Thalamocortical axons regulate neurogenesis and laminar fates in the early sensory cortex.

Area-specific axonal projections from the mammalian thalamus shape unique cellular organization in target areas in the adult neocortex. How these axons control neurogenesis and early neuronal fate specification is poorly understood. By using mutant mice lacking the majority of thalamocortical axons, we show that these axons are required for the production and specification of the proper number of layer 4 neurons in primary sensory areas by the neonatal stage. Part of these area-specific roles is played by the thalamus-derived molecule, VGF. Our work reveals that extrinsic cues from sensory thalamic projections have an early role in the formation of cortical cytoarchitecture by enhancing the production and specification of layer 4 neurons.

Heme oxygenase-1 inhibits DENV-induced endothelial hyperpermeability and serves as a potential target against dengue hemorrhagic fever.

Dengue virus (DENV) is a cause of vascular endothelial dysfunction and vascular leakage, which are characterized as hallmarks of dengue hemorrhagic fever or dengue shock syndrome, which become a severe global health emergency with substantial morbidity and mortality. Currently, there are still no promising therapeutics to alleviate the dengue-associated vascular hemorrhage in a clinical setting. In the present study, we first observed that heme oxygenase-1 (HO-1) expression level was highly suppressed in severe DENV-infected patients. In contrast, the overexpression of HO-1 could attenuate DENV-induced pathogenesis, including plasma leakage and thrombocytopenia, in an AG129 mouse model. Our data indicate that overexpression of HO-1 or its metabolite biliverdin can maintain endothelial integrity upon DENV infection in vitro and in vivo. We further characterized the positive regulatory effect of HO-1 on the endothelial adhesion factor vascular endothelial-cadherin to decrease DENV-induced endothelial hyperpermeability. Subsequently, we confirmed that two medicinal plant-derived compounds, andrographolide, and celastrol, widely used as a nutritional or medicinal supplement are useful to attenuate DENV-induced plasma leakage through induction of the HO-1 expression in DENV-infected AG129 mice. In conclusion, our findings reveal that induction of the HO-1 signal pathway is a promising option for the treatment of DENV-induced vascular pathologies.

Potential Vasculoprotective Effects of Blackcurrant (Ribes nigrum) Extract in Diabetic KK-Ay Mice.

Polyphenols are bioactive compounds found naturally in fruits and vegetables; they are widely used in disease prevention and health maintenance. Polyphenol-rich blackcurrant extract (BCE) exerts beneficial effects on vascular health in menopausal model animals. However, the vasculoprotective effects in diabetes mellitus (DM) and atherosclerotic vascular disease secondary to DM are unknown. Therefore, we investigated whether BCE is effective in preventing atherosclerosis using KK-Ay mice as a diabetes model. The mice were divided into three groups and fed a high-fat diet supplemented with 1% BCE (BCE1), 3% BCE (BCE2), or Control for 9 weeks. The mice in the BCE2 group showed a considerable reduction in the disturbance of elastic lamina, foam cell formation, and vascular remodeling compared to those in the BCE1 and Control groups. Immunohistochemical staining indicated that the score of endothelial nitric oxide synthase staining intensity was significantly higher in both BCE2 (2.9) and BCE1 (1.9) compared to that in the Control (1.1). Furthermore, the score for the percentage of alpha-smooth muscle actin was significantly lower in the BCE2 (2.9%) than in the Control (2.1%). Our results suggest that the intake of anthocyanin-rich BCE could have beneficial effects on the blood vessels of diabetic patients.

A recessive PRDM13 mutation results in congenital hypogonadotropic hypogonadism and cerebellar hypoplasia.

The positive regulatory (PR) domain containing 13 (PRDM13) putative chromatin modifier and transcriptional regulator functions downstream of the transcription factor PTF1A, which controls GABAergic fate in the spinal cord and neurogenesis in the hypothalamus. Here, we report a recessive syndrome associated with PRDM13 mutation. Patients exhibited intellectual disability, ataxia with cerebellar hypoplasia, scoliosis, and delayed puberty with congenital hypogonadotropic hypogonadism (CHH). Expression studies revealed Prdm13/PRDM13 transcripts in the developing hypothalamus and cerebellum in mouse and human. An analysis of hypothalamus and cerebellum development in mice homozygous for a Prdm13 mutant allele revealed a significant reduction in the number of Kisspeptin (Kiss1) neurons in the hypothalamus and PAX2+ progenitors emerging from the cerebellar ventricular zone. The latter was accompanied by ectopic expression of the glutamatergic lineage marker TLX3. Prdm13-deficient mice displayed cerebellar hypoplasia and normal gonadal structure, but delayed pubertal onset. Together, these findings identify PRDM13 as a critical regulator of GABAergic cell fate in the cerebellum and of hypothalamic kisspeptin neuron development, providing a mechanistic explanation for the cooccurrence of CHH and cerebellar hypoplasia in this syndrome. To our knowledge, this is the first evidence linking disrupted PRDM13-mediated regulation of Kiss1 neurons to CHH in humans.

