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Neurofibromatosis type 1 results from loss-of-function NF1 pathogenic variants (PVs). Up to 30% of all NF1 PVs disrupt mRNA splicing, including deep intronic variants. Here, we retrospectively investigated the spectrum of NF1 deep intronic PVs in a cohort of 8,090 unrelated individuals from the University of Alabama at Birmingham (UAB) dataset with a molecularly confirmed neurofibromatosis type 1. All variants were identified through their effect on the NF1 transcript, followed by variant characterization at the DNA-level. A total of 68 distinct variants, which were ≥ 20 nucleotides away from the closest exon–intron junction, were identified in 2.5% unrelated individuals with NF1 (200/8,090). Nine different pathogenic splice variants, identified in 20 probands, led to exonization of different parts of intron 30 [23.2] or 31 [23a]. The two major NF1 transcript isoforms, distinguished by the absence (type I) or presence (type II) of the alternatively spliced cassette exon 31 [23a], are equally expressed in blood in control individuals without NF1 or NF1-affected individuals carrying their PV not in the introns flanking exon 31 [23a]. By fragment and cloning analysis we demonstrated that the exonization of intron 31 [23a] sequences due to deep intronic PV predominantly affects the NF1 isoform II. Seven additional (likely) pathogenic NF1 deep intronic variants not observed in the UAB dataset were found by classification of 36 variants identified by a literature search. Hence, the unique list of these 75 deep intronic (likely) PVs should be included in any comprehensive NF1 testing strategy.

Art exhibitions are for promoting social aesthetic education. As the information carrier of exhibition culture, exhibition materials play a role in the dissemination of aesthetic education between the tangible and the intangible. Based on the long-life design philosophy, the textual research continued the exhibition’s lifecycle through innovative creative design after the exhibition. Reconstructing the design symbols of cultural derivatives guided the development of cultural derivative design experiments with PVC inkjet cloth as the object. The evolution from “waste product” to “product” and then to “commodity” was completed, which opened up an innovative path for exhibition culture to further enhance social aesthetic education, while also broadening the opportunities for researchers engaged in long-life design and green design.

Purpose Biallelic germline mismatch repair (MMR) gene pathogenic variants (PVs) cause constitutional MMR deficiency (CMMRD), a highly penetrant childhood cancer syndrome phenotypically overlapping with neurofibromatosis type 1 (NF1). CMMRD testing in suspected NF1 children without NF1/SPRED1 PVs enables inclusion of CMMRD positives into monitoring programs prior to tumor onset. However, testing is associated with potential harms and the prevalence of CMMRD among these children is unknown. Methods Using a simple and scalable microsatellite instability (MSI) assay of non-neoplastic leukocyte DNA to detect CMMRD, we retrospectively screened >700 children suspected of sporadic NF1 but lacking NF1/SPRED1 PVs. Results For three of seven MSI-positive patients germline MMR gene PVs confirmed the diagnosis of CMMRD. Founder variants NM_000535.5(PMS2):c.736_741delinsTGTGTGTGAAG, prevalent in Europe and North America, and NM_000179.2(MSH6):c.10C>G, affecting 1:400 French Canadians, represented two of five PVs. The prevalence of CMMRD was 3/735 (0.41%, 95% confidence interval [CI]: 0.08–1.19%). Conclusion Our empirical data provide reliable numbers for genetic counseling and confirm previous prevalence estimations, on which Care for CMMRD consortium guidelines are based. These advocate CMMRD testing of preselected patients rather than offering reflex testing to all suspected sporadic NF1 children lacking NF1/SPRED1 PVs. The possibility of founder effects should be considered alongside these testing guidelines.

Hidradenitis Suppurativa (HS) is a proinflammatory painful skin disorder. This chronic disease is often associated with aggressive squamous cell carcinoma (SCC). The molecular pathogenesis of this disease progression remains undefined. The translation initiation factor eIF4E/4G/4A1 complex is overexpressed in a variety of human malignancies. In this study, we found that the expression of eIF4E/4G/4A1 as well as phosphorylated eIF4E were upregulated in HS skin. In the global transcription profiles derived from two public database, we were able to enrich 734 eIF4F-related genes. GSEA pathway enrichment analysis further demonstrated that RAS/MEK/ERK oncogene signaling pathway associated with inflammation signaling were significantly activated in HS lesion. The increase expression of eIF4 protein components was associated with enhanced eIF4E translation targets Cyclin D1 and c-Myc. Confocal fluorescence microscopy analysis further revealed that Cyclin D1 and c-Myc specifically co-localized in nuclei of certain cells in HS epithelium. We also found that many of the PCNA positive hyperproliferative cells were also positive for c-Myc expression. These data demonstrate that 5’-cap□dependent translation is a potential pathway underlying the SCC pathogenesis in chronic HS lesions. Furthermore, being a druggable target, inhibition of eIF4F may block lesion-associated lethal SCCs in HS patients.

