A new therapeutic target for systemic lupus erythematosus: the current landscape for drug development of a toll-like receptor 7/8 antagonist through academia-industry-government collaboration.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by inflammation in multiple organs. A few treatments for SLE currently exist, including antimalarials, glucocorticoids, immunosuppressants, and two recently approved antibody agents; however, an unmet medical need remains for SLE. In addition, developing new drugs targeting SLE is a challenge since no specific biomarkers exist for the prediction of disease progression or drug response. A new drug candidate, E6742, is a specific antagonist of the toll-like receptors 7/8. To address the challenges for drug development in SLE, the process of developing E6742 utilizes a unique system of the Japan Agency for Medical Research and Development (AMED), the Cyclic Innovation for Clinical Empowerment (CiCLE) program. In the CiCLE program, a Phase 1 study in healthy adults was completed (NCT04683185) and a Phase 1/2 study in patients with SLE is on-going (NCT05278663). One of the potential benefits of this program is to conduct academia-led clinical research to identify specific biomarkers for E6742 in parallel with clinical studies (UMIN000042037). The aim of this review is to present current progress within the strategic collaboration of the AMED CiCLE program that optimize clinical development for patients with SLE.

Structures of dengue virus RNA replicase complexes.

Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), a member of the flaviviruses. The DENV genome is a 5'-capped positive-sense RNA with a unique 5'-stem-loop structure (SLA), which is essential for RNA replication and 5' capping. The virus-encoded proteins NS5 and NS3 are responsible for viral genome replication, but the structural basis by which they cooperatively conduct the required tasks has remained unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of SLA-bound NS5 (PC), NS3-bound PC (PC-NS3), and an RNA-elongating NS5-NS3 complex (EC). While SLA bridges the NS5 methyltransferase and RNA-dependent RNA polymerase domains in PC, the NS3 helicase domain displaces it in elongation complex (EC). The SLA- and NS3-binding sites overlap with that of human STAT2. These structures illuminate the key steps in DENV genome replication, namely, SLA-dependent replication initiation, processive RNA elongation, and 5' capping of the nascent genomic RNA, thereby providing foundations to combat flaviviruses.

Structural basis of the transcription termination factor Rho engagement with transcribing RNA polymerase from Thermus thermophilus.

Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the adenosine triphosphate-dependent RNA translocase/helicase Rho, which causes RNA/DNA dissociation from the RNAP elongation complex (EC). However, the structural basis of the interplay between Rho and RNAP remains obscure. Here, we report the cryo-electron microscopy structure of the Thermus thermophilus RNAP EC engaged with Rho. The Rho hexamer binds RNAP through the carboxyl-terminal domains, which surround the RNA exit site of RNAP, directing the nascent RNA seamlessly from the RNA exit to its central channel. The ß-flap tip at the RNA exit is critical for the Rho-dependent RNA release, and its deletion causes an alternative Rho-RNAP binding mode, which is irrelevant to termination. The Rho binding site overlaps with the binding sites of other macromolecules, such as ribosomes, providing a general basis of gene regulation.

Intra-pituitary follicle-stimulating hormone signaling regulates hepatic lipid metabolism in mice.

Inter-organ communication is a major hallmark of health and is often orchestrated by hormones released by the anterior pituitary gland. Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to regulate gonadal function and control fertility. Whether FSH and LH also act on organs other than the gonads is debated. Here, we find that gonadotrope depletion in adult female mice triggers profound hypogonadism, obesity, glucose intolerance, fatty liver, and bone loss. The absence of sex steroids precipitates these phenotypes, with the notable exception of fatty liver, which results from ovary-independent actions of FSH. We uncover paracrine FSH action on pituitary corticotropes as a mechanism to restrain the production of corticosterone and prevent hepatic steatosis. Our data demonstrate that functional communication of two distinct hormone-secreting cell populations in the pituitary regulates hepatic lipid metabolism.

Safety of linezolid in patients with decreased renal function and trough monitoring: a systematic review and meta-analysis.

