonist of cellular transformation. In addition, a novel isoform may develop new roles for Prune2 at the synapse in olfactory systems.

Cayman ataxia-related protein is a presynapse-specific caspase-3 substrate.

Caspase plays an important role in apoptosis and physiological processes such as synaptic plasticity. However, the caspase substrate at the synapse is still unknown. Here we used an in vitro cleavage assay with a small-pool human brain cDNA library. We identified the presynaptic protein Caytaxin as a substrate of caspase-3 and caspase-7. Deficiency in Caytaxin causes Cayman ataxia, a disorder characterized by cerebellar dysfunction and mental retardation. Caytaxin cleavage in cerebellar granule neurons is dependent on caspase-3 activation. The cleavage site is upstream of the cellular retinal and the TRIO guanine exchange factor domain, producing a C-terminal fragment that may play an alternative role in inhibiting MEK2 signaling. Thus, we concluded that Caytaxin is a novel substrate of caspase-3 at the presynapse.

Cellular composition and organization of the subventricular zone and rostral migratory stream in the adult and neonatal common marmoset brain.

The adult subventricular zone (SVZ) of the lateral ventricle contains neural stem cells. In rodents, these cells generate neuroblasts that migrate as chains toward the olfactory bulb along the rostral migratory stream (RMS). The neural-stem-cell niche at the ventricular wall is conserved in various animal species, including primates. However, it is unclear how the SVZ and RMS organization in nonhuman primates relates to that of rodents and humans. Here we studied the SVZ and RMS of the adult and neonatal common marmoset (Callithrix jacchus), a New World primate used widely in neuroscience, by electron microscopy, and immunohistochemical detection of cell-type-specific markers. The marmoset SVZ contained cells similar to type B, C, and A cells of the rodent SVZ in their marker expression and morphology. The adult marmoset SVZ had a three-layer organization, as in the human brain, with ependymal, hypocellular, and astrocyte-ribbon layers. However, the hypocellular layer was very thin or absent in the adult-anterior and neonatal SVZ. Anti-PSA-NCAM staining of the anterior SVZ in whole-mount ventricular wall preparations of adult marmosets revealed an extensive network of elongated cell aggregates similar to the neuroblast chains in rodents. Time-lapse recordings of marmoset SVZ explants cultured in Matrigel showed the neuroblasts migrating in chains, like rodent type A cells. These results suggest that some features of neurogenesis and neuronal migration in the SVZ are common to marmosets, humans, and rodents. This basic description of the adult and neonatal marmoset SVZ will be useful for future studies on adult neurogenesis in primates.

Nova2 regulates neuronal migration through an RNA switch in disabled-1 signaling.

Neuronal migration leads to a highly organized laminar structure in the mammalian brain, and its misregulation causes lissencephaly and behavioral and cognitive defects. Reelin signaling, which is mediated in part by a key adaptor, disabled-1 (Dab1), plays a critical but incompletely understood role in this process. We found that the neuron-specific RNA-binding protein Nova2 regulates neuronal migration in late-generated cortical and Purkinje neurons. An unbiased HITS-CLIP and exon junction array search for Nova-dependent reelin-pathway RNAs at E14.5 revealed only one candidate-an alternatively spliced isoform of Dab1 (Dab1.7bc). In utero electroporation demonstrated that Dab1.7bc was sufficient to induce neuronal migration defects in wild-type mice and exacerbate defects when Dab1 levels were reduced, whereas Dab1 overexpression mitigates defects in Nova2 null mice. Thus, Nova2 regulates an RNA switch controlling the ability of Dab1 to mediate neuronal responsiveness to reelin signaling and neuronal migration, suggesting new links between splicing regulation, brain disease, and development.

Autophagy impairment stimulates PS1 expression and gamma-secretase activity.

Gamma-secretase plays an important role in the development of Alzheimer disease (AD). Gamma-secretase activity is enriched in autophagic vacuoles and it augments amyloid-beta (Abeta) synthesis. Autophagy-lysosomal dysfunction has been implicated in AD, but whether gamma-secretase activity is affected by autophagy remains unclear. Here we report that gamma-secretase activity is enhanced in basal autophagy-disturbed cells through the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) kinase, general control nonderepressible 2 (GCN2). Presenilin-1 (PS1) expression was increased even in the presence of nutrients in autophagy-related 5 knockdown (Atg5KD) human embryonic kidney (HE K293) cells expressing a short hairpin RNA as well as in chloroquine-treated HE K293 cells. However, PS1 expression induction was prevented in GCN2KD and ATF4KD cells. Furthermore, Atg5KD cells showed an increase in Abeta production and Notch1 cleavage. These were reduced by an autophagy inducer, resveratrol. Thus, we conclude that the autophagy-lysosomal system regulates gamma-secretase activity through GCN2.

An alternative spliced mouse presenilin-2 mRNA encodes a novel gamma-secretase inhibitor.

The gamma-secretase, composed of presenilin-1 (PS1) or presenilin-2 (PS2), nicastrin (NCT), anterior pharynx-defective phenotype 1 (APH-1), and PEN-2, is critical for the development of Alzheimer's disease (AD). PSs are autoproteolytically cleaved, producing an N-terminal fragment (NTF) and a hydrophilic loop domain-containing C-terminal fragment. However, the role of the loop domain in the gamma-secretase complex assembly remains unknown. Here, we report a novel PS2 isoform generated by alternative splicing, named PS2beta, which is composed of an NTF with a hydrophilic loop domain. PS2beta disturbed the interaction between NCT and APH-1, resulting in the inhibition of amyloid-beta production. We concluded that PS2beta may inhibit gamma-secretase activity by affecting the gamma-secretase complex assembly.

Epidermal growth factor signaling mediated by grb2 associated binder1 is required for the spatiotemporally regulated proliferation of olig2-expressing progenitors in the embryonic spinal cord.

Gab1 (Grb2 associated binder1) has been identified as an adaptor molecule downstream of many growth factors, including epidermal growth factor (EGF), fibroblast growth factor, and platelet-derived growth factor, which have been shown to play crucial roles as mitotic signals for a variety of neural progenitor cells, including stem cells, both in vitro and in vivo. Here, we show that Gab1 deficiency results in a reduction in the number of Olig2-positive (Olig2(+)) progenitor cells in the developing mouse spinal cord after embryonic day 12.5 (E12.5), when gliogenesis starts in the pMN domain where the EGF receptor (EGFR) is expressed predominantly. Our in vitro analysis further revealed that Gab1 is essential for EGF-dependent proliferation of Olig2(+) progenitor cells derived from the E12.5 ventral and E14.5 dorsal but not ventral spinal cord, whereas Gab1 is always required for the activation of Akt1 but not of ERK1/2. Moreover, we found that the action of the Gab1/Akt pathway is context-dependent, since constitutively active Akt1 could rescue the proliferation defect only in the E12.5 spinal cord of the Gab1-deficient mouse in vitro. Finally, we demonstrated that EGFR-deficient mice and Gab1-deficient mice showed a similar reduction in the number of Olig2(+) progenitor cells in the developing spinal cord. These findings indicate that EGFR-mediated signaling through Gab1/Akt contributes to the sufficient expansion of Olig2(+) progenitor cells in a spatiotemporally regulated manner, which represents the origin of glial cells in the developing spinal cord. Disclosure of potential conflicts of interest is found at the end of this article.