Constitutional LZTR1 or SMARCB1 pathogenic variants (PVs) have been found in ∼86% of familial and ∼40% of sporadic schwannomatosis cases. Hence, we performed massively parallel sequencing of the entire LZTR1, SMARCB1, and NF2 genomic loci in 35 individuals with schwannomas negative for constitutional first-hit PVs in the LZTR1/SMARCB1/NF2 coding sequences; however, with 22q deletion and/or a different NF2 PV in each tumor, including six cases with only one tumor available. Furthermore, we verified whether any other LZTR1/SMARCB1/NF2 (likely) PVs could be found in 16 cases carrying a SMARCB1 constitutional variant in the 3'-untranslated region (3'-UTR) c.*17C>T, c.*70C>T, or c.*82C>T. As no additional variants were found, functional studies were performed to clarify the effect of these 3'-UTR variants on the transcript. The 3'-UTR variants c.*17C>T and c.*82C>T showed pathogenicity by negatively affecting the SMARCB1 transcript level. Two novel deep intronic SMARCB1 variants, c.500+883T>G and c.500+887G>A, resulting in out-of-frame missplicing of intron 4, were identified in two unrelated individuals. Further resequencing of the entire repeat-masked genomics sequences of chromosome 22q in individuals negative for PVs in the SMARCB1/LZTR1/NF2 coding- and noncoding regions revealed five potential schwannomatosis-predisposing candidate genes, that is, MYO18B, NEFH, SGSM1, SGSM3, and SBF1, pending further verification.

We report 281 individuals carrying a pathogenic recurrent NF1 missense variant at p.Met1149, p.Arg1276, or p.Lys1423, representing three nontruncating NF1 hotspots in the University of Alabama at Birmingham (UAB) cohort, together identified in 1.8% of unrelated NF1 individuals. About 25% (95% confidence interval: 20.5-31.2%) of individuals heterozygous for a pathogenic NF1 p.Met1149, p.Arg1276, or p.Lys1423 missense variant had a Noonan-like phenotype, which is significantly more compared with the "classic" NF1-affected cohorts (all p < .0001). Furthermore, p.Arg1276 and p.Lys1423 pathogenic missense variants were associated with a high prevalence of cardiovascular abnormalities, including pulmonic stenosis (all p < .0001), while p.Arg1276 variants had a high prevalence of symptomatic spinal neurofibromas (p < .0001) compared with "classic" NF1-affected cohorts. However, p.Met1149-positive individuals had a mild phenotype, characterized mainly by pigmentary manifestations without externally visible plexiform neurofibromas, symptomatic spinal neurofibromas or symptomatic optic pathway gliomas. As up to 0.4% of unrelated individuals in the UAB cohort carries a p.Met1149 missense variant, this finding will contribute to more accurate stratification of a significant number of NF1 individuals. Although clinically relevant genotype-phenotype correlations are rare in NF1, each affecting only a small percentage of individuals, together they impact counseling and management of a significant number of the NF1 population.

Posttraumatic stress disorder (PTSD) after the pandemic has emerged as a major neuropsychiatric component of post-acute COVID-19 syndrome, yet the current pharmacotherapy for PTSD is limited. The use of adrenergic drugs to treat PTSD has been suggested; however, it is hindered by conflicting clinical results and a lack of mechanistic understanding of drug actions. Our studies, using both genetically modified mice and human induced pluripotent stem cell-derived neurons, reveal a novel α