Mindfulness intervention improves cognitive function in older adults by enhancing the level of miRNA-29c in neuron-derived extracellular vesicles.

Although mindfulness-based stress reduction (MBSR) improves cognitive function, the mechanism is not clear. In this study, people aged 65 years and older were recruited from elderly communities in Chitose City, Japan, and assigned to a non-MBSR group or a MBSR group. Before and after the intervention, the Japanese version of the Montreal Cognitive Assessment (MoCA-J) was administered, and blood samples were collected. Then, neuron-derived extracellular vesicles (NDEVs) were isolated from blood samples, and microRNAs, as well as the target mRNAs, were evaluated in NDEVs. A linear mixed model analysis showed significant effects of the MBSR x time interaction on the MoCA-J scores, the expression of miRNA(miR)-29c, DNA methyltransferase 3 alpha (DNMT3A), and DNMT3B in NDEVs. These results indicate that MBSR can improve cognitive function by increasing the expression of miR-29c and decreasing the expression of DNMT3A, as well as DNMT3B, in neurons. It was also found that intracerebroventricular injection of miR-29c mimic into 5xFAD mice prevented cognitive decline, as well as neuronal loss in the subiculum area, by down-regulating Dnmt3a  and Dnmt3b  in the hippocampus. The present study suggests that MBSR can prevent neuronal loss and cognitive impairment by increasing the neuronal expression of miR-29c.

Cochlear outer hair cell electromotility enhances organ of Corti motion on a cycle-by-cycle basis at high frequencies in vivo.

Mammalian hearing depends on an amplification process involving prestin, a voltage-sensitive motor protein that enables cochlear outer hair cells (OHCs) to change length and generate force. However, it has been questioned whether this prestin-based somatic electromotility can operate fast enough in vivo to amplify cochlear vibrations at the high frequencies that mammals hear. In this study, we measured sound-evoked vibrations from within the living mouse cochlea and found that the top and bottom of the OHCs move in opposite directions at frequencies exceeding 20 kHz, consistent with fast somatic length changes. These motions are physiologically vulnerable, depend on prestin, and dominate the cochlea's vibratory response to high-frequency sound. This dominance was observed despite mechanisms that clearly low-pass filter the in vivo electromotile response. Low-pass filtering therefore does not critically limit the OHC's ability to move the organ of Corti on a cycle-by-cycle basis. Our data argue that electromotility serves as the primary high-frequency amplifying mechanism within the mammalian cochlea.

Variably methylated retrotransposons are refractory to a range of environmental perturbations.

The agouti viable yellow (Avy) allele is an insertional mutation in the mouse genome caused by a variably methylated intracisternal A particle (VM-IAP) retrotransposon. Avy expressivity is sensitive to a range of early-life chemical exposures and nutritional interventions, suggesting that environmental perturbations can have long-lasting effects on the methylome. However, the extent to which VM-IAP elements are environmentally labile with phenotypic implications is unknown. Using a recently identified repertoire of VM-IAPs, we assessed the epigenetic effects of different environmental contexts. A longitudinal aging analysis indicated that VM-IAPs are stable across the murine lifespan, with only small increases in DNA methylation detected for a subset of loci. No significant effects were observed after maternal exposure to the endocrine disruptor bisphenol A, an obesogenic diet or methyl donor supplementation. A genetic mouse model of abnormal folate metabolism exhibited shifted VM-IAP methylation levels and altered VM-IAP-associated gene expression, yet these effects are likely largely driven by differential targeting by polymorphic KRAB zinc finger proteins. We conclude that epigenetic variability at retrotransposons is not predictive of environmental susceptibility.

Decrease in DHA and other fatty acids correlates with photoreceptor degeneration in retinitis pigmentosa.

