Administration of prosaposin-derived neurotrophic factor to neural tube defects facilitates regeneration and restores neurological functions.

Neural tube defects (NTDs) cause fetal and pediatric deaths or lifelong neurological disabilities. No effective treatment is currently available for NTDs. We attempted to elucidate the pathogenesis of NTDs and propose a therapeutic strategy. Intra-amniotic treatment with prosaposin-derived 18-mer peptide (PS18) protected the spinal cord from secondary damage and rescued neurological function in an established chicken model of spina bifida aperta (SBA), the severe type of NTDs. PS18 promoted the formation of a neuroectodermal covering over the defective neural tube within 24-h after treatment, enhanced the regeneration/restoration process, and decreased apoptotic activity in the developing spinal cord. PS18 reduced the SBA wound and almost completely formed the spinal cord. SBA chicks that received PS18 exhibited relatively normal walking and sensorimotor responses, and reduced pain-associated behavior in postnatal life. In conclusion, PS18 is a promising therapeutic agent for NTDs and may be useful for treating other types of spinal cord injuries.

Cadaveric and CT angiography study of vessels around the transverse colon mesentery.

BACKGROUND: Laparoscopic and robotic surgery for transverse colon cancer are difficult due to complex fusion of the foregut and midgut and variation of the vessels of the transverse colon. Although the vessels of the right colon have been investigated, middle colic artery (MCA) variation and the relationship with vessels around the transvers colon are unknown. We investigated variation of the MCA using computed tomography angiography (CTA) and cadaver specimen and the relationship between the superior mesenteric vein (SMV) and MCA using CTA. The classification of vessels around the transverse colon may lead to safer and reliable surgery. METHODS: This study included 505 consecutive patients who underwent CTA in our institution from 2014 to 2020 and 44 cadaver specimens. Vascular anatomical classifications and relationships were analyzed using CT images. RESULTS: The MCA was defined as the arteries arising from the superior mesenteric artery (SMA) that flowed into the transverse colon at the distal ends. The classifications were as follows: type I, branching right and left from common trunk; type II, the right and left branches bifurcated separately from the SMA; and type III, the MCA branched from a vessel other than the SMA. Type II was subclassified into two subtypes, type IIa with one left branch and type IIb with two or more left branches from SMA. In the CTA and cadaver studies, respectively, the classifications were as follows: type I, n = 290 and n = 31; type IIa, n = 211 and n = 13; type IIb, n = 3 and n = 0; and type III, n = 1 and n = 0. We classified the relationship between the MCA and left side of the SMV into three types: type A, a common trunk runs along the left edge of the SMV (n = 173; 59.7%); type B, a right branch of the MCA runs along the left edge of the SMV (n = 116; 40.0%); and type C, the MCA runs dorsal of the SMV (n = 1; 0.3%). CONCLUSIONS: This study revealed that The MCA branching classifications and relationship between the SMV and MCA. Preoperative CT angiography may be able to reliably identify vessel variation, which may be useful in clinical practice.

Degradable optical resonators as in situ microprobes for microscopy-based observation of enzymatic hydrolysis.

Optical resonators work as precise physical and chemical sensors. Here, we assemble a whispering gallery mode resonator from a natural polymer, fibroin protein, and successfully observe its catalytic degradation reaction as a spectral shift. This methodology will contribute to the precise in situ observation of biological reactions by optical microscopy.

Antagonist of sphingosine 1-phosphate receptor 3 reduces cold injury of rat donor hearts for transplantation.

Cold storage is widely used to preserve an organ for transplantation; however, a long duration of cold storage negatively impacts graft function. Unfortunately, the mechanisms underlying cold exposure remain unclear. Based on the sphingosine-1-phosphate (S1P) signal involved in cold tolerance in hibernating mammals, we hypothesized that S1P signal blockage reduces damage from cold storage. We used an in vitro cold storage and rewarming model to evaluate cold injury and investigated the relationship between cold injury and S1P signal. Compounds affecting S1P receptors (S1PR) were screened for their protective effect in this model and its inhibitory effect on S1PRs was measured using the NanoLuc Binary Technology (NanoBiT)-ß-arrestin recruitment assays. The effects of a potent antagonist were examined via heterotopic abdominal rat heart transplantation. The heart grafts were transplanted after 24-hour preservation and evaluated on day 7 after transplantation. Cold injury increased depending on the cold storage time and was induced by S1P. The most potent antagonist strongly suppressed cold injury consistent with the effect of S1P deprivation in vitro. In vivo, this antagonist enabled 24-hour preservation, and drastically improved the beating score, cardiac size, and serological markers. Pathological analysis revealed that it suppressed the interstitial edema, inflammatory cell infiltration, myocyte lesion, TUNEL-positive cell death, and fibrosis. In conclusion, S1PR3 antagonist reduced cold injury, extended the cold preservation time, and improved graft viability. Cold preservation strategies via S1P signaling may have clinical applications in organ preservation for transplantation and contribute to an increase in the donor pool.