The adenosine A1 receptor (A1R) is a key mediator of the neuroprotective effect by endogenous adenosine. Yet targeting this receptor for neuroprotection is challenging due to its broad expression throughout the body. A mechanistic understanding of the regulation of A1R signaling is necessary for the future design of therapeutic agents that can selectively enhance A1R-mediated responses in the nervous system. In this study, we demonstrate that A1R activation leads to a sustained localization of regulator of G protein signaling 4 (RGS4) at the plasma membrane, a process that requires neurabin (a neural tissue-specific protein). A1R and RGS4 interact with the overlapping regions of neurabin. In addition, neurabin domains required for oligomerization are essential for formation of the A1R/neurabin/RGS4 ternary complex, as well as for stable localization of RGS4 at the plasma membrane and attenuation of A1R signaling. Thus, A1R and RGS4 each likely interact with one neurabin molecule in a neurabin homo-oligomer to form a ternary complex, representing a novel mode of regulation of G protein–coupled receptor signaling by scaffolding proteins. Our mechanistic analysis of neurabin-mediated regulation of A1R signaling in this study will be valuable for the future design of therapeutic agents that can selectively enhance A1R-mediated responses in the nervous system.

Alternative splicing (AS) is involved in cell fate decisions and embryonic development. However, regulation of these processes is poorly understood. Here, we have identified the serine threonine kinase receptor-associated protein (STRAP) as a putative spliceosome-associated factor. Upon Strap deletion, there are numerous AS events observed in mouse embryoid bodies (EBs) undergoing a neuroectoderm-like state. Global mapping of STRAP-RNA binding in mouse embryos by enhanced-CLIP sequencing (eCLIP-seq) reveals that STRAP preferably targets transcripts for nervous system development and regulates AS through preferred binding positions, as demonstrated for two neuronal-specific genes, Nnat and Mark3. We have found that STRAP involves in the assembly of 17S U2 snRNP proteins. Moreover, in Xenopus, loss of Strap leads to impeded lineage differentiation in embryos, delayed neural tube closure, and altered exon skipping. Collectively, our findings reveal a previously unknown function of STRAP in mediating the splicing networks of lineage commitment, alteration of which may be involved in early embryonic lethality in mice., STRAP (serine threonine kinase receptor-associated protein) promotes tumorigenicity. Here the authors report that STRAP associates with spliceosome and regulates alternative splicing during embryonic stem cell lineage commitment and early mouse embryo organogenesis.

The brain noradrenergic system is critical for normal cognition and is affected at early stages in Alzheimer's disease (AD). Here, we reveal a previously unappreciated direct role of norepinephrine signaling in connecting β-amyloid (Aβ) and tau, two key pathological components of AD pathogenesis. Our results show that Aβ oligomers bind to an allosteric site on α2A adrenergic receptor (α2AAR) to redirect norepinephrine-elicited signaling to glycogen synthase kinase 3β (GSK3β) activation and tau hyperphosphorylation. This norepinephrine-dependent mechanism sensitizes pathological GSK3β/tau activation in response to nanomolar accumulations of extracellular Aβ, which is 50- to 100-fold lower than the amount required to activate GSK3β by Aβ alone. The significance of our findings is supported by in vivo evidence in two mouse models, human tissue sample analysis, and longitudinal clinical data. Our study provides translational insights into mechanisms underlying Aβ proteotoxicity, which might have strong implications for the interpretation of Aβ clearance trial results and future drug design and for understanding the selective vulnerability of noradrenergic neurons in AD.

PURPOSE: Neurofibromatosis type 1 (NF1) is characterized by a highly variable clinical presentation, but almost all NF1-affected adults present with cutaneous and/or subcutaneous neurofibromas. Exceptions are individuals heterozygous for the NF1 in-frame deletion, c.2970_2972del (p.Met992del), associated with a mild phenotype without any externally visible tumors. METHODS: A total of 135 individuals from 103 unrelated families, all carrying the constitutional NF1 p.Met992del pathogenic variant and clinically assessed using the same standardized phenotypic checklist form, were included in this study. RESULTS: None of the individuals had externally visible plexiform or histopathologically confirmed cutaneous or subcutaneous neurofibromas. We did not identify any complications, such as symptomatic optic pathway gliomas (OPGs) or symptomatic spinal neurofibromas; however, 4.8% of individuals had nonoptic brain tumors, mostly low-grade and asymptomatic, and 38.8% had cognitive impairment/learning disabilities. In an individual with the NF1 constitutional c.2970_2972del and three astrocytomas, we provided proof that all were NF1-associated tumors given loss of heterozygosity at three intragenic NF1 microsatellite markers and c.2970_2972del. CONCLUSION: We demonstrate that individuals with the NF1 p.Met992del pathogenic variant have a mild NF1 phenotype lacking clinically suspected plexiform, cutaneous, or subcutaneous neurofibromas. However, learning difficulties are clearly part of the phenotypic presentation in these individuals and will require specialized care. ispartof: GENETICS IN MEDICINE vol:21 issue:4 pages:867-876 ispartof: location:United States status: published