BACKGROUND: Linezolid causes hematological toxicity, mostly thrombocytopenia, which leads to treatment discontinuation and failure. Recent studies revealed that during linezolid therapy, the incidence of treatment-related hematological toxicity is significantly higher in patients with decreased renal function (DRF) than in those with normal renal function. Linezolid monitoring is necessary due to the high frequency of hematological toxicity in patients with DRF and the relationship between blood concentration and safety. We performed a systematic review and meta-analysis to evaluate the safety correlation between DRF and trough monitoring. METHODS: Articles published before June 24, 2022, on MEDLINE, Web of Sciences, Cochrane Register of Controlled Trials, and ClinicalTrials.gov were systematically analyzed. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the Mantel-Haenszel method and the variable effects model. RESULTS: The incidence of hematological toxicity was significantly higher in patients with DRF than in those without DRF (OR = 2.37; p < 0.001). Subgroup analysis, performed according to hematotoxicity classification, including thrombocytopenia, anemia, and pancytopenia, revealed a significantly higher incidence of thrombocytopenia (OR = 2.45; p < 0.001) and anemia (OR = 2.31; p = 0.006) in patients with DRF than in those without; pancytopenia (OR = 1.41; p = 0.80) incidences were not significantly higher. Based on a systematic review, linezolid trough concentrations > 6-7 µg/mL may be associated with an increased incidence of thrombocytopenia. However, no confidential threshold values for the development of thrombocytopenia were found in the area under the concentration curve values for children or adults. CONCLUSION: We observed a high frequency of hematological toxicity during linezolid therapy in patients with DRF. To ensure safety, linezolid trough concentrations should be ≤6-7 µg/mL.

CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation.

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.

Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.

Prolactin-sensitive olfactory sensory neurons regulate male preference in female mice by modulating responses to chemosensory cues.

Chemosensory cues detected in the nose need to be integrated with the hormonal status to trigger appropriate behaviors, but the neural circuits linking the olfactory and the endocrine system are insufficiently understood. Here, we characterize olfactory sensory neurons in the murine nose that respond to the pituitary hormone prolactin. Deletion of prolactin receptor in these cells results in impaired detection of social odors and blunts male preference in females. The prolactin-responsive olfactory sensory neurons exhibit a distinctive projection pattern to the brain that is similar across different individuals and express a limited subset of chemosensory receptors. Prolactin modulates the responses within these neurons to discrete chemosensory cues contained in male urine, providing a mechanism by which the hormonal status can be directly linked with distinct olfactory cues to generate appropriate behavioral responses.

Widespread Cell-Specific Prolactin Receptor Expression in Multiple Murine Organs.

The prolactin receptor (Prlr) mediates not only the multiple effects of prolactin, but also those of the placental lactogens and, in humans, some actions of growth hormone. Although Prlr expression has been reported to be widespread in the body, specific cellular expression patterns within tissues are undefined for many organs. One persisting problem in investigating Prlr function is that the protein is difficult to detect using conventional methods. To allow investigation of Prlr expression with a single cell resolution, we have recently developed a knock-in mouse strain in which Cre recombinase is expressed together with the long isoform of the Prlr using an internal ribosome entry site. When crossed to a Cre-dependent reporter mouse strain, Cre-mediated recombination will genetically label cells that acutely express the Prlr as well as cells that have transiently expressed the Prlr during development. We report here the anatomical distribution of cells which express the fluorescent reporter τ green fluorescent protein in a total of 38 organs prepared from young adult male and female Prlr reporter mice. Our results establish a resource for dissecting the functional role of Prlr in multiple murine tissues.

Primary dendrites of mitral cells synapse unto neighboring glomeruli independent of their odorant receptor identity.