Identification of Qk as a Glial Precursor Cell Marker that Governs the Fate Specification of Neural Stem Cells to a Glial Cell Lineage.

During brain development, neural stem cells (NSCs) initially produce neurons and change their fate to generate glias. While the regulation of neurogenesis is well characterized, specific markers for glial precursor cells (GPCs) and the master regulators for gliogenesis remain unidentified. Accumulating evidence suggests that RNA-binding proteins (RBPs) have significant roles in neuronal development and function, as they comprehensively regulate the expression of target genes in a cell-type-specific manner. We systematically investigated the expression profiles of 1,436 murine RBPs in the developing mouse brain and identified quaking (Qk) as a marker of the putative GPC population. Functional analysis of the NSC-specific Qk-null mutant mouse revealed the key role of Qk in astrocyte and oligodendrocyte generation and differentiation from NSCs. Mechanistically, Qk upregulates gliogenic genes via quaking response elements in their 3' untranslated regions. These results provide crucial directions for identifying GPCs and deciphering the regulatory mechanisms of gliogenesis from NSCs.

Multilateral Bioinformatics Analyses Reveal the Function-Oriented Target Specificities and Recognition of the RNA-Binding Protein SFPQ.

RNA-binding proteins (RBPs) recognize consensus sequences and regulate specific target mRNAs. However, large-scale CLIP-seq revealed loose and broad binding of RBPs to larger proportion of expressed mRNAs: e.g. SFPQ binds to >10,000 pre-mRNAs but distinctly regulates <200 target genes during neuronal development. Identification of crucial binding for regulation and rules of target recognition is highly anticipated for systemic understanding of RBP regulations. For a breakthrough solution, we developed a bioinformatical method for CLIP-seq and transcriptome data by adopting iterative hypothesis testing. Essential binding was successfully identified in C-rich sequences close to the 5' splice sites of long introns, which further proposed target recognition mechanism via association between SFPQ and splicing factors during spliceosome assembly. The identified features of functional binding enabled us to predict regulons and also target genes in different species. This multilateral bioinformatics approach facilitates the elucidation of functionality, regulatory mechanism, and regulatory networks of RBPs.

Loss of RNA-Binding Protein Sfpq Causes Long-Gene Transcriptopathy in Skeletal Muscle and Severe Muscle Mass Reduction with Metabolic Myopathy.

Growing evidences are suggesting that extra-long genes in mammals are vulnerable for full-gene length transcription and dysregulation of long genes is a mechanism underlying human genetic disorders. How long-distance transcription is achieved is a fundamental question to be elucidated. In previous study, we had discovered that RNA-binding protein SFPQ preferentially binds to long pre-mRNAs and specifically regulates the cluster of neuronal genes >100 kbp. Here we investigated the roles of SFPQ for long gene expression, target specificities, and also physiological functions in skeletal muscle. Loss of Sfpq selectively downregulated genes >100 kbp including Dystrophin, which is 2.26 Mbp in length. Sfpq knockout (KO) mice showed progressive muscle mass reduction and metabolic myopathy characterized by glycogen accumulation and decreased abundance of mitochondrial oxidative phosphorylation complexes. Functional clustering analysis identified energy metabolism pathway genes as SFPQ's targets. These findings indicate target gene specificities and tissue-specific physiological functions of SFPQ in skeletal muscle.

Loss of Sfpq Causes Long-Gene Transcriptopathy in the Brain.

Genes specifically expressed in neurons contain members with extended long introns. Longer genes present a problem with respect to fulfilment of gene length transcription, and evidence suggests that dysregulation of long genes is a mechanism underlying neurodegenerative and psychiatric disorders. Here, we report the discovery that RNA-binding protein Sfpq is a critical factor for maintaining transcriptional elongation of long genes. We demonstrate that Sfpq co-transcriptionally binds to long introns and is required for sustaining long-gene transcription by RNA polymerase II through mediating the interaction of cyclin-dependent kinase 9 with the elongation complex. Phenotypically, Sfpq disruption caused neuronal apoptosis in developing mouse brains. Expression analysis of Sfpq-regulated genes revealed specific downregulation of developmentally essential neuronal genes longer than 100 kb in Sfpq-disrupted brains; those genes are enriched in associations with neurodegenerative and psychiatric diseases. The identified molecular machinery yields directions for targeted investigations of the association between long-gene transcriptopathy and neuronal diseases.

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-ß assembly.

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aß-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.

Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia.

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by missplicing of exon 20, resulting from an intronic mutation in the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene encoding IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1). A newly established splicing reporter assay allowed us to visualize pathogenic splicing in cells and to screen small chemicals for the ability to correct the aberrant splicing of IKBKAP. Using this splicing reporter, we screened our chemical libraries and identified a compound, rectifier of aberrant splicing (RECTAS), that rectifies the aberrant IKBKAP splicing in cells from patients with FD. Here, we found that the levels of modified uridine at the wobble position in cytoplasmic tRNAs are reduced in cells from patients with FD and that treatment with RECTAS increases the expression of IKAP and recovers the tRNA modifications. These findings suggest that the missplicing of IKBKAP results in reduced tRNA modifications in patients with FD and that RECTAS is a promising therapeutic drug candidate for FD.

Fgfr1 and the IIIc isoform of Fgfr2 play critical roles in the metanephric mesenchyme mediating early inductive events in kidney development.

Fibroblast growth factor receptors (Fgfrs) have critical roles in kidney development. FgfrIIIb is thought to act in epithelium, while FgfrIIIc functions in mesenchyme. We aimed to determine roles of Fgfr2IIIc in kidney development. Mice with deletion of Fgfr2IIIc (Fgfr2IIIc-/-) had normal kidneys. Combination of Fgfr2IIIc-/- with conditional deletion of Fgfr1 in metanephric mesenchyme (MM) (Fgfr1(Mes-/-)Fgfr2IIIc-/-) had small but identifiable MM at embryonic day (E) 10.5, expressing mesenchymal markers including Eya1, Six2, Pax2, and Gdnf (unlike Fgfr1/2(Mes-/-) mice that have no obvious MM). E11.5 Fgfr1(Mes-/-)Fgfr2IIIc-/- mice had rudimentary MM expressing only Eya1. Control, Fgfr2IIIc-/-, and Fgfr1(Mes-/-)Fgfr2IIIc-/- kidney mesenchymal tissues also express Fgfr2IIIb. In ureteric lineages, E10.5 Fgfr1(Mes-/-)Fgfr2IIIc-/- embryos had ureteric outgrowth (sometimes multiple buds); however, by E11.5 Gdnf absence lead to no ureteric elongation or branching (similar to Fgfr1/2(Mes-/-) mice). Beyond E12.5, Fgfr1(Mes-/-)Fgfr2IIIc-/- mice had no renal tissue. In conclusion, Fgfr2IIIc and Fgfr1 in kidney mesenchyme (together) are critical for normal early renal development.

Splicing reporter mice revealed the evolutionally conserved switching mechanism of tissue-specific alternative exon selection.

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of "splice code". So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this "balance" model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the "switch-like" mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved "splice code," in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.

Novel IgCAM, MDGA1, expressed in unique cortical area- and layer-specific patterns and transiently by distinct forebrain populations of Cajal-Retzius neurons.

The laminar and area patterning of the mammalian neocortex are two organizing principles that define its functional architecture. Members of the immunoglobulin (Ig) superfamily of cell adhesion molecules influence neural development by regulating cell adhesion, migration, and process growth. Here we describe the dynamic expression of the unique Ig-containing cell adhesion molecule, MAM domain-containing glycosylphosphatidylinositol anchor 1 (MDGA1), during forebrain development in mice and compare it with other markers. We show that MDGA1 is a layer-specific marker and an area-specific marker, being expressed in layers 2/3 throughout the neocortex, but within the primary somatosensory area (S1), MDGA1 is also uniquely expressed in layers 4 and 6a. Comparisons with other markers, including cadherins, serotonin, cytochrome oxidase, ROR beta, and COUP-TF1, reveal unique features of patterned expression of MDGA1 within cortex and S1 barrels. Further, our findings indicate that at earlier stages of development, MDGA1 is expressed by Reelin- and Tbr1-positive Cajal-Retzius neurons that originate from multiple sources outside of neocortex and emigrate into it. At even earlier stages, MDGA1 is expressed by the earliest diencephalic and mesencephalic neurons, which appear to migrate from a MDGA1-positive domain of progenitors in the diencephalon and form a "preplate." These findings show that MDGA1 is a unique marker for studies of cortical lamination and area patterning and together with recent reports suggest that MDGA1 has critical functions in forebrain/midbrain development.

Regulation of laminar and area patterning of mammalian neocortex and behavioural implications.