Fatty acids, and especially docosahexaenoic acid (DHA), are essential for photoreceptor cell integrity and are involved in the phototransduction cascade. In this study, we analyzed the changes in the fatty acid profile in the retina of the rd10 mouse, model of retinitis pigmentosa, in order to identify potential risk factors for retinal degeneration and possible therapeutic approaches. Fatty acids from C57BL/6J and rd10 mouse retinas were extracted with Folch's method and analyzed by gas chromatography/mass spectrometry. Changes in retinal morphology were evaluated by immunohistochemistry. The rd10 mouse retina showed a decreased number of photoreceptor rows and alterations in photoreceptor morphology compared to C57BL/6J mice. The total amount of fatty acids dropped by 29.4% in the dystrophic retinas compared to C57BL/6J retinas. A positive correlation was found between the retinal content of specific fatty acids and the number of photoreceptor rows. We found that the amount of several short-chain and long-chain saturated fatty acids, as well as monounsaturated fatty acids, decreased in the retina of rd10 mice. Moreover, the content of the n-6 polyunsaturated fatty acid arachidonic acid and the n-3 polyunsaturated DHA decreased markedly in the dystrophic retina. The fall of DHA was more pronounced, hence the n-6/n-3 ratio was significantly increased in the diseased retina. The content of specific fatty acids in the retina decreased with photoreceptor degeneration in retinitis pigmentosa mice, with a remarkable reduction in DHA and other saturated and unsaturated fatty acids. These fatty acids could be essential for photoreceptor cell viability, and they should be evaluated for the design of therapeutical strategies and nutritional supplements.

Folate Intake Alters Mutation Frequency and Profiles in a Tissue- and Dose-Specific Manner in MutaMouse Male Mice.

BACKGROUND: While cancer is common, its incidence varies widely by tissue. These differences are attributable to variable risk factors, such as environmental exposure, genetic inheritance, and lifetime number of stem cell divisions in a tissue. Folate deficiency is generally associated with increased risk for colorectal cancer (CRC) and acute lymphocytic leukemia (ALL). Conversely, high folic acid (FA) intake has also been associated with higher CRC risk. OBJECTIVE: Our objective was to compare the effect of folate intake on mutant frequency (MF) and types of mutations in the colon and bone marrow of mice. METHODS: Five-week-old MutaMouse male mice were fed a deficient (0 mg FA/kg), control (2 mg FA/kg), or supplemented (8 mg FA/kg) diet for 20 wk. Tissue MF was assessed using the lacZ mutant assay and comparisons made by 2-factor ANOVA. LacZ mutant plaques were sequenced using next-generation sequencing, and diet-specific mutation profiles within each tissue were compared by Fisher's exact test. RESULTS: In the colon, the MF was 1.5-fold and 1.3-fold higher in mice fed the supplemented diet compared with mice fed the control (P = 0.001) and deficient (P = 0.008) diets, respectively. This contrasted with the bone marrow MF in the same mice where the MF was 1.7-fold and 1.6-fold higher in mice fed the deficient diet compared with mice fed the control (P = 0.02) and supplemented (P = 0.03) diets, respectively. Mutation profiles and signatures (mutation context) were tissue-specific. CONCLUSIONS: Our data indicate that dietary folate intake affects mutagenesis in a tissue- and dose-specific manner in mice. Mutation profiles were generally tissue- but not dose-specific, suggesting that altered cellular folate status appears to interact with endogenous mutagenic mechanisms in each tissue to create a permissive context in which specific mutation types accumulate. These data illuminate potential mechanisms underpinning differences in observed associations between folate intake/status and cancer.

An abundant biliary metabolite derived from dietary omega-3 polyunsaturated fatty acids regulates triglycerides.

Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified, despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here, we identified in bile a class of hypotriglyceridemic omega-3 fatty acid-derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid-containing (DHA-containing) NAT C22:6 NAT was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, whereas selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high-fat diet-induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT is a negative feedback mediator that limits excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid-based therapeutics.

A new insulin-sensitive enhancer from Silene viscidula, WPTS, treats type 2 diabetes by ameliorating insulin resistance, reducing dyslipidemia, and promoting proliferation of islet ß cells.

