We previously identified spinophilin as a regulator of α₂ adrenergic receptor (α₂AR) trafficking and signaling in vitro and in vivo (Science304:1940–1944, 2004). To assess the generalized role of spinophilin in regulating α₂AR functions in vivo, the present study examined the impact of eliminating spinophilin on α₂AR-evoked cardiovascular and hypnotic responses, previously demonstrated to be mediated by the α_2AAR subtype, after systemic administration of the α₂-agonists 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304) and clonidine in spinophilin-null mice. Mice lacking spinophilin expression display dramatically enhanced and prolonged hypotensive, bradycardic, and sedative-hypnotic responses to α₂AR stimulation. Whereas these changes in sensitivity to α₂AR agonists occur independent of any changes in α_2AAR density or intrinsic affinity for agonist in the brains of spinophilin-null mice compared with wild-type control mice, the coupling of the α_2AAR to cognate G proteins is enhanced in spinophilin-null mice. Thus, brain preparations from spinophilin-null mice demonstrate enhanced guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of α_2AAR in the guanine-nucleotide-sensitive higher affinity state compared with those from wild-type mice. These findings suggest that eliminating spinophilin expression in native tissues leads to an enhanced receptor/G protein coupling efficiency that contributes to sensitization of receptor mediated responses in vivo.

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder caused by loss-of-function variants in the NF1 gene. As of 20 November 2023, over 5000 distinct pathogenic or likely pathogenic variants have been reported in public databases. However, only a few NF1 genotype–phenotype correlations have been established so far. In this study, we present findings on 40 individuals with NF1, comprising 26 unrelated probands and 14 affected relatives, who carry one of nine NF1 heterozygous pathogenic splicing variants, all of which result in the in-frame skipping of exon 24 [19a] (NM_000267.3:r.3114_3197del, p.Asn1039_Arg1066del). These variants include c.3114-2A>G, c.3114-1G>A, c.3196A>G, c.3197G>A, c.3197G>T, c.3197+1G>A, c.3197+1G>T, c.3197+2T>C, and c.3197+3A>T. Among individuals with these variants, none exhibit externally visible plexiform neurofibromas, histopathologically confirmed cutaneous or subcutaneous neurofibromas, symptomatic spinal neurofibromas, or symptomatic optic pathway gliomas. The most prevalent, and sometimes sole, clinical feature observed in this cohort is multiple café-au-lait macules, with or without skinfold freckles: 85% and 60.5% of the individuals display six or more café-au-lait macules and freckles, respectively. In comparison to established NF1 genotype–phenotype correlations, these patients demonstrate highly similar clinical presentations to those associated with the NF1 pathogenic variant c.2970_2972del (p.Met992del), known for resulting in the mildest clinical features. Despite the generally mild phenotype, cognitive impairment, developmental delay, and/or learning difficulties are still observed in 33.3% of these patients, suggesting that learning challenges remain a prominent aspect of the phenotypic presentation in these individuals and necessitate specialized care. This newly established genotype–phenotype correlation will assist clinicians in improving the management of patients harboring NF1 exon 24 [19a] skipping variants and provide a new therapeutic target for NF1 treatment.

Epileptic seizures are common sequelae of stroke, acute brain injury, and chronic neurodegenerative diseases, including Alzheimer’s disease (AD), and cannot be effectively controlled in approximately 40% of patients, necessitating the development of novel therapeutic agents. Activation of the A1 receptor (A1R) by endogenous adenosine is an intrinsic mechanism to self-terminate seizures and protect neurons from excitotoxicity. However, targeting A1R for neurological disorders has been hindered by side effects associated with its broad expression outside the nervous system. Here we aim to target the neural-specific A1R/neurabin/regulator of G protein signaling 4 (A1R/neurabin/RGS4) complex that dictates A1R signaling strength and response outcome in the brain. We developed a peptide that blocks the A1R-neurabin interaction to enhance A1R activity. Intracerebroventricular or i.n. administration of this peptide shows marked protection against kainate-induced seizures and neuronal death. Furthermore, in an AD mouse model with spontaneous seizures, nasal delivery of this blocking peptide reduces epileptic spike frequency. Significantly, the anticonvulsant and neuroprotective effects of this peptide are achieved through enhanced A1R function in response to endogenous adenosine in the brain, thus, avoiding side effects associated with A1R activation in peripheral tissues and organs. Our study informs potentially new anti-seizure therapy applicable to epilepsy and other neurological illness with comorbid seizures.

Background: Expression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as these domains/fragments. Furthermore, a HTP cloning pipeline incorporated with bioinformatics domain/fragment selection methods will be beneficial to studies of structure and function genomics/proteomics. Results: With bioinformatics tools, we developed a domain/domain boundary prediction (DDBP) method, which was trained by available experimental data. Combined with an improved cloning strategy, DDBP had been applied to 57 proteins from C. elegans. Expression and purification results showed there was a 10-fold increase in terms of obtaining purified proteins. Based on the DDBP method, the improved GATEWAY cloning strategy and a robotic platform, we constructed a high throughput (HTP) cloning pipeline, including PCR primer design, PCR, BP reaction, transformation, plating, colony picking and entry clones extraction, which have been successfully applied to 90 C. elegans genes, 88 Brucella genes, and 188 human genes. More than 97% of the targeted genes were obtained as entry clones. This pipeline has a modular design and can adopt different operations for a variety of cloning/expression strategies. Conclusion: The DDBP method and improved cloning strategy were satisfactory. The cloning pipeline, combined with our recombinant protein HTP expression pipeline and the crystal screening robots, constitutes a complete platform for structure genomics/proteomics. This platform will increase the success rate of purification and crystallization dramatically and promote the further advancement of structure genomics/proteomics.

This series reports 3 unrelated individuals with features of neurofibromatosis type 1 (NF1) and/or schwannomatosis (SWN) in whom variants of uncertain significance (VUSs) were resolved through RNA testing and tissue analysis, highlighting the clinical impact of these tests. Each patient's genetic testing identified a VUS that could have explained their clinical presentation. Because diagnostic criteria were not met, we performed VUS resolution. For patient 1, we conducted tissue analysis and RNA-based Sanger sequencing. For patients 2 and 3, RNA-based Sanger sequencing was pursued. Tissue analysis and RNA testing reclassified the NF1 c.8050 + 93A>T VUS in patient 1 as pathogenic, confirming diagnosis of NF1. RNA testing of the NF2 c.448-5T>C VUS in patient 2 reclassified the variant as likely benign, ruling out diagnosis of germline neurofibromatosis type 2 (NF2). RNA testing of the SPRED1 c.377-13T>A VUS in patient 3 reclassified the variant as likely benign, ruling out diagnosis of germline Legius syndrome. Because diagnostic criteria for NF1 and SWN include genetic testing, resolving VUSs in those who do not meet criteria is crucial for clinical care. In this article, we report 3 cases in which RNA testing and tissue analysis clarified pathogenicity of VUSs, thereby affecting clinical care, and in one case providing a definitive diagnosis.