We will focus on describing our recent studies on the laminar and area patterning of the mammalian neocortex. We describe a novel IgCAM, MDGA1, that is a unique laminar and area specific marker, and functional studies showing its influence on radial migration. We also describe time-lapse imaging studies showing that the pre-plate and its derivative, the subplate, is a cellular protomap of the cortical ventricular zone, and the implications of this finding for mechanisms of arealization and development of area-specific TCA projections. We will summarize studies of each of the four transcription factors, Emx2, Pax6, Couptfl and Sp8, expressed by cortical progenitors and involved in specifying area patterning. Finally, we will describe studies showing that area size dictates performance at modality-specific behaviours.

Radial migration of superficial layer cortical neurons controlled by novel Ig cell adhesion molecule MDGA1.

MAM (meprin/A5 protein/receptor protein tyrosine phosphatase mu) domain glycosylphosphatidylinositol anchor 1 (MDGA1), a unique cell surface glycoprotein, is similar to Ig-containing cell adhesion molecules that influence neuronal migration and process outgrowth. We show in postnatal mice that MDGA1 is expressed by layer 2/3 neurons throughout the neocortex. During development, MDGA1 is expressed in patterns consistent with its expression by migrating layer 2/3 neurons, suggesting a role for MDGA1 in controlling their migration and settling in the superficial cortical plate. To test this hypothesis, we performed loss-of-function studies using RNA interference (RNAi) targeting different sequences of mouse MDGA1. RNAi or empty vectors were coelectroporated with an enhanced green fluorescent protein reporter in utero into the lateral ventricle at embryonic day 15.5 to transfect progenitors of superficial layer neurons; the distributions of transfected neurons were analyzed late on postnatal day 0. We found a direct correlation between effectiveness of an RNAi in suppressing MDGA1 expression and disrupting migration of superficial layer neurons. An RNAi with no effect on MDGA1 expression has no effect on the migration. In contrast, an RNAi that suppresses MDGA1 expression also blocks proper migration of transfected superficial layer neurons, with essentially all transfected cells found deep in the cortical plate or beneath it. This migration defect is rescued by cotransfection of a rat MDGA1 expression construct along with the effective RNAi, confirming that the RNAi effect is specific to diminishing mouse MDGA1 expression. RNAi transfections of deep layer neurons that do not express MDGA1 do not significantly affect their migration. We conclude that MDGA1 acts cell autonomously to control the migration of MDGA1-expressing superficial layer cortical neurons.

The function of GADD34 is a recovery from a shutoff of protein synthesis induced by ER stress: elucidation by GADD34-deficient mice.

GADD34 is a protein that is induced by stresses such as DNA damage. The function of mammalian GADD34 has been proposed by in vitro transfection, but its function in vivo has not yet been elucidated. Here we generated and analyzed GADD34 knockout mice. Despite their embryonic stage- and tissue-specific expressions, GADD34 knockout mice showed no abnormalities at fetal development and in early adult life. However, in GADD34-/- mouse embryonic fibroblasts (MEFs), recovery from a shutoff of protein synthesis was delayed when MEFs were exposed to endoplasmic reticulum (ER) stress. The phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2alpha) at Ser51 induced by thapsigargin or DTT was prolonged in GADD34-/- MEF, although following treatment with tunicamycin, the eIF2alpha phosphorylation level did not change in either GADD34+/+ or GADD34-/- cells. ER stress stimuli induced expressions of Bip (binding Ig protein) and CHOP (C/EBP homologous protein) in MEF of wild-type mice. These expressions were strongly reduced in GADD34-/- MEF, which suggests that GADD34 up-regulates Bip and CHOP. These results indicate that GADD34 works as a sensor of ER stress stimuli and recovers cells from shutoff of protein synthesis.

Heterozygosity with respect to Zfp148 causes complete loss of fetal germ cells during mouse embryogenesis.

Zfp148 belongs to a large family of C2H2-type zinc-finger transcription factors. Zfp148 is expressed in fetal germ cells in 13.5-d-old (E13.5) mouse embryos. Germ-line transmission of mutations were not observed in chimeric Zfp148(+/-) mice, and some of these mice completely lacked spermatogonia. The number of primordial germ cells in Zfp148(+/-) tetraploid embryos was normal until E11.5, but declined from E11.5 to E13.5 and continued to decline until few germ cells were present at E18.5. This phenotype was not rescued by wild-type Sertoli or stromal cells, and is therefore a cell-autonomous phenotype. These results indicate that two functional alleles of Zfp148 are required for the normal development of fetal germ cells. Recent studies have shown that Zfp148 activates p53, which has an important role in cell-cycle regulation. Primordial germ cells stop proliferating at approximately E13.5, which correlates with induction of phosphorylation of p53 and its translocation to the nucleus. Phosphorylation of p53 is impaired in Zfp148(+/-) embryonic stem cells and in fetal germ cells from chimeric Zfp148(+/-) embryos. Thus, Zfp148 may be required for regulating p53 in the development of germ cells.