G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α, β, and γ subunits, with 16 Gα, 5 Gβ and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gβγs activated by a given GPCR and that activate each effector in vivo is not known. Here, we examined the in vivo Gβγ specificity of presynaptic α2a-adrenergic receptors (α2aARs) in both adrenergic (auto-α2aARs) and non-adrenergic neurons (hetero-α2aARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits, and co-immunoprecipitation of tagged α2aARs from mouse models including transgenic FLAG-α2aARs and knock-in HA-α2aARs, we investigated the in vivo specificity of Gβ and Gγ subunits to auto-α2aARs and hetero-α2aARs activated with epinephrine to understand the role of Gβγ specificity in diverse physiological functions such as anesthetic sparing, and working memory enhancement. We detected Gβ2, Gγ2, Gγ3, and Gγ4 with activated auto α2aARs, whereas we found Gβ4 and Gγ12 preferentially interacted with activated hetero-α2aARs. Further understanding of in vivo Gβγ specificity to various GPCRs offers new insights into the multiplicity of genes for Gβ and Gγ, and the mechanisms underlying GPCR signaling through Gβγ subunits.

Background Teeth treated endodontically are more susceptible to vertical root fracture (VRF). Some studies have suggested that obturating the root canals with Gutta-percha or Resilon can reinforce endodontically treated teeth, but a few others have presented conflicting results. These inconsistent results cannot guide clinicians in determining clinical approaches. The objective of this meta-analysis is to evaluate and compare the vertical fracture resistance of endodontically treated root canals obturated with Gutta-percha/AH plus and the Resilon system. Methods Comprehensive literature searches were performed in the PubMed, Cochrane Library, ScienceDirect, Web of Science and Embase databases. The titles and abstracts of all of the retrieved articles were independently assessed by two authors according to predefined selection criteria. Data in the included articles were independently extracted. Statistical analyses were conducted using Review Manager 5.3 and Stata 12.0 software. The pooled standardized mean differences (SMDs) with 95% confidence intervals (CIs) were calculated for the outcome indicators. The level of statistical significance was set at p

The underlying defect in cystic fibrosis is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed at the apical surface of lung epithelia. In addition to its export and maintenance at the cell surface, CFTR regulation involves repeated cycles of transport through the endosomal trafficking system, including endocytosis and recycling. Many of the known disease mutations cause CFTR intracellular trafficking defects that result in failure of ion channel delivery to the apical plasma membrane. Corrective maneuvers directed at improving transport to the plasma membrane are thwarted by rapid internalization and degradation of the mutant CFTR proteins. The molecular mechanisms involved in these processes are not completely understood but may involve protein-protein interactions with the C-terminal type I PDZ-binding motif of CFTR. Using a proteomic approach, we identify sorting nexin 27 (SNX27) as a novel CFTR binding partner in human airway epithelial Calu-3 cells. SNX27 and CFTR interact directly, with the SNX27 PDZ domain being both necessary and sufficient for this interaction. SNX27 co-localizes with internalized CFTR at sub-apical endosomal sites in polarized Calu-3 cells, and either knockdown of the endogenous SNX27, or over-expression of a dominant-negative SNX27 mutant, resulted in significant decreases in cell surface CFTR levels. CFTR internalization was not affected by SNX27 knockdown, but defects were observed in the recycling arm of CFTR trafficking through the endosomal system. Furthermore, knockdown of SNX27 in Calu-3 cells resulted in significant decreases in CFTR protein levels, consistent with degradation of the internalized pool. These data identify SNX27 as a physiologically significant regulator of CFTR trafficking and homeostasis in epithelial cells.

