Serotonin synthesis in mammals is initiated by 2 distinct tryptophan hydroxylases (TPH), TPH1 and TPH2. By genetically ablating TPH2, we created mice (Tph2 ؊/؊ ) that lack serotonin in the central nervous system. Surprisingly, these mice can be born and survive until adulthood. However, depletion of serotonin signaling in the brain leads to growth retardation and 50% lethality in the first 4 weeks of postnatal life. Telemetric monitoring revealed more extended daytime sleep, suppressed respiration, altered body temperature control, and decreased blood pressure (BP) and heart rate (HR) during nighttime in Tph2 ؊/؊ mice. Moreover, Tph2 ؊/؊ females, despite being fertile and producing milk, exhibit impaired maternal care leading to poor survival of their pups. These data confirm that the majority of central serotonin is generated by TPH2. TPH2-derived serotonin is involved in the regulation of behavior and autonomic pathways but is not essential for adult life.growth retardation ͉ maternal care ͉ respiration ͉ serotonin ͉ sleep S erotonin (5-hydroxytryptamine, 5-HT) is an extracellular signaling molecule with a multitude of functions in the central nervous system (CNS) and in the periphery. 5-HT effects are conveyed by at least 13 receptors classified in 7 families, 5-HT1 to 5-HT7. Serotonin synthesis from tryptophan is initiated by the enzyme tryptophan hydroxylase (TPH) generating 5-hydroxytryptophan followed by aromatic amino acid decarboxylase (AADC), which produces 5-HT. We have recently discovered that 2 TPH isoenzymes exist in all vertebrates, TPH1 and TPH2, encoded by 2 distinct genes (1, 2). Tph1 is mainly expressed in the gut, generating serotonin that is distributed into the whole body by thrombocytes, and in the pineal gland, where the resulting 5-HT is metabolized to melatonin. The Tph1-deficient mice generated by us (2) and others (3, 4) revealed that 95% of peripheral 5-HT is produced by TPH1. They also revealed that 5-HT in platelets and other peripheral cells is involved in such diverse processes as thrombosis (5), liver regeneration (6), hepatitis (7), colon cancer (8), mammary gland plasticity (9), pulmonary hypertension (10), and bone formation (11). TPH2, on the other hand, is responsible for the synthesis of serotonin in the raphé nuclei of the brainstem, from where all central serotonergic projections originate (12). Accordingly, polymorphisms and functional mutations in the human and mouse genes for this enzyme have been linked to neurological and behavioral abnormalities (13)(14)(15)(16).In this study, we generated mice lacking TPH2 by gene targeting and analyzed the physiological consequences resulting from a lack of brain serotonin. Results and DiscussionGeneration and Basic Characteristics of Tph2-Deficient Mice. Tph2-deficient (Tph2 Ϫ/Ϫ ) mice were generated by deleting the coding sequence in exons 1 and 2 (supporting information (SI) Fig. S1 A and B). In the resulting Tph2 Ϫ/Ϫ mice, no Tph2 mRNA could be found by RT-PCR (Fig. S1C) and in situ hybridization in the brain (Fig. 1A). Immu...

Background-Hypertensive target organ damage, especially cardiac hypertrophy with heart failure and arrhythmia, is a major source of morbidity and mortality. Angiotensin II, a major mediator of hypertension and cardiac damage, has proinflammatory properties. Inflammation and activation of the immune system play a pivotal role in pathogenesis of hypertensive target organ damage. However, the role of immunosuppressive CD4

The prorenin receptor (PRR) is highly expressed in podocytes, but its role in the maintenance of podocyte function is unknown. Here we generated podocyte-specific PRR-knockout mice and found that these animals died between 2 to 3 wk after birth. Within 14 d, PRR-knockout mice developed nephrotic syndrome, albuminuria with podocyte foot-process fusion, and cytoskeletal changes. Podocyte-specific PRR deletion also led to disturbed processing of multivesicular bodies and enrichment of autophagosomal (LC3) and lysosomal (LAMP2) markers, indicating a functional block in autophagosome-lysosome fusion and an overload of the proteasomal protein-degradation machinery. In vitro, PRR knockdown and pharmacologic blockade of vacuolar H ϩ -ATPases, which associate with the PRR, increased vesicular pH, led to accumulation of LC3-positive and LAMP2-positive vesicles and altered the cytoskeleton. Taken together, these results suggest that the PRR is essential for podocyte function and survival by maintaining autophagy and protein-turnover machinery. Furthermore, PRR contributes to the control of lysosomal pH, which is important for podocyte survival and cytoskeletal integrity.