In the mouse olfactory bulb, neural map topography is largely established by axon-axon interactions of olfactory sensory neurons (OSNs). However, to make the map functional, the OSNs must make proper connections to second-order neurons, the mitral cells. How do the mitral-cell dendrites find their partner glomeruli for synapse formation with OSN axons? Here, we analyze dendrite connections of mitral cells in various mutant mice in which glomerular formation is perturbed. Our present results support the proximity model, whereby mitral cells tend to connect primary dendrites to the nearest neighboring glomeruli regardless of their odorant receptor identities. The physical location of glomeruli rather than the odorant-receptor specificity appears to play a key role in matching mitral cells with their partner OSN axons.

Structural insight into microtubule stabilization and kinesin inhibition by Tau family MAPs.

The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin-dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin-dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin-dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.

Analysis of prolactin receptor expression in the murine brain using a novel prolactin receptor reporter mouse.

Prolactin influences a wide range of physiological functions via actions within the central nervous system, as well as in peripheral tissues. A significant limitation in studies investigating these functions is the difficulty in identifying prolactin receptor (Prlr) expression, particularly in the brain. We have developed a novel mouse line using homologous recombination within mouse embryonic stem cells to produce a mouse in which an internal ribosome entry site (IRES) followed by Cre recombinase cDNA is inserted immediately after exon 10 in the Prlr gene, thereby targeting the long isoform of the Prlr. By crossing this Prlr-IRES-Cre mouse with a ROSA26-CAGS-tauGFP (τGFP) reporter mouse line, and using immunohistochemistry to detect τGFP, we were able to generate a detailed map of the distribution of individual Prlr-expressing neurones and fibres throughout the brain of adult mice without the need for amplification of the GFP signal. Because the τGFP is targeted to neurotubules, the labelling detected not only cell bodies, but also processes of prolactin-sensitive neurones. In both males and females, Cre-dependent τGFP expression was localised, with varying degrees of abundance, in a number of brain regions, including the lateral septal nucleus, bed nucleus of the stria terminalis, preoptic and hypothalamic nuclei, medial habenula, posterodorsal medial amygdala, and brainstem regions such as the periaqueductal grey and parabrachial nucleus. The labelling was highly specific, occurring only in cells where we could also detect PrlrmRNA by in situ hybridisation. Apart from two brain areas, the anteroventral periventricular nucleus and the medial preoptic nucleus, the number and distribution of τGFP-immunopositive cells was similar in males and females, suggesting that prolactin may have many equivalent functions in both sexes. These mice provide a valuable tool for investigating the neural circuits underlying the actions of prolactin.

Structural Insights into the Altering Function of CRMP2 by Phosphorylation.

Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity by controlling microtubule dynamics. CRMP2 activity is regulated by semaphorin-induced phosphorylation at the C-terminal tail domain. Unphosphorylated CRMP2 induces effective axonal microtubule formation to give the axonal characteristics to a neurite, whereas phosphorylated CRMP2 leads to the apparently opposite effect, growth cone collapse. We have recently characterized the structural detail of CRMP2-induced axonal microtubule formation (Niwa et al. (2017) Sci. Rep., 7: 10681). CRMP2 forms the hetero-trimer with GTP-tubulin to induce effective axonal microtubule formation in the future axon. Phosphorylation of CRMP2 has been reported to decrease the affinity between CRMP2 and the microtubule, albeit the molecular mechanisms of how the phosphorylation of CRMP2 changes the structure to achieve distinct effects from unphosphorylated CRMP2 is not well understood. Here we performed a series of biochemical and structural analyses of phospho-mimic CRMP2. Phosphorylation of CRMP2 undergoes small conformational changes at the C-terminal tail with shifting the surface charge, which not only alters the interactions within the CRMP2 tetramer but also alters the interactions with GTP-tubulin. Consequently, phospho-mimic CRMP2 fails to form a hetero-trimer with GTP-tubulin, thus losing the ability to establish and maintain the axonal microtubules.Key words: CRMP2, phosphorylation, microtubule, axon, crystal structure.

Female sexual behavior in mice is controlled by kisspeptin neurons.