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Neurofibromatosis type 1 (NF1), one of the most common genetic disorders with an estimated prevalence of 1:3000 live births, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and an in-frame 1-amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 patients (129 unrelated probands and 33 affected relatives) carrying a constitutional missense mutation affecting one of five neighboring NF1 codons Leu844, Cys845, Ala846, Leu847 and Gly848, located in the Cysteine-Serine-Rich Domain (CSRD). These recurrent missense mutations affect ~0.8% of unrelated NF1 mutation-positive probands in the UAB cohort. A substantial fraction of these patients presented with a severe phenotype, including plexiform and/or spinal neurofibromas, symptomatic optic pathway gliomas, malignant neoplasms or osseous lesions. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent compared with classic NF1 cohorts (both p

The amyloid precursor protein (APP) has long been appreciated for its role in Alzheimer's disease (AD) pathology. However, less is known about the physiologic function of APP outside of AD. Particularly, whether and how APP may regulate functions of cell surface receptors, including GPCRs, remains largely unclear. In this study, we identified a novel direct interaction between APP and the α2A-adrenergic receptor (α2AAR) that occurs at the intracellular domains of both proteins. The APP interaction with α2AAR is promoted by agonist stimulation and competes with arrestin 3 binding to the receptor. Consequently, the presence of APP attenuates α2AAR internalization and desensitization, which are arrestin-dependent processes. Furthermore, in neuroblastoma neuro-2A cells and primary superior cervical ganglion neurons, where APP is highly expressed, the lack of APP leads to a dramatic increase in plasma membrane recruitment of endogenous arrestin 3 following α2AAR activation. Concomitantly, agonist-induced internalization of α2AAR is significantly enhanced in these neuronal cells. Our study provided the first evidence that APP fine tunes GPCR signaling and trafficking. Given the important role of α2AAR in controlling norepinephrine release and response, this novel regulation of α2AAR by APP may have an impact on modulation of noradrenergic activity and sympathetic tone.-Zhang, F., Gannon, M., Chen, Y., Zhou, L., Jiao, K., Wang, Q. The amyloid precursor protein modulates α2A-adrenergic receptor endocytosis and signaling through disrupting arrestin 3 recruitment.

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Accumulation of amyloid β (Aβ) peptides in the brain is the key pathogenic factor driving Alzheimer's disease (AD). Endocytic sorting of amyloid precursor protein (APP) mediated by the vacuolar protein sorting (Vps10) family of receptors plays a decisive role in controlling the outcome of APP proteolytic processing and Aβ generation. Here we report for the first time to our knowledge that this process is regulated by a G protein-coupled receptor, the α(2A) adrenergic receptor (α(2A)AR). Genetic deficiency of the α(2A)AR significantly reduces, whereas stimulation of this receptor enhances, Aβ generation and AD-related pathology. Activation of α(2A)AR signaling disrupts APP interaction with a Vps10 family receptor, sorting-related receptor with A repeat (SorLA), in cells and in the mouse brain. As a consequence, activation of α(2A)AR reduces Golgi localization of APP and concurrently promotes APP distribution in endosomes and cleavage by β secretase. The α(2A)AR is a key component of the brain noradrenergic system. Profound noradrenergic dysfunction occurs consistently in patients at the early stages of AD. α(2A)AR-promoted Aβ generation provides a novel mechanism underlying the connection between noradrenergic dysfunction and AD. Our study also suggests α(2A)AR as a previously unappreciated therapeutic target for AD. Significantly, pharmacological blockade of the α(2A)AR by a clinically used antagonist reduces AD-related pathology and ameliorates cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α(2A)R antagonists would be an effective therapeutic strategy for AD.

