A Genetically Defined Compartmentalized Striatal Direct Pathway for Negative Reinforcement.

The striosome compartment within the dorsal striatum has been implicated in reinforcement learning and regulation of motivation, but how striosomal neurons contribute to these functions remains elusive. Here, we show that a genetically identified striosomal population, which expresses the Teashirt family zinc finger 1 (Tshz1) and belongs to the direct pathway, drives negative reinforcement and is essential for aversive learning in mice. Contrasting a "conventional" striosomal direct pathway, the Tshz1 neurons cause aversion, movement suppression, and negative reinforcement once activated, and they receive a distinct set of synaptic inputs. These neurons are predominantly excited by punishment rather than reward and represent the anticipation of punishment or the motivation for avoidance. Furthermore, inhibiting these neurons impairs punishment-based learning without affecting reward learning or movement. These results establish a major role of striosomal neurons in behaviors reinforced by punishment and moreover uncover functions of the direct pathway unaccounted for in classic models.

Transcriptional Architecture of Synaptic Communication Delineates GABAergic Neuron Identity.

Understanding the organizational logic of neural circuits requires deciphering the biological basis of neuronal diversity and identity, but there is no consensus on how neuron types should be defined. We analyzed single-cell transcriptomes of a set of anatomically and physiologically characterized cortical GABAergic neurons and conducted a computational genomic screen for transcriptional profiles that distinguish them from one another. We discovered that cardinal GABAergic neuron types are delineated by a transcriptional architecture that encodes their synaptic communication patterns. This architecture comprises 6 categories of ∼40 gene families, including cell-adhesion molecules, transmitter-modulator receptors, ion channels, signaling proteins, neuropeptides and vesicular release components, and transcription factors. Combinatorial expression of select members across families shapes a multi-layered molecular scaffold along the cell membrane that may customize synaptic connectivity patterns and input-output signaling properties. This molecular genetic framework of neuronal identity integrates cell phenotypes along multiple axes and provides a foundation for discovering and classifying neuron types.

Brain-wide Maps Reveal Stereotyped Cell-Type-Based Cortical Architecture and Subcortical Sexual Dimorphism.

The stereotyped features of neuronal circuits are those most likely to explain the remarkable capacity of the brain to process information and govern behaviors, yet it has not been possible to comprehensively quantify neuronal distributions across animals or genders due to the size and complexity of the mammalian brain. Here we apply our quantitative brain-wide (qBrain) mapping platform to document the stereotyped distributions of mainly inhibitory cell types. We discover an unexpected cortical organizing principle: sensory-motor areas are dominated by output-modulating parvalbumin-positive interneurons, whereas association, including frontal, areas are dominated by input-modulating somatostatin-positive interneurons. Furthermore, we identify local cell type distributions with more cells in the female brain in 10 out of 11 sexually dimorphic subcortical areas, in contrast to the overall larger brains in males. The qBrain resource can be further mined to link stereotyped aspects of neuronal distributions to known and unknown functions of diverse brain regions.

The calcium-dependent protein kinase CPK16 regulates hypoxia-induced ROS production by phosphorylating the NADPH oxidase RBOHD in Arabidopsis.

Reactive oxygen species (ROS) production is a key event in modulating plant responses to hypoxia and post-hypoxia reoxygenation. However, the molecular mechanism by which hypoxia-associated ROS homeostasis is controlled remains largely unknown. Here, we showed that the calcium-dependent protein kinase CPK16 regulates plant hypoxia tolerance by phosphorylating the plasma membrane-anchored NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) to regulate ROS production in Arabidopsis (Arabidopsis thaliana). In response to hypoxia or reoxygenation, CPK16 was activated through phosphorylation of its Ser274 residue. The cpk16 knockout mutant displayed enhanced hypoxia tolerance, whereas CPK16-overexpressing (CPK16-OE) lines showed increased sensitivity to hypoxic stress. In agreement with these observations, hypoxia and reoxygenation both induced ROS accumulation in the rosettes of CPK16-OEs more strongly than in rosettes of the cpk16-1 mutant or the wild type. Moreover, CPK16 interacted with and phosphorylated the N terminus of RBOHD at four serine residues (Ser133, Ser148, Ser163, and Ser347) that were necessary for hypoxia- and reoxygenation-induced ROS accumulation. Furthermore, the hypoxia-tolerant phenotype of cpk16-1 was fully abolished in the cpk16 rbohd double mutant. Thus, we have uncovered a regulatory mechanism by which the CPK16-RBOHD module shapes ROS production during hypoxia and reoxygenation in Arabidopsis.

Genetic dissection of the glutamatergic neuron system in cerebral cortex.