Wacao pentacyclic triterpenoid saponins (WPTS) is a newly discovered insulin sensitivity enhancer. It is a powerful hypoglycemic compound derived from Silene viscidula, which has a hypoglycemic effect similar to that of insulin. It can rapidly reduce blood glucose levels, normalizing them within 3 days of administration. However, its mechanism of action is completely different from that of insulin. Thus, we aimed to determine the pharmacological effects and mechanism of activity of WPTS on type 2 diabetes to elucidate the main reasons for its rapid effects. The results showed that WPTS could effectively improve insulin resistance in KKAy diabetic mice. Comparative transcriptomics showed that WPTS could upregulate the expression of insulin resistance-related genes such as glucose transporter type 4 (Glut4), insulin receptor substrate 1 (Irs1), Akt, and phosphoinositide 3-kinase (PI3K), and downregulate the expression of lipid metabolism-related genes such as monoacylglycerol O-acyltransferase 1 (Moat1), lipase C (Lipc), and sphingomyelin phosphodiesterase 4 (Smpd4). The results indicated that the differentially expressed genes could regulate lipid metabolism via the PI3K/AKT metabolic pathway, and it is noteworthy that WPTS was found to upregulate Glut4 expression, decrease blood glucose levels, and attenuate insulin resistance via the PI3K/AKT pathway. Q-PCR and western blotting further validated the transcriptomics findings at the mRNA and protein levels, respectively. We believe that WPTS can achieve a rapid hypoglycemic effect by improving the lipid metabolism and insulin resistance of the diabetic KKAy mice. WPTS could be a very promising candidate drug for the treatment of diabetes and deserves further research.

Mechanical allodynia triggered by cold exposure in mice with the Scn11a p.R222S mutation: a novel model of drug therapy for neuropathic pain related to NaV1.9.

Mutations within the SCN11A gene which encodes the voltage-gated sodium channel NaV1.9 mainly expressed in small fiber sensory neurons have been associated with neuropathic disorders; however, suitable medications have not been fully investigated. To develop drug therapies against NaV1.9-related neuropathic pain, we aimed to establish a novel model using mice carrying the Scn11a p.R222S mutation initially identified in patients with familial episodic limb pain that is characterized by paroxysmal pain induced by fatigue or bad weather conditions. We investigated the influence of cold exposure (4 °C, overnight) on the behavioral and biochemical phenotypes of Scn11a p.R222S mutant (R222S) and wild type C57BL/6N (WT) mice. We also tested the effects of acetaminophen (125, 250 mg/kg, perorally, p.o.) and traditional Japanese medicine, goshajinkigan (0.5 or 1.0 g/kg, p.o.), which are analgesic drugs prescribed to patients with neuropathic pain, in this model of cold-induced mechanical allodynia in R222S mice.Cold-exposed R222S mice exhibited enhanced mechanical allodynia and thermal hypersensitivity compared with WT mice. The decrease of the mechanical withdrawal threshold in R222S mice was reversible 24 h after housing at room temperature. There was no significant change in the levels of interleukin-1ß, interleukin-6, tumor necrosis factor-α, or interferon-γ in the plasma or spinal cords of WT and R222S mice after cold exposure. Both acetaminophen (250 mg/kg) and goshajinkigan (1.0 g/kg) significantly attenuated mechanical allodynia in R222S mice. The model of cold-induced mechanical allodynia in mice with the Scn11a p.R222S mutation is novel and useful for evaluating analgesic drugs for intractable neuropathies related to NaV1.9.

Catechin-Rich Green Tea Extract and the Loss-of-TLR4 Signaling Differentially Alter the Hepatic Metabolome in Mice with Nonalcoholic Steatohepatitis.

SCOPE: Catechin-rich green tea extract (GTE) limits inflammation in nonalcoholic steatohepatitis (NASH) consistent with a Toll-like receptor 4 (TLR4)-dependent mechanism. It is hypothesized that GTE supplementation during NASH will shift the hepatic metabolome similar to that attributed to the loss-of-TLR4 signaling. METHODS AND RESULTS: Wild-type (WT) and loss-of-function TLR4-mutant (TLR4mut ) mice are fed a high-fat diet containing 0% or 2% GTE for 8 weeks prior to performing untargeted mass spectrometry-based metabolomics on liver tissue. The loss-of-TLR4 signaling and GTE shift the hepatic metabolome away from that of WT mice. However, relatively few metabolites are altered by GTE in WT mice to the same extent as the loss-of-TLR4 signaling in TLR4mut mice. GTE increases acetyl-coenzyme A precursors and spermidine to a greater extent than the loss-of-TLR4 signaling. Select metabolites associated with thiol metabolism are similarly affected by GTE and the loss-of-TLR4 signaling. Glycerophospholipid catabolites are decreased by GTE, but are unaffected in TLR4mut mice. Conversely, the loss-of-TLR4 signaling but not GTE increases several bile acid metabolites. CONCLUSION: GTE limitedly alters the hepatic metabolome consistent with a TLR4-dependent mechanism. This suggests that the anti-inflammatory activities of GTE and loss-of-TLR4 signaling that regulate hepatic metabolism to abrogate NASH are likely due to distinct mechanisms.