Sexual behavior is essential for the survival of many species. In female rodents, mate preference and copulatory behavior depend on pheromones and are synchronized with ovulation to ensure reproductive success. The neural circuits driving this orchestration in the brain have, however, remained elusive. Here, we demonstrate that neurons controlling ovulation in the mammalian brain are at the core of a branching neural circuit governing both mate preference and copulatory behavior. We show that male odors detected in the vomeronasal organ activate kisspeptin neurons in female mice. Classical kisspeptin/Kiss1R signaling subsequently triggers olfactory-driven mate preference. In contrast, copulatory behavior is elicited by kisspeptin neurons in a parallel circuit independent of Kiss1R involving nitric oxide signaling. Consistent with this, we find that kisspeptin neurons impinge onto nitric oxide-synthesizing neurons in the ventromedial hypothalamus. Our data establish kisspeptin neurons as a central regulatory hub orchestrating sexual behavior in the female mouse brain.

Prolactin action in the medial preoptic area is necessary for postpartum maternal nursing behavior.

Pregnancy hormones, such as prolactin, sensitize neural circuits controlling parental interactions to induce timely activation of maternal behaviors immediately after parturition. While the medial preoptic area (MPOA) is known to be critical for maternal behavior, the specific role of prolactin in this brain region has remained elusive. Here, we evaluated the role of prolactin action in the MPOA using complementary genetic strategies in mice. We characterized prolactin-responsive neurons within the MPOA at different hormonal stages and delineated their projections in the brain. We found that MPOA neurons expressing prolactin receptors (Prlr) form the nexus of a complex prolactin-responsive neural circuit, indicating that changing prolactin levels can act at multiple sites and thus, impinge on the overall activity of a distributed network of neurons. Conditional KO of Prlr from neuronal subpopulations expressing the neurotransmitters GABA or glutamate within this circuit markedly reduced the capacity for prolactin action both in the MPOA and throughout the network. Each of these manipulations, however, produced only subtle impacts on maternal care, suggesting that this distributed circuit is robust with respect to alterations in prolactin signaling. In contrast, acute deletion of Prlr in all MPOA neurons of adult female mice resulted in profound deficits in maternal care soon after birth. All mothers abandoned their pups, showing that prolactin action on MPOA neurons is necessary for the normal expression of postpartum maternal behavior in mice. Our data establish a critical role for prolactin-induced behavioral responses in the maternal brain, ensuring survival of mammalian offspring.

Structural basis for CRMP2-induced axonal microtubule formation.

Microtubule associated protein Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity in developing neurons through interactions with tubulins or microtubules. However, how CRMP2 promotes axonal formation by affecting microtubule behavior remains unknown. This study aimed to obtain the structural basis for CRMP2-tubulin/microtubule interaction in the course of axonogenesis. The X-ray structural studies indicated that the main interface to the soluble tubulin-dimer is the last helix H19 of CRMP2 that is distinct from the known C-terminal tail-mediated interaction with assembled microtubules. In vitro structural and functional studies also suggested that the H19-mediated interaction promoted the rapid formation of GTP-state microtubules directly, which is an important feature of the axon. Consistently, the H19 mutants disturbed axon elongation in chick neurons, and failed to authorize the structural features for axonal microtubules in Caenorhabditis elegans. Thus, CRMP2 induces effective axonal microtubule formation through H19-mediated interactions with a soluble tubulin-dimer allowing axonogenesis to proceed.

Novel ALK5 inhibitor TP0427736 reduces TGF-ß　induced growth inhibition in human outer root sheath cells and elongates anagen phase in mouse hair follicles.