Diverse types of glutamatergic pyramidal neurons mediate the myriad processing streams and output channels of the cerebral cortex1,2, yet all derive from neural progenitors of the embryonic dorsal telencephalon3,4. Here we establish genetic strategies and tools for dissecting and fate-mapping subpopulations of pyramidal neurons on the basis of their developmental and molecular programs. We leverage key transcription factors and effector genes to systematically target temporal patterning programs in progenitors and differentiation programs in postmitotic neurons. We generated over a dozen temporally inducible mouse Cre and Flp knock-in driver lines to enable the combinatorial targeting of major progenitor types and projection classes. Combinatorial strategies confer viral access to subsets of pyramidal neurons defined by developmental origin, marker expression, anatomical location and projection targets. These strategies establish an experimental framework for understanding the hierarchical organization and developmental trajectory of subpopulations of pyramidal neurons that assemble cortical processing networks and output channels.

RIPK1 activation in Mecp2-deficient microglia promotes inflammation and glutamate release in RTT.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder primarily caused by mutations in the methyl-CpG binding protein 2 (Mecp2) gene. Here, we found that inhibition of Receptor-Interacting Serine/Threonine-Protein Kinase 1 (RIPK1) kinase ameliorated progression of motor dysfunction after onset and prolonged the survival of Mecp2-null mice. Microglia were activated early in myeloid Mecp2-deficient mice, which was inhibited upon inactivation of RIPK1 kinase. RIPK1 inhibition in Mecp2-deficient microglia reduced oxidative stress, cytokines production and induction of SLC7A11, SLC38A1, and GLS, which mediate the release of glutamate. Mecp2-deficient microglia release high levels of glutamate to impair glutamate-mediated excitatory neurotransmission and promote increased levels of GluA1 and GluA2/3 proteins in vivo, which was reduced upon RIPK1 inhibition. Thus, activation of RIPK1 kinase in Mecp2-deficient microglia may be involved both in the onset and progression of RTT.

Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.

CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3'-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3'-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3'-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.

Sacral Neural Crest-Independent Origin of the Enteric Nervous System in Mouse.

BACKGROUND & AIMS: The enteric nervous system (ENS), the gut's intrinsic nervous system critical for gastrointestinal function and gut-brain communication, is believed to mainly originate from vagal neural crest cells (vNCCs) and partially from sacral NCCs (sNCCs). Resolving the exact origins of the ENS is critical for understanding congenital ENS diseases but has been confounded by the inability to distinguish between both NCC populations in situ. Here, we aimed to resolve the exact origins of the mammalian ENS. METHODS: We genetically engineered mouse embryos facilitating comparative lineage-tracing of either all (pan-) NCCs including vNCCs or caudal trunk and sNCCs (s/tNCCs) excluding vNCCs. This was combined with dual-lineage tracing and 3-dimensional reconstruction of pelvic plexus and hindgut to precisely pinpoint sNCC and vNCC contributions. We further used coculture assays to determine the specificity of cell migration from different neural tissues into the hindgut. RESULTS: Both pan-NCCs and s/tNCCs contributed to established NCC derivatives but only pan-NCCs contributed to the ENS. Dual-lineage tracing combined with 3-dimensional reconstruction revealed that s/tNCCs settle in complex patterns in pelvic plexus and hindgut-surrounding tissues, explaining previous confusion regarding their contributions. Coculture experiments revealed unspecific cell migration from autonomic, sensory, and neural tube explants into the hindgut. Lineage tracing of ENS precursors lastly provided complimentary evidence for an exclusive vNCC origin of the murine ENS. CONCLUSIONS: sNCCs do not contribute to the murine ENS, suggesting that the mammalian ENS exclusively originates from vNCCs. These results have immediate implications for comprehending (and devising treatments for) congenital ENS disorders, including Hirschsprung's disease.

NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication.

Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and liver cancer, despite strong prevention and treatment efforts. The study of the epigenetic modification of HBV has become a research hotspot, including the N6-methyladenosine (m6A) modification of HBV RNA, which plays complex roles in the HBV life cycle. In addition to m6A modification, 5-methylcytosine (m5C) is another major modification of eukaryotic mRNA. In this study, we explored the roles of m5C methyltransferase and demethyltransferase in the HBV life cycle. The results showed that m5C methyltransferase NSUN2 deficiency could negatively regulate the expression of HBV while m5C demethyltransferase TET2 deficiency positively regulates the expression of HBV. Subsequently, we combined both in vitro bisulfite sequencing and high-throughput bisulfite sequencing methods to determine the distribution and stoichiometry of m5C modification in HBV RNA. Two sites: C2017 and C131 with the highest-ranking methylation rates were identified, and mutations at these two sites could lead to the decreased expression and replication of HBV, while the mutation of the "fake" m5C site had no effect. Mechanistically, NSUN2-mediated m5C modification promotes the stability of HBV RNA. In addition, compared with wild-type HepG2-NTCP cells and primary human hepatocytes, the replication level of HBV after NSUN2 knockdown decreased, and the ability of the mutant virus to infect and replicate in wild-type HepG2-NTCP cells and PHHs was substantially impaired. Similar results were found in the experiments using C57BL/6JGpt-Nsun2+/- mice. Interestingly, we also found that HBV expression and core protein promoted the endogenous expression of NSUN2, which implied a positive feedback loop. In summary, our study provides an accurate and high-resolution m5C profile of HBV RNA and reveals that NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication by maintaining RNA stability.

CCDC50 promotes tumor growth through regulation of lysosome homeostasis.

The maintenance of lysosome homeostasis is crucial for cell growth. Lysosome-dependent degradation and metabolism sustain tumor cell survival. Here, we demonstrate that CCDC50 serves as a lysophagy receptor, promoting tumor progression and invasion by controlling lysosomal integrity and renewal. CCDC50 monitors lysosomal damage, recognizes galectin-3 and K63-linked polyubiquitination on damaged lysosomes, and specifically targets them for autophagy-dependent degradation. CCDC50 deficiency causes the accumulation of ruptured lysosomes, impaired autophagic flux, and superfluous reactive oxygen species, consequently leading to cell death and tumor suppression. CCDC50 expression is associated with malignancy, progression to metastasis, and poor overall survival in human melanoma. Targeting CCDC50 suppresses tumor growth and lung metastasis, and enhances the effect of BRAFV600E inhibition. Thus, we demonstrate critical roles of CCDC50-mediated clearance of damaged lysosomes in supporting tumor growth, hereby identifying a potential therapeutic target of melanoma.

Mycn deficiency underlies the development of orofacial clefts in mice and humans.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most common subphenotype of non-syndromic orofacial clefts arising from genetic and/or environmental perturbations during embryonic development. We previously identified 2p24.2 as a risk locus associated with NSCL/P in the Chinese Han population, and MYCN is a candidate risk gene in this region. To understand the potential function of MYCN in craniofacial development, we generated Wnt1-Cre;Mycnflox/flox mice that exhibited cleft palate, microglossia and micrognathia, resembling the Pierre Robin sequence (PRS) in humans. Further analyses indicated that the cleft palate was secondary to the delayed elevation of palatal shelves caused by micrognathia. The micrognathia resulted from impaired chondrogenic differentiation in Merkel's cartilage, which limited tongue development, leading to microglossia. In terms of mechanism, Mycn deficiency in cranial neural crest cells (CNCCs) downregulated Sox9 expression by inhibiting Wnt5a in a CNCC-derived chondrogenic lineage in Merkel's cartilage. To investigate whether MYCN deficiency contributed to NSCL/P, we performed direct sequencing targeting all exons and exon-intron boundaries of MYCN in 104 multiplex families with Mendelian NSCL/P and identified a novel pathogenic variant in MYCN. Taken together, our data indicate that ablation of Mycn in mouse CNCCs could resemble PRS by suppressing the Wnt5a-Sox9 signaling pathway in Merkel's cartilage and that mutations in MYCN may be novel potential causes of NSCL/P.

Bi-allelic variants in the ER quality-control mannosidase gene EDEM3 cause a congenital disorder of glycosylation.

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.

The small nucleolar RNA SNORA51 enhances breast cancer stem cell-like properties via the RPL3/NPM1/c-MYC pathway.

Breast cancer stem cells (BCSCs) are key players in carcinogenesis and development. Small nucleolar RNAs (snoRNAs) seem to have a crucial influence on regulating stem cell-like properties in various cancers, but the underlying mechanism in breast cancer has not been determined. In this study, we first found that the expression of SNORA51 might be strongly and positively related to BCSCs-like properties. SNORA51 expression was assessed in breast cancer tissues (n = 158 patients) by in situ hybridization. Colony formation, cell counting kit-8, and sphere formation assays were used to detect cell proliferation and self-renewal, respectively. Wound healing and transwell assays were used to detect cell migration. Coimmunoprecipitation and molecular docking were used to determine the underlying mechanism through which SNORA51 regulates BCSCs-like properties. High SNORA51 expression was associated with a worse prognosis, overall survival, and disease-free survival, in 158 breast cancer patients and was also closely related to lymph node status, ER status, the Ki-67 index, histological grade, and TNM stage. Further analysis proved that SNORA51 could enhance and maintain stem cell-like properties, including cell proliferation, self-renewal, and migration, in breast cancer. Moreover, high SNORA51 expression could reduce nucleolar RPL3 expression, induce changes in the expression of NPM1 in the nucleolus and nucleoplasm, and ultimately increase c-MYC expression. Taken together, our findings demonstrated that SNORA51 could enhance BCSCs-like properties via the RPL3/NPM1/c-MYC pathway both in vitro and in vivo. Therefore, SNORA51 might be a significant biomarker and potential therapeutic target and might even provide a new viewpoint on the regulatory mechanism of snoRNAs in breast cancer or other malignant tumors.