Celastrol ameliorates Propionibacterium acnes/LPS-induced liver damage and MSU-induced gouty arthritis via inhibiting K63 deubiquitination of NLRP3.

BACKGROUND: Celastrol, a pentacyclic triterpenoid quinonemethide isolated from several spp. of Celastraceae family, exhibits anti-inflammatory activities in a variety of diseases including arthritis. PURPOSE: This study aims to investigate whether the inhibition of NLRP3 inflammasome is engaged in the anti-inflammatory activities of celastrol and delineate the underlying mechanism. METHODS: The influence of celastrol on NLRP3 inflammasome activation was firstly studied in lipopolysaccharide (LPS)-primed mouse bone marrow-derived macrophages (BMDMs) and phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells treated with nigericin. Reconstituted inflammasome was also established by co-transfecting NLRP3, ASC, pro-caspase-1 and pro-IL-1ß in HEK293T cells. The changes of inflammasome components including NLRP3, ASC, pro-caspase-1/caspase-1 and pro-IL-1ß/IL-1ß were examined by enzyme-linked immunosorbent assay (ELISA), western blotting and immunofluorescence. Furthermore, Propionibacterium acnes (P. acnes)/LPS-induced liver injury and monosodium urate (MSU)-induced gouty arthritis in mice were employed in vivo to validate the inhibitory effect of celastrol on NLRP3 inflammasome. RESULTS: Celastrol significantly suppressed the cleavage of pro-caspase-1 and pro-IL-1ß, while not affecting the protein expressions of NLRP3, ASC, pro-caspase-1 and pro-IL-1ß in THP-1 cells, BMDMs and HEK293T cells. Celastrol suppressed NLRP3 inflammasome activation and alleviated P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis. Mechanism study revealed that celastrol could interdict K63 deubiquitination of NLRP3, which may concern interaction of celastrol and BRCA1/BRCA2-containing complex subunit 3 (BRCC3), and thereby prohibited the formation of NLRP3, ASC and pro-caspase-1 complex to block the generation of mature IL-1ß. CONCLUSION: Celastrol suppresses NLRP3 inflammasome activation in P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis via inhibiting K63 deubiquitination of NLRP3, which presents a novel insight into inhibition of celastrol on NLRP3 inflammasome and provides more evidences for its application in the therapy of inflammation-related diseases.

Blockade of Retinal Oscillations by Benzodiazepines Improves Efficiency of Electrical Stimulation in the Mouse Model of RP, rd10.

Purpose: In RP, photoreceptors degenerate. Retinal prostheses are considered a suitable strategy to restore vision. In animal models of RP, a pathologic rhythmic activity seems to compromise the efficiency of retinal ganglion cell stimulation by an electrical prosthesis. We, therefore, strove to eliminate this pathologic activity. Methods: Electrophysiologic recordings of local field potentials and spike activity of retinal ganglion cells were obtained in vitro from retinae of wild-type and rd10 mice using multielectrode arrays. Retinae were stimulated electrically. Results: The efficiency of electrical stimulation was lower in rd10 retina than in wild-type retina and this was highly correlated with the presence of oscillations in retinal activity. Glycine and GABA, as well as the benzodiazepines diazepam, lorazepam, and flunitrazepam, abolished retinal oscillations and, most important, increased the efficiency of electrical stimulation to values similar to those in wild-type retina. Conclusions: Treatment of patients with these benzodiazepines may offer a way to improve the performance of retinal implants in cases with poor implant proficiency. This study may open the way to a therapy that supports electrical stimulation by prostheses with pharmacologic treatment.

Anti-CD40 monoclonal antibody ameliorates experimental autoimmune uveoretinitis in mice.

