NOVA1 acts on Impact to regulate hypothalamic function and translation in inhibitory neurons.

We describe a patient haploinsufficient for the neuronal RNA binding protein NOVA1 who developed a behavioral motor hyperactivity disorder, suggesting a role of NOVA1 in postnatal motor inhibition. To investigate Nova1's action in adult Gad2+ inhibitory neurons, we generated a conditional Nova1-null mouse (Nova1-cKOGad2-cre). Strikingly, the phenotypes of these mice show many similarities to the NOVA1 haploinsufficient patient and identify a function of Nova1 in the hypothalamus. Molecularly, Nova1 loss in Gad2-positive neurons alters downstream expression of Impact mRNA, along with a subset of RNAs encoding electron transport chain-related factors and ribosomal proteins. NOVA1 stabilizes Impact mRNA by binding its 3' UTR, antagonizing the actions of miR-138 and miR-124. Together, these studies demonstrate actions of NOVA1 in adult hypothalamic neurons, mechanisms by which it functions in translation and metabolism, including through direct binding to Impact mRNA, and illuminate its role in human neurologic disease.

Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which ß-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the ß-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.

Differential NOVA2-Mediated Splicing in Excitatory and Inhibitory Neurons Regulates Cortical Development and Cerebellar Function.

RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell types in vivo is unknown. We developed NOVA2 cTag-crosslinking and immunoprecipitation (CLIP) to generate functional RBP-RNA maps from different neuronal populations in the mouse brain. Combining cell type datasets from Nova2-cTag and Nova2 conditional knockout mice revealed differential NOVA2 regulatory actions on alternative splicing (AS) on the same transcripts expressed in different neurons. This includes functional differences in transcripts expressed in cortical and cerebellar excitatory versus inhibitory neurons, where we find NOVA2 is required for, respectively, development of laminar structure, motor coordination, and synapse formation. We also find that NOVA2-regulated AS is coupled to NOVA2 regulation of intron retention in hundreds of transcripts, which can sequester the trans-acting splicing factor PTBP2. In summary, cTag-CLIP complements single-cell RNA sequencing (RNA-seq) studies by providing a means for understanding RNA regulation of functional cell diversity.

cTag-PAPERCLIP Reveals Alternative Polyadenylation Promotes Cell-Type Specific Protein Diversity and Shifts Araf Isoforms with Microglia Activation.

Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell types, but obtaining APA maps from individual cell types typically requires prior purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP, that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes and microglia, regulated in part by neuron-specific RNA-binding proteins NOVA2 and PTBP2. We further identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell types and cellular states within the brain.

An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors.

Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

Continuous cell supply from Krt7-expressing hematopoietic stem cells during native hematopoiesis revealed by targeted in vivo gene transfer method.

The nature of hematopoietic stem cells under normal hematopoiesis remained largely unknown due to the limited assays available to monitor their behavior in situ. Here, we develop a new mouse model to transfer genes specifically into the primitive hematopoietic stem cell compartment through the utilization of a modified Rcas/TVA system. We succeeded in transferring a GFP reporter gene into adult hematopoietic stem cells in vivo, which are predominantly quiescent, by generating pseudotyped-lentivirus. Furthermore, we demonstrate the utility of this system to study neonatal hematopoiesis, a developmental stage that has been difficult to analyze to date. Using the system developed in this study, we observed continuous multi-lineage hematopoietic cell supply in peripheral blood from Krt7-positive hematopoietic stem cells during unperturbed homeostatic condition. This powerful experimental system could provide a new standard tool to analyze hematopoiesis under physiological condition without transplantation.

Gene targeting study reveals unexpected expression of brain-expressed X-linked 2 in endocrine and tissue stem/progenitor cells in mice.

Identification of genes specifically expressed in stem/progenitor cells is an important issue in developmental and stem cell biology. Genome-wide gene expression analyses in liver cells performed in this study have revealed a strong expression of X-linked genes that include members of the brain-expressed X-linked (Bex) gene family in stem/progenitor cells. Bex family genes are expressed abundantly in the neural cells and have been suggested to play important roles in the development of nervous tissues. However, the physiological role of its individual members and the precise expression pattern outside the nervous system remain largely unknown. Here, we focused on Bex2 and examined its role and expression pattern by generating knock-in mice; the enhanced green fluorescence protein (EGFP) was inserted into the Bex2 locus. Bex2-deficient mice were viable and fertile under laboratory growth conditions showing no obvious phenotypic abnormalities. Through an immunohistochemical analysis and flow cytometry-based approach, we observed unique EGFP reporter expression patterns in endocrine and stem/progenitor cells of the liver, pyloric stomach, and hematopoietic system. Although Bex2 seems to play redundant roles in vivo, these results suggest the significance and potential applications of Bex2 in studies of endocrine and stem/progenitor cells.

KISS1 gene expression in the developing brain of female pigs in pre- and peripubertal periods.

Puberty is associated with an increase in gonadotropin secretion as a result of an increase in gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is considered to play a key role in puberty onset in many mammalian species, including rodents, ruminants and primates. The present study aimed to determine if changes in hypothalamic expression of the KISS1 gene, encoding kisspeptin, are associated with the onset of puberty in pigs. The animals (n=4 in each group) were perfused with 4% paraformaldehyde at 0, 1, 2, 3 and 4 months old, as prepubertal stages, and at 5 months old, as the peripubertal stage, following each blood sampling. KISS1 gene expressions in coronal sections of brains were visualized by in situ hybridization. Plasma luteinizing hormone (LH) was measured by radioimmunoassay. KISS1 mRNA signals were observed in the arcuate nucleus (ARC) at all ages examined without any significant difference in the number of KISS1-expressing cells, indicating that the KISS1 gene is constantly expressed in the ARC throughout pubertal development in pigs. The plasma LH concentration was the highest in 0-month-old piglets and significantly decreased in the 1- and 2 month-old groups (P<0.05), suggesting a developing negative feedback mechanism affecting gonadotropin release during the prepubertal period. Considering the potent stimulating effect of kisspeptin on gonadotropin release in prepubertal pigs, kisspeptin secretion rather than kisspeptin synthesis may be responsible for the onset of puberty in pigs.