Abstract-The renin-angiotensin system (RAS) participates in preeclampsia; however, the relative contributions from the circulating RAS and the tissue-based, uteroplacental RAS are unknown. We hypothesized that the tissue-based uteroplacental RAS is dysregulated in preeclampsia. We performed microarray and gene expression studies and confirmed the findings on the protein level by immunohistochemistry in ureteroplacental units from 10 preeclamptic women and 10 women with uneventful pregnancies. All of the women were delivered by cesarean section. We also analyzed plasma renin activity and circulating agonistic angiotensin II type 1 (AT 1 ) receptor autoantibodies. In preeclampsia, we found that the angiotensin II AT 1 receptor gene was 5-fold upregulated in decidua (maternal origin). We also found AT 1 autoantibodies in preeclamptic women and in their offspring by neonatal cardiomyocyte bioassay compared with women with normal pregnancies and their infants (mother: 17.5Ϯ2.2 versus 0.05Ϯ0.4; fetus: 14.5Ϯ1.8 versus 0.5Ϯ0.5 ⌬bpm). Gene expressions for renin (35.0-fold), angiotensin-converting enzyme (2.9-fold), and angiotensinogen (8.9-fold) were higher in decidua than placenta (fetal origin) in both control and preeclamptic women, whereas the AT 1 receptor was expressed 10-fold higher in placenta than in decidua in both groups. Our findings elucidate the ureteroplacental unit RAS in preeclamptic and normal pregnancies. We found that, in preeclampsia, the AT 1 receptor expression is particularly high in decidua, combined with pregnancy-specific tissue RAS involving decidual angiotensin II production and AT 1 autoantibodies. We also showed that AT 1 autoantibodies cross the ureteroplacental barrier. These components could participate in the pathophysiology of preeclampsia.

Receptor-activator of NF-kappaB ligand (TNFSF11, also known as RANKL, OPGL, TRANCE and ODF) and its tumour necrosis factor (TNF)-family receptor RANK are essential regulators of bone remodelling, lymph node organogenesis and formation of a lactating mammary gland. RANKL and RANK are also expressed in the central nervous system. However, the functional relevance of RANKL/RANK in the brain was entirely unknown. Here we report that RANKL and RANK have an essential role in the brain. In both mice and rats, central RANKL injections trigger severe fever. Using tissue-specific Nestin-Cre and GFAP-Cre rank(floxed) deleter mice, the function of RANK in the fever response was genetically mapped to astrocytes. Importantly, Nestin-Cre and GFAP-Cre rank(floxed) deleter mice are resistant to lipopolysaccharide-induced fever as well as fever in response to the key inflammatory cytokines IL-1beta and TNFalpha. Mechanistically, RANKL activates brain regions involved in thermoregulation and induces fever via the COX2-PGE(2)/EP3R pathway. Moreover, female Nestin-Cre and GFAP-Cre rank(floxed) mice exhibit increased basal body temperatures, suggesting that RANKL and RANK control thermoregulation during normal female physiology. We also show that two children with RANK mutations exhibit impaired fever during pneumonia. These data identify an entirely novel and unexpected function for the key osteoclast differentiation factors RANKL/RANK in female thermoregulation and the central fever response in inflammation.