OBJECTIVE: To investigate the effects of CD40 on ocular inflammation in experimental autoimmune uveoretinitis (EAU) in B10.RIII mice. ANIMALS STUDIED: EAU-susceptible B10.RIII mice were subcutaneously immunized with interphotoreceptor retinoid-binding protein (IRBP) 161-180 in complete Freund's adjuvant and evaluated clinically and pathologically on days 7, 14, 21, 28, and 35 postimmunization. Anti-CD40 antibody was intraperitoneally injected into mice every other day from days 7 to 14 postimmunization. Phosphate-buffered saline (PBS)-injected EAU mice were used as the controls. PROCEDURES: The frequencies of CD11c+ CD40+ dendritic cells (DCs), CD11c+ MHC-II+ DCs, and CD11c+ CD40+ MHC-II+ DCs in splenocytes were evaluated by flow cytometry on days 0, 7, 14, and 21 after immunization. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in CD11c+ DCs was assessed by ELISA. IRBP-specific lymphocyte proliferation was assessed using a modified MTT cell proliferation assay. RESULTS: The number of CD11c+ CD40+ DCs, CD11c+ MHC-II+ DCs, and CD11c+ CD40+ MHC-II+ DCs increased at the onset of EAU, peaked at the height of disease severity, and was sustained at a high level until day 21. Treatment with anti-CD40 antibody significantly alleviated clinical and pathological activities related to EAU. Compared with the control mice, antibody-treated EAU mice showed few CD11c+ CD40+ DC and CD11c+ CD40+ MHC-II+ DC frequencies in splenocytes. The anti-CD40 antibody significantly suppressed IRBP-specific lymphocyte proliferation and TNF-α and IL-6 production by DCs in EAU mice. CONCLUSIONS: The increased expression of CD40 and major histocompatibility complex (MHC) class II molecules in the splenocytes of EAU mice were correlated with inflammatory activity. Anti-CD40 treatment can significantly attenuate EAU activity by inhibiting systemic IRBP-specific immune responses.

Vcp Overexpression and Leucine Supplementation Increase Protein Synthesis and Improve Fear Memory and Social Interaction of Nf1 Mutant Mice.

Neurofibromatosis type 1 (NF1) is a dominant genetic disorder manifesting, in part, as cognitive defects. Previous study indicated that neurofibromin (NF1 protein) interacts with valosin-containing protein (VCP)/P97 to control dendritic spine formation, but the mechanism is unknown. Here, using Nf1+/- mice and transgenic mice overexpressing wild-type Vcp/p97, we demonstrate that neurofibromin acts with VCP to control endoplasmic reticulum (ER) formation and consequent protein synthesis and regulates dendritic spine formation, thereby modulating contextual fear memory and social interaction. To validate the role of protein synthesis, we perform leucine supplementation in vitro and in vivo. Our results suggest that leucine can effectively enter the brain and increase protein synthesis and dendritic spine density of Nf1+/- neurons. Contextual memory and social behavior of Nf1+/- mice are also restored by leucine supplementation. Our study suggests that the "ER-protein synthesis" pathway downstream of neurofibromin and VCP is a critical regulator of dendritic spinogenesis and brain function.

Nicotinamide riboside supplementation corrects deficits in oxytocin, sociability and anxiety of CD157 mutants in a mouse model of autism spectrum disorder.

Oxytocin (OT) is a critical molecule for social recognition and memory that mediates social and emotional behaviours. In addition, OT acts as an anxiolytic factor and is released during stress. Based on the activity of CD38 as an enzyme that produces the calcium-mobilizing second messenger cyclic ADP-ribose (cADPR), CD157, a sister protein of CD38, has been considered a candidate mediator for the production and release of OT and its social engagement and anti-anxiety functions. However, the limited expression of CD157 in the adult mouse brain undermined confidence that CD157 is an authentic and/or actionable molecular participant in OT-dependent social behaviour. Here, we show that CD157 knockout mice have low levels of circulating OT in cerebrospinal fluid, which can be corrected by the oral administration of nicotinamide riboside, a recently discovered vitamin precursor of nicotinamide adenine dinucleotide (NAD). NAD is the substrate for the CD157- and CD38-dependent production of cADPR. Nicotinamide riboside corrects social deficits and fearful and anxiety-like behaviours in CD157 knockout males. These results suggest that elevating NAD levels with nicotinamide riboside may allow animals with cADPR- and OT-forming deficits to overcome these deficits and function more normally.

Odiparcil, a potential glycosaminoglycans clearance therapy in mucopolysaccharidosis VI-Evidence from in vitro and in vivo models.