BACKGROUND: Androgenic alopecia (AGA) occurs as a result of the contraction of the anagen phase because of the action of androgens on hair follicles. TGF-ß production from dermal papillae is enhanced by androgens, and growth inhibition of hair-follicle cells is induced by TGF-ß, and the hair cycle progresses from the anagen phase to the catagen phase. We investigated both the in vitro and in vivo potency of the newly identified ALK5 inhibitor TP0427736 {6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole}. METHODS: For in vitro study, kinase inhibitory activity was evaluated with ELISA, and inhibitory activity against TGF-ß-induced Smad2/3 phosphorylation in A549 cells and TGF-ß-induced growth inhibition of human outer root sheath cells were assayed using ELISA. For in vivo study, we used a mouse model that had been synchronized through dorsal hair depilation. RESULTS: TP0427736 inhibited ALK5 kinase activity with an IC50 of 2.72nM; this effect was 300-fold higher than the inhibitory effect on ALK3. In cell-based assays, TP0427736 inhibited Smad2/3 phosphorylation in A549 cells and decreased the growth inhibition of human outer root sheath cells. The topical application of TP0427736 significantly decreased Smad2 phosphorylation in mouse skin, and its repeated application suppressed the shortening of average hair follicle length during the transition from the late anagen phase to the catagen phase. CONCLUSIONS: TP0427736, a potent ALK5 inhibitor with appropriate in vitro and in vivo profiles, may serve as a potential new therapy for AGA.　.

Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility.

Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motility assay, we show that in addition to the neck helix, the conserved five residues at the C-terminal region in kinesin-14, namely the neck mimic, are necessary to give kinesin-1 an ability to reverse its directionality toward the minus end of microtubules. Our structural analyses further demonstrate that the C-terminal neck mimic, in cooperation with conformational changes in the catalytic core during ATP binding, forms a kinesin-14 bundle with the N-terminal neck helix to swing toward the minus end of microtubules. Thus, the neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14.

Serum anticholinergic activity: a possible peripheral marker of the anticholinergic burden in the central nervous system in Alzheimer's disease.

We review the utility of serum anticholinergic activity (SAA) as a peripheral marker of anticholinergic activity (AA) in the central nervous system (CAA). We hypothesize that the compensatory mechanisms of the cholinergic system do not contribute to SAA if their system is intact and that if central cholinergic system deteriorates alone in conditions such as Alzheimer's disease or Lewy body dementia, CAA and SAA are caused by way of hyperactivity of inflammatory system and SAA is a marker of the anticholinergic burden in CNS. Taking into account the diurnal variations in the plasma levels of corticosteroids, which are thought to affect SAA, it should be measured at noon or just afterward.

Plasma thiobarbituric acid reactive substances, vitamin A and vitamin E levels and resumption of postpartum ovarian activity in dairy cows.

Vitamins with antioxidative functions are commonly used as supplements to improve fertility in dairy cows. However, according to field test results uncertainty exists about the effect of these vitamins, especially in vitamin A and vitamin E, on ovarian functional activity. This study was performed to reveal the physiological characteristics of cows receiving enough feed and the ovaries of which were activated in the early postpartum period. Six of 12 primiparous cows showing the corpus luteum on 25 to 27 days after parturition were classified as early responders (PER); the remaining six were classified as late responders (PLR). Among 11 multiparous cows, nine were early responders (MER), and the remaining two were late responders (MLR). Plasma concentration of thiobarbituric acid reactive substances (TBARS) in the PER were lower than those in the PLR (P<0.01). The ratio of plasma all-trans-retinol to intake α-tocopherol or ß-carotene were increased in the following order: MER

Possible roles of Robo1+ ensheathing cells in guiding dorsal-zone olfactory sensory neurons in mouse.

In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) correlate with their axonal projection sites along the dorsoventral axis of the olfactory bulb (OB). We have previously reported that Neuropilin-2 expressed by ventral-zone OSNs contributes to the segregation of dorsal and ventral OSN axons, and that Slit is acting as a negative land mark to restrict the projection of Robo2+, early-arriving OSN axons to the embryonic OB. Here, we report that another guidance receptor, Robo1, also plays an important role in guiding OSN axons. Knockout mice for Robo1 demonstrated defects in targeting of OSN axons to the OB. Although Robo1 is colocalized with dorsal-zone OSN axons, it is not produced by OSNs, but instead by olfactory ensheathing cells. These findings indicate a novel strategy of axon guidance in the mouse olfactory system during development.