The role of the hypoxia-inducible factor (HIF) subunits 1alpha and 1beta in cellular response to hypoxia is well established, whereas little is known about HIF-2alpha and HIF-3alpha with respect to organ distribution and transcriptional regulation by hypoxia. We investigated mRNA levels of all HIF subunits and of their target genes erythropoietin (EPO) and glucose-transporter 1 (GLUT1) in rats undergoing systemic hypoxia for 30 or 120 min by quantitative real-time RT-PCR. In normoxia, persistently high mRNA levels of all HIF subunits were detected in cerebral cortex, hippocampus, and lung; the heart contained the lowest amounts. Hypoxia did not affect mRNA levels of HIF-1alpha, -1beta, and -2alpha. HIF-3alpha mRNA levels increased in all organs examined after 2 h of hypoxia. A significant rise of EPO and GLUT1 mRNA levels occurred in cortex, heart, liver, and kidney after 2 h of hypoxia, indicating activation of the HIF system. Protein levels of all HIF subunits, determined in brain and lung by immunoblotting, showed a marked increase corresponding to the duration of hypoxia. Our results suggest that induction at the transcriptional level is a unique feature of HIF-3alpha, which therefore may represent a rapidly reacting component of the HIF system in protection against hypoxic damage.

Abstract-Angiotensin (Ang) II induces vascular injury in part by activating innate and adaptive immunity; however, the mechanisms are unclear. We investigated the role of interferon (IFN

All Mendelian hypertension syndromes described to date involve increased sodium reabsorption in the distal nephron. 5 The sole exception is autosomal-dominant hypertension with BDE (HTNB, OMIM #112410), first reported in a Turkish kindred. 2,6 HTNB was linked to chromosome 12p in six unrelated families. 2,7,8 The locus accounts for a ~50 mm Hg mean blood pressure difference at age 50 years. 2 The penetrance is 100% (Fig. 1a). Previously, we reported a rearrangement on chromosome 12p common to all families. 8,9 A linkage study in Chinese hypertensive families without BDE coincided with the HTNB locus, supporting relevance to essential hypertension. 10 Whole-genome sequencing of Turkish family members revealed a heterozygous missense mutation in PDE3A (Gene ID: 5139), a gene encoding a cGMP/cAMP phosphodiesterase with a prominent role in the heart, VSMC, oocytes and platelets. 11 Resequencing of all 48 affected persons in six unrelated families identified six independently clustered heterozygous missense mutations in exon 4 (Fig. 1a, b Supplementary Fig. 1).We detected none of the previously described chromosomal breakpoints on chromosome 12p12.2-12.1, perhaps due to high repetitive content in the breakpoint regions Fig. 2a-c). 4 A haplotype analysis identified a novel recombination that reduced the linkage interval and eliminated an inversion common to all affected individuals in the six families (Fig. 2c). 9 In contrast, the affected mother's haplotype showed co-segregation with the more severe brachydactyly phenotype.PDEs are involved during early stages of osteogenesis. 12 PDE4D mutations have been associated with severe brachydactyly in acrodysostosis. 13,14 In mice, Pde3a was expressed in the developing limbs, consistent with a role during chondrogenesis (Fig. 2d, Supplementary Fig. 3a, b). Chondrogenic downregulation of PTHLH encoding PTHrP was associated with BDE. 15 We also observed PTHLH downregulation in chondrogenically induced fibroblasts from affected persons (Fig. 2e, Supplementary Fig. 3c).We addressed the functional consequences of the identified PDE3A mutations in HeLa cells expressing the six mutations. Forskolin or L-arginine stimulated the adenylate or guanylate cyclases to enhance cellular cAMP or cGMP levels, respectively. 16,17 We detected significantly reduced cAMP levels, consistent with gain-of-function mutations with no change in cGMP levels for the PDE3A mutations ( Supplementary Fig. 4a, b). Three PDE3A isoforms, PDE3A1 (microsomal), PDE3A2 and PDE3A3 (microsomal and cytosolic), have been identified in human myocardium. 18,19 PDE3A3 does not contain the sequence harboring the detected mutations. The predominant isoform in VSMC is PDE3A2. 18,20 To directly elucidate the mutations' effects, we compared the Michaelis-Menten kinetics of cAMPhydrolytic activity for recombinant T445N FLAG-tagged PDE3A1 and PDE3A1-WT and the tagged A2 isoforms purified from transfected cells (Fig. 3a, b, Supplementary Fig. 4d-k). The T445N mutation increased the affinity of both enzyme's isoforms for cAM...