Mucopolysaccharidoses are a class of lysosomal storage diseases, characterized by enzymatic deficiency in the degradation of specific glycosaminoglycans (GAG). Pathological accumulation of excess GAG leads to multiple clinical symptoms with systemic character, most severely affecting bones, muscles and connective tissues. Current therapies include periodic intravenous infusion of supplementary recombinant enzyme (Enzyme Replacement Therapy-ERT) or bone marrow transplantation. However, ERT has limited efficacy due to poor penetration in some organs and tissues. Here, we investigated the potential of the ß-D-xyloside derivative odiparcil as an oral GAG clearance therapy for Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI, MPS VI). In vitro, in bovine aortic endothelial cells, odiparcil stimulated the secretion of sulphated GAG into culture media, mainly of chondroitin sulphate (CS) /dermatan sulphate (DS) type. Efficacy of odiparcil in reducing intracellular GAG content was investigated in skin fibroblasts from MPS VI patients where odiparcil was shown to reduce efficiently the accumulation of intracellular CS with an EC50 in the range of 1 µM. In vivo, in wild type rats, after oral administrations, odiparcil was well distributed, achieving µM concentrations in MPS VI disease-relevant tissues and organs (bone, cartilage, heart and cornea). In MPS VI Arylsulphatase B deficient mice (Arsb-), after chronic oral administration, odiparcil consistently stimulated the urinary excretion of sulphated GAG throughout the treatment period and significantly reduced tissue GAG accumulation in liver and kidney. Furthermore, odiparcil diminished the pathological cartilage thickening observed in trachea and femoral growth plates of MPS VI mice. The therapeutic efficacy of odiparcil was similar in models of early (treatment starting in juvenile, 4 weeks old mice) or established disease (treatment starting in adult, 3 months old mice). Our data demonstrate that odiparcil effectively diverts the synthesis of cellular glycosaminoglycans into secreted soluble species and this effect can be used for reducing cellular and tissue GAG accumulation in MPS VI models. Therefore, our data reveal the potential of odiparcil as an oral GAG clearance therapy for MPS VI patients.

Circadian clock regulates metabolism and toxicity of Fuzi(lateral root of Aconitum carmichaeli Debx) in mice.

BACKGROUND: Therapeutic applications of Fuzi (lateral root of Aconitum carmichaeli Debx) are seriously concerned with its toxic effects. Strategies and approaches to reducing toxicity are of great interest. PURPOSE: We aimed to characterize the diurnal rhythm of Fuzi toxicity, and to determine the role of metabolism and pharmacokinetics in generating toxicity rhythmicity. METHODS: Toxicity was determined based on assessment of heart injury and animal survival after dosing mice with Fuzi decoction at different circadian time points. Circadian clock control of pharmacokinetics and toxicity was investigated using Bmal1-deficient (Bmal1-/-) mice. RESULTS: Fuzi exhibited a diurnal rhythmicity in cardiotoxicity (reflected by plasma CK-MB and LDH levels). The highest level of toxicity was observed at ZT10 (5 PM), while the lowest level of toxicity occurred at ZT22 (5 AM). Also, a higher mortality rate was observed at ZT10 and lower mortality rates at other times of the day. ZT10 dosing of Fuzi generated higher systemic exposures of three toxic alkaloid ingredients aconitine (AC), hypaconitine (HA) and mesaconitine (MA) compared to ZT22. This was accompanied by reduced the formation of the metabolites (N-deethyl-AC, didemethyl-HA and 2­hydroxyl­MA) at ZT10. Bmal1 ablation resulted in an increased level of Fuzi toxicity at ZT22, while having no influences when drug was dosed at ZT10. As a consequence, circadian time-dependent toxicity of Fuzi was lost in Bmal1-deficient mice. In addition, Bmal1 ablation increased the plasma concentrations of AC, HA and MA in mice after oral gavage of Fuzi, and reduced formation of their metabolites (N-deethyl-AC, didemethyl-HA and 2­hydroxyl­MA). Moreover, Fuzi metabolism in wild-type liver microsomes was more extensive at ZT22 than at ZT10. Bmal1 ablation abrogated circadian time-dependency of hepatic Fuzi metabolism. CONCLUSIONS: Fuzi chronotoxicity in mice was attributed to time-varying hepatic metabolism and systemic exposure regulated by circadian clock. The findings may have implications in reducing Fuzi toxicity with a chronotherapeutic approach.