Objective Autosomal dominant cortical myoclonus and epilepsy (ADCME) is characterized by distal, fairly rhythmic myoclonus and epilepsy with variable severity. We have previously mapped the disease locus on chromosome 2p11.1-q12.2 by genome-wide linkage analysis. Additional pedigrees affected by similar forms of epilepsy have been associated with chromosomes 8q, 5p, and 3q, but none of the causing genes has been identified. We aim to identify the mutant gene responsible for this form of epilepsy. Methods Genes included in the ADCME critical region were directly sequenced. Coimmunoprecipitation, immunofluorescent, and electrophysiologic approaches to transfected human cells have been utilized for testing the functional significance of the identified mutation. Results Here we show that mutation in the α2-adrenergic receptor subtype B (α2B-AR) is associated with ADCME by identifying a novel in-frame insertion/deletion in 2 Italian families. The mutation alters several conserved residues of the third intracellular loop, hampering neither the α2B-AR plasma membrane localization nor the arrestin-mediated internalization capacity, but altering the binding with the scaffolding protein spinophilin upon neurotransmitter activation. Spinophilin, in turn, regulates interaction of G protein coupled receptors with regulator of G protein signaling proteins. Accordingly, the mutant α2B-AR increases the epinephrine-stimulated calcium signaling. Interpretation The identified mutation is responsible for ADCME, as the loss of α2B-AR/spinophilin interaction causes a gain of function effect. This work implicates for the first time the α-adrenergic system in human epilepsy and opens new ways of understanding the molecular pathway of epileptogenesis, widening the spectrum of possible therapeutic targets. ANN NEUROL 2014;75:77–87

To establish the Chinese Han national criteria for the thickness of upper lip in Chongqing and to investigate the effect of lip thickness on lip retraction in orthodontic treatment. A total of 240 adult patients (male=120, female=120) of Chinese Han nationality aged 18-35 years in Chongqing were treated with cephalograms and measured the thickness of upper lip. We established the Chinese Han national criteria for the thickness of upper lip in Chongqing according to the results. Sixty-eight female patients with Angle II 1 from Department of Orthodontics Stomatological Hospital of Chongqing Medical University were treated with maxillary premolars extraction, and pre- and post-treated with lateral cephalograms, then divided into 3 groups according to our criteria: a thin lips group, a normal lips group and a thick lips group. The ratio of the incisors retraction and the upper lip retraction were measured and calculated. The correlation between incisors retraction and the upper lip retraction was analyzed by the Pearson correlation method. There was statistical significance in the thickness of upper lip between males and females (P0.01). The ratio of maxillary inscior retraction to the upper lip soft tissue retraction was 1.6:1, 2.2:1 and 2.9:1 in the thin lips group, the normal lips group and the thick lips group, respectively. Gender differences exist in the thickness of upper lip. Upper lip retraction with the incisors was negatively correlated with the soft tissue thickness of the upper lip in females with Angle II 1.目的：通过头颅侧位片对重庆地区汉族人群唇部厚度进行测量分析，建立重庆地区唇部厚度分类标准，同时探索唇部软组织厚度对唇部回收的影响及不同唇厚度的切牙上唇回收比。方法：招募240名18~35岁重庆地区汉族成年人，其中男女各120 名。每个样本拍摄头颅侧位片，测量唇部软组织厚度，并根据测量结果建立唇厚度分类标准，同时对男女唇厚度进行t检验。选取2009年1月至2012年12月就诊于重庆医科大学附属口腔医院的安氏II类I分类成年女性患者68例，术前术后均拍摄头颅侧位片，测量唇部软组织厚度并根据第一部分测量结果分为薄唇型、均衡型和厚唇型，求出各组切牙上唇回收比例。用Pearson相关分析法对唇厚度与切牙上唇回收比进行分析。结果：成年男性患者唇部厚度大于成年女性患者(P0.01)；安氏II类I分类成年女性患者切牙上唇回收比例在薄唇型为1.6:1，均衡型为2.2:1，厚唇型为2.9:1。结论：上唇软组织厚度存在性别差异；上唇软组织厚度对安氏II类I分类女性患者上唇回收有影响，上唇随切牙回收量与上唇软组织厚度呈负相关。.

The neurobiological mechanisms of action underlying antidepressant drugs remain poorly understood. Desipramine (DMI) is an antidepressant classically characterized as an inhibitor of norepinephrine reuptake. Available evidence, however, suggests a mechanism more complex than simple reuptake inhibition. In the present study, we have characterized the direct interaction between DMI and the α(2A)-adrenergic receptor (α(2A)AR), a key regulator of noradrenergic neurotransmission with altered expression and function in depression. DMI alone was found to be sufficient to drive receptor internalization acutely and a robust down-regulation of α(2A)AR expression and signaling following prolonged stimulation in vitro. These effects are achieved through arrestin-biased regulation of the receptor, as DMI selectively induces recruitment of arrestin but not activation of heterotrimeric G proteins. Meanwhile, a physiologically relevant concentration of endogenous agonist (norepinephrine) was unable to sustain a down-regulation response. Prolonged in vivo administration of DMI resulted in significant down-regulation of synaptic α(2A)AR expression, a response that was lost in arrestin3-null animals. We contend that direct DMI-driven arrestin-mediated α(2A)AR down-regulation accounts for the therapeutically desirable but mechanistically unexplained adaptive alterations in receptor expression associated with this antidepressant. Our results provide novel insight into both the pharmacology of this antidepressant drug and the targeting of the α(2A)AR in depression.