Clock gene Bmal1 is dispensable for intrinsic properties of murine hematopoietic stem cells.

BACKGROUND: Circadian rhythms are known to influence a variety of biological phenomena such as cell cycle, sleep-wake rhythm, hormone release and other important physiological functions. Given that cell cycle entry of hibernating hematopoietic stem cells (HSCs) plays a critical role in controlling hematopoiesis, we asked functional significance of the clock gene Bmal1, which plays a central role in regulating circadian rhythms as a transcription factor. Here we investigated the necessity of Bmal1 for HSC functions using Bmal1 deficient (Bmal1⁻/⁻) mice. FINDINGS: Using colony-forming assays in vitro, we found that the frequency of mixed colony formation between Bmal1⁺/⁺ and Bmal1⁻/⁻ CD34-KSL cells does not differ significantly. Competitive bone marrow assays also revealed that Bmal1⁻/⁻ bone marrow cells competed normally with wild-type cells and displayed long-term multi-hematopoietic lineage reconstitution. In addition, there were no significant differences in the frequencies and hibernation state of bone marrow HSCs between Bmal1⁺/⁺ and Bmal1⁻/⁻ mice, suggesting that they are independent of circadian rhythms. CONCLUSIONS: This paper discusses the necessity of circadian rhythms for HSC functions. Our data clearly shows that a key circadian clock gene Bmal1 is dispensable for intrinsic functions of HSCs, such as differentiation, proliferation and repopulating ability.

[Biomarker exploration and its clinical use].

Biomarkers are useful tools as indicators/predictors of disease severity and drug responsiveness, and thus, are expected to make drug development more efficient and to accelerate proper use of approved drugs. Many academic achievements on biomarkers have been reported, but only several biomarkers are used in drug development and clinical settings. We first show our results on the pharmacogenomic analysis of the anti-cancer drug irinotecan and of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). UGT1A1*6 and *28 were significantly associated with altered pharmacokinetics of an irinotecan metabolite, SN-38, and with increased frequency of severe neutropenia. HLA* 58:01 and HLA-B*15:11/HLA-A*31:01 were associated with SJS/TEN by allopurinol and carbamazepine, respectively. Our papers have been cited in the package inserts of irinotecan and allopurinol. In addition to these genomic biomarkers, metabolomic biomarkers, which can reflect the disease phenotype and drug responsiveness, have been exploring for 12 major diseases in Japan, as a part of a multi-omics team with multi-national centers. In animal models of dilated cardiomyopathy and Alzheimer's disease, we found several changes in lipid metabolite levels in the diseased tissues. Moreover, two oxidized fatty acids were correlatively changed in the brain and plasma from Alzheimer's model mice before its onset, and thus, could be candidates for predictive biomarkers. Finally, we propose/discuss several key issues for academic researches on biomarker discovery and development, especially for newly coming researchers in the field of pharmaceutical sciences. We hope that this review would help novel biomarker identification and qualification in Japan.

Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation.

Adoptive immunotherapy with functional T cells is potentially an effective therapeutic strategy for combating many types of cancer and viral infection. However, exhaustion of antigen-specific T cells represents a major challenge to this type of approach. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8(+) T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells were then redifferentiated into CD8(+) T cells that had a high proliferative capacity and elongated telomeres. These "rejuvenated" cells possessed antigen-specific killing activity and exhibited T cell receptor gene-rearrangement patterns identical to those of the original T cell clone from the patient. We also found that this method can be effective for generating specific T cells for other pathology-associated antigens. Thus, this type of approach may have broad applications in the field of adoptive immunotherapy.

Static and dynamic characterization of nanodiscs with apolipoprotein A-I and its model peptide.

The class A amphipathic α-helical peptide 18A is known to form discoidal phospholipid complexes (nanodiscs) similar to that formed by apolipoprotein A-I (apoA-I). To reveal the structural differences in nanodiscs formed with this protein and peptide, we prepared nanodiscs with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and applied fluorescence techniques to these nanoparticles. Fluorescence resonance energy transfer between 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) in nanodiscs revealed that lipid exchange with 18A nanodiscs is mediated by collisions between nanodiscs. The fluorescence lifetime of dansyl phosphatidylethanolamine and excimer fluorescence of 1,2-bis(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine showed that the degree of hydration of the membrane surface and lateral pressure of acyl chains in 18A nanodiscs are independent of the disc size, suggesting that 18A nanodiscs form planar lipid bilayers irrespective of their size, which differs from apoA-I nanodiscs, whose bilayer deforms to a saddle surface with decreasing size. These results suggest that the flexible structure of a chain of helices in apoA-I is crucial for the formation of saddle surfaces in nanodiscs.

Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6.

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA4. Here we report that p2y5 is a novel LPA receptor coupling to the G13-Rho signaling pathway. "LPA receptor-null" RH7777 and B103 cells exogenously expressing p2y5 showed [3H]LPA binding, LPA-induced [35S]guanosine 5'-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and Gs/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA6.