D-mannose as a new therapy for fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG).

INTRODUCTION: Fucokinase deficiency-related congenital disorder of glycosylation (FCSK-CDG) is a rare autosomal recessive inborn error of metabolism characterized by a decreased flux through the salvage pathway of GDP-fucose biosynthesis due to a block in the recycling of L-fucose that exits the lysosome. FCSK-CDG has been described in 5 individuals to date in the medical literature, with a phenotype comprising global developmental delays/intellectual disability, hypotonia, abnormal myelination, posterior ocular disease, growth and feeding failure, immune deficiency, and chronic diarrhea, without clear therapeutic recommendations. PATIENT AND METHODS: In a so far unreported FCSK-CDG patient, we studied proteomics and glycoproteomics in vitro in patient-derived fibroblasts and also performed in vivo glycomics, before and after treatment with either D-Mannose or L-Fucose. RESULTS: We observed a marked increase in fucosylation after D-mannose supplementation in fibroblasts compared to treatment with L-Fucose. The patient was then treated with D-mannose at 850 mg/kg/d, with resolution of the chronic diarrhea, resolution of oral aversion, improved weight gain, and observed developmental gains. Serum N-glycan profiles showed an improvement in the abundance of fucosylated glycans after treatment. No treatment-attributed adverse effects were observed. CONCLUSION: D-mannose is a promising new treatment for FCSK-CDG.

ALG13-Congenital Disorder of Glycosylation (ALG13-CDG): Updated clinical and molecular review and clinical management guidelines.

ALG13-Congenital Disorder of Glycosylation (CDG), is a rare X-linked CDG caused by pathogenic variants in ALG13 (OMIM 300776) that affects the N-linked glycosylation pathway. Affected individuals present with a predominantly neurological manifestation during infancy. Epileptic spasms are a common presenting symptom of ALG13-CDG. Other common phenotypes include developmental delay, seizures, intellectual disability, microcephaly, and hypotonia. Current management of ALG13-CDG is targeted to address patients' symptoms. To date, less than 100 individuals have been reported with ALG13-CDG. In this article, an international group of experts in CDG reviewed all reported individuals affected with ALG13-CDG and suggested diagnostic and management guidelines for ALG13-CDG. The guidelines are based on the best available data and expert opinion. Neurological symptoms dominate the phenotype of ALG13-CDG where epileptic spasm is confirmed to be the most common presenting symptom of ALG13-CDG in association with hypotonia and developmental delay. We propose that ACTH/prednisolone treatment should be trialed first, followed by vigabatrin, however ketogenic diet has been shown to have promising results in ALG13-CDG. In order to optimize medical management, we also suggest early cardiac, gastrointestinal, skeletal, and behavioral assessments in affected patients.

The prevalence of Human Immunodeficiency Virus infection among voluntary blood donors in mainland China: A systematic review and meta-analysis.

Human immunodeficiency virus (HIV) infection through transfusion has been an imperative challenge for blood safety. Despite the implementation of screening strategies, there was still the residual risk of transfusion-transmitted HIV. Considering that the prevalence of HIV infection in blood donors is significant for evaluating blood safety and potential risks to the population, meta-analysis was applied to investigate the HIV prevalence among voluntary blood donors during the past 27 years to characterize the epidemiology and related risk factors of HIV in blood donors. The literature concerning the HIV screening reactive rate and prevalence in Chinese voluntary blood donors was collected through the systematic searching of four electronic databases. After integrating data, following the Preferred Reporting of Items for Systematic Reviews and Meta-Analyses guidelines, data manipulation and statistical analyses were conducted by Stata 12.0. The results indicated that overall HIV prevalence was 0.0178% (95% confidence interval [CI], 0.0169%-0.0187%) with a remarkable rise, which varied from 2000 (0.0034%) to 2015 (0.027%). The HIV window period infection rate was 0.0475‱ (95% CI, 0.0304‱-0.0646‱). Importantly, subgroup analysis revealed the heterogeneity in gender, occupations, education and donation frequency. With the effective control of HIV transmission through blood, HIV prevalence declined in China to some extent in recent years, and the characteristics of HIV epidemic in some provinces have drastically changed. However, remaining relatively high HIV prevalence and overall increased trend of HIV prevalence since the 21th century demonstrates the potential residual risk of blood transfusion, and the whole society is supposed to pay close attention to HIV infection.