The genetic regulation necessary for the formation of a four-chambered heart is tightly regulated by transcription factors such as TBX20, a member of the T-box (TBX) transcription factor family. TBX20 is critical for proper cardiogenesis and is expressed in the heart throughout development. Missense mutations in TBX20 have been found in patients with congenital heart defects (CHD). Characterization of modifiers of TBX20 activity will help elucidate the genetic mechanisms of heart development and CHD. A yeast two-hybrid assay screening an embryonic mouse heart cDNA library with TBX20b as bait was used to identify potential modifiers of TBX20 activity and identified an interaction with muskelin (MKLN1), a primarily cytoplasmic protein with potential roles in signal transduction machinery scaffolding and nucleocytoplasmic protein shuttling. In cellular studies, MKLN1 directly binds to the T-box DNA-binding domain of only the TBX20b isoform by its kelch repeats domain. Immunostaining of mammalian cells transfected with tagged TBX20b and MKLN1 revealed colocalization primarily in the cytoplasm. Immunohistochemistry analysis of embryonic mouse hearts reveals coexpression in the developing endocardial valvular and myocardial interventricular cells. This novel interaction between TBX20b and MKLN1 may help elucidate new regulatory mechanisms within heart development.

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged alpha(2A)-adrenergic receptor (AR) expression was driven by the endogenous mouse alpha(2A)AR gene locus. Exploiting this mouse line, we evaluated alpha(2A)AR trafficking and alpha(2A)AR-mediated inhibition of Ca(2+) currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the alpha(2A)AR subtype. We discovered a more rapid desensitization of Ca(2+) current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of alpha(2A)AR evoked by clonidine than by guanfacine. Clonidine-induced alpha(2A)AR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of alpha(2A)AR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-alpha(2A)AR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.

Spinophilin plays critical roles in regulating trafficking and signaling of the alpha(2)-adrenergic receptor (AR) both in vitro and in vivo (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940-1944). In the present study, we demonstrate that protein kinase A (PKA) phosphorylation of spinophilin modulates the spinophilin-alpha(2A)AR interaction to regulate alpha(2A)AR internalization. Activation of PKA by forskolin abolishes the agonist-enhanced interaction between spinophilin and the alpha(2A)AR, and this event can be blocked by Ser --> Ala mutations at the PKA phosphorylation sites of spinophilin. In addition, a Ser --> Asp mutation that mimics the phosphorylated state at the PKA phosphorylation site Ser-177, which is located within the alpha(2A)AR binding region of spinophilin, is sufficient to block the spinophilin-alpha(2A)AR interaction in intact cells. In cells expressing mutant spinophilin carrying the S177D mutation, agonist-induced internalization of the alpha(2A)AR is accelerated and enhanced, as revealed by both intact cell enzyme-linked immunosorbent assay and quantitative immunofluorescent studies. Furthermore, activation of PKA by forskolin enhances agonist-induced internalization of the alpha(2A)AR in cells expressing wild type spinophilin, but not in cells lacking spinophilin or expressing the spinophilin mutant Sp177D. These results strongly support that PKA phosphorylation of spinophilin is functionally relevant in regulating alpha(2A)AR trafficking. Therefore, modulation of spinophilin-receptor interaction through phosphorylation of spinophilin may represent a novel mechanism whereby PKA regulates G protein-coupled receptor trafficking.

The brain noradrenergic system supplies the neurotransmitter norepinephrine throughout the brain via widespread efferent projections, and plays a pivotal role in modulating cognitive activities in the cortex. Profound noradrenergic degeneration in Alzheimer's disease (AD) patients has been observed for decades, with recent research suggesting that the locus coeruleus (where noradrenergic neurons are mainly located) is a predominant site where AD-related pathology begins. Mounting evidence indicates that the loss of noradrenergic innervation greatly exacerbates AD pathogenesis and progression, although the precise roles of noradrenergic components in AD pathogenesis remain unclear. The aim of this review is to summarize current findings on noradrenergic dysfunction in AD, as well as to point out deficiencies in our knowledge where more research is needed.

To increase the coverage of secreted protein prediction, we describe a combination strategy. Instead of using a single method, we combine Hidden Markov Model (HMM)-based methods CJ-SPHMM and TMHMM with PSORT in secreted protein prediction. CJ-SPHMM is an HMM-based signal peptide prediction method, while TMHMM is an HMM-based transmembrane (TM) protein prediction algorithm. With CJ-SPHMM and TMHMM, proteins with predicted signal peptide and without predicted TM regions are taken as putative secreted proteins. This HMM-based approach predicts secreted protein with Ac (Accuracy) at 0.82 and Cc (Correlation coefficient) at 0.75, which are similar to PSORT with Ac at 0.82 and Cc at 0.76. When we further complement the HMM-based method, i.e., CJ-SPHMM + TMHMM with PSORT in secreted protein prediction, the Ac value is increased to 0.86 and the Cc value is increased to 0.81. Taking this combination strategy to search putative secreted proteins from the International Protein Index (IPI) maintained at the European Bioinformatics Institute (EBI), we constructed a putative human secretome with 5235 proteins. The prediction system described here can also be applied to predicting secreted proteins from other vertebrate proteomes.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple cafe-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P, We thank the patients, their family and their referral physicians for the information. We thank the genetics counselor students who helped to re-contact all referral physicians for confirming information. This work was supported though the Children's Tumor Foundation by the Isaac and Sadie Fuchs Genotype-Phenotype Study (to L.M.) and by internal funds from the Medical Genomics Laboratory at UAB.

In women, arterial pressure generally increases after menopause, but several studies suggest that women who eat large amounts of plant estrogens (phytoestrogens) experience a slower rise in the incidence of postmenopausal hypertension. This suggests that both ovarian hormones (principally estrogen) and phytoestrogens may protect at least some women from hypertension. The present study tests the hypothesis that phytoestrogens blunt hypertension in estrogen-depleted female spontaneously hypertensive rats (SHR). Three-week-old ovariectomized SHR were fed one of four diets that contained basal (0.6%) or high (8%) NaCl with or without dietary phytoestrogens for 9 wk. In SHR on the basal NaCl diet, arterial pressure was unaffected by the removal of dietary phytoestrogens. In contrast, in SHR on the high-NaCl diet, arterial pressure was significantly higher in rats on the phytoestrogen-free (204 ± 4 mmHg) compared with the phytoestrogen-replete (153 ± 4 mmHg) diet. Ganglionic blockade resulted in reductions in arterial pressure that were directly related to the dietary NaCl-induced increases in arterial pressure. Together, these data indicate that dietary phytoestrogens protect ovariectomized female SHR from dietary NaCl-sensitive hypertension and that the sympathetic nervous system plays an important role in this effect. Furthermore, these results demonstrate that dietary phytoestrogens can have a major impact on the interpretation of studies into the physiological role of estrogen in females.

Haploinsufficiency for CHD7, an ATP-dependent nucleosome remodeling factor, is the leading cause of CHARGE syndrome. While congenital heart defects (CHDs) are major clinical features of CHARGE syndrome, affecting >75% of patients, it remains unclear whether CHD7 can directly regulate cardiogenic genes in embryos. Our complementary yeast two-hybrid and biochemical assays reveal that CHD7 is a novel interaction partner of canonical BMP signaling pathway nuclear mediators, SMAD1/5/8, in the embryonic heart. Moreover, CHD7 associates in a BMP-dependent manner with the enhancers of a critical cardiac transcription factor, Nkx2.5, that contain functional SMAD1-binding elements. Both the active epigenetic signature of Nkx2.5 regulatory elements and its proper expression in cardiomyocytes require CHD7. Finally, inactivation of Chd7 in mice impairs multiple BMP signaling-regulated cardiogenic processes. Our results thus support the model that CHD7 is recruited by SMAD1/5/8 to the enhancers of BMP-targeted cardiogenic genes to epigenetically regulate their expression. Impaired BMP activities in embryonic hearts may thus have a major contribution to CHDs in CHARGE syndrome.