Reconstitution of RNA cap methylation reveals different features of SARS-CoV-2 and SARS-CoV methyltransferases.

Cap RNA methylations play important roles in the replication, evasion of host RNA sensor recognition, and pathogenesis. Coronaviruses possess both guanine N7- and 2'-O-ribose methyltransferases (N7-MTase and 2'-O-MTase) encoded by nonstructural protein (nsp) 14 and nsp16/10 complex, respectively. In this study, we reconstituted the two-step RNA methylations of N7-MTase and 2'-O-MTase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and demonstrated its common and different features in comparison with that of SARS-CoV. We revealed that the nsp16/10 2'-O-MTase of SARS-CoV-2 has a broader substrate selectivity than the counterpart of SARS-CoV and can accommodate both unmethylated and uncapped RNA substrates in a sequence-independent manner. Most intriguingly, the substrate selectivity of nsp16/10 complex is not determined by the apoenzyme of nsp16 MTase but by its cofactor nsp10. These results provide insight into the unique features of SARS-CoV-2 MTases and may help develop strategies to precisely intervene in the methylation pathway and pathogenesis of SARS-CoV-2.

Syntaxin6 contributes to hepatocellular carcinoma tumorigenesis via enhancing STAT3 phosphorylation.

BACKGROUND: Syntaxin6 (STX6) is a SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein complex located in the trans-Golgi network and endosomes, which is closely associated with a variety of intracellular membrane transport events. STX6 has been shown to be overexpressed in a variety of human malignant tumors such as esophageal, colorectal, and renal cell carcinomas, and participates in tumorigenesis and development. METHODS: Based on clinical public database and clinical liver samples analysis, the expression of STX6 in hepatocellular carcinoma (HCC) tissues was investigated. The effects of STX6 on proliferation, migration and invasion of HCC cell in vitro and in vivo were evaluated through gain- and loss-of-function studies. We further performed RNA-seq analysis and protein interactome analysis, to further decifer the detailed mechanisms of STX6 in the regulation of the JAK-STAT pathway in HCC. RESULTS: STX6 expression was upregulated in HCC tissues and its expression was highly correlated with the high histological grade of the tumor. STX6 promoted HCC cell proliferation, migration and invasion both in vitro and in vivo. Mechanistically, STX6 mediated tumor progression depending on promoting the activation of JAK-STAT signaling pathway. Receptor for activated protein kinase C (RACK1) as an essential adaptor protein mediating STX6 regulation of JAK-STAT pathway. Specifically, STX6 interacted with RACK1 and then recruited signal transducer and activator of transcription 3 (STAT3) to form a protein-binding complex and activates STAT3 transcriptional activity. CONCLUSIONS: This study provided a novel concept that STX6 exerted oncogenic effects by activating the STAT3 signaling pathway, and STX6 might be a promising therapeutic target for HCC.

CCDC50 suppresses NLRP3 inflammasome activity by mediating autophagic degradation of NLRP3.

The NLRP3-directed inflammasome complex is crucial for the host to resist microbial infection and monitor cellular damage. However, the hyperactivation of NLRP3 inflammasome is implicated in pathogenesis of inflammatory diseases, including inflammatory bowel disease (IBD). Autophagy and autophagy-related genes are closely linked to NLRP3-mediated inflammation in these inflammatory disorders. Here, we report that CCDC50, a novel autophagy cargo receptor, negatively regulates NLRP3 inflammasome assembly and suppresses the cleavage of pro-caspase-1 and interleukin 1ß (IL-1ß) release by delivering NLRP3 for autophagic degradation. Transcriptome analysis showed that knockdown of CCDC50 results in upregulation of signaling pathways associated with autoinflammatory diseases. CCDC50 deficiency leads to enhanced proinflammatory cytokine response triggered by a wide range of endogenous and exogenous NLRP3 stimuli. Ccdc50-deficient mice are more susceptible to dextran sulfate (DSS)-induced colitis and exhibit more severe gut inflammation with elevated NLRP3 inflammasome activity. These results illustrate the physiological significance of CCDC50 in the pathogenicity of inflammatory diseases, suggesting protective roles of CCDC50 in keeping gut inflammation under control.

Deficient glycan extension and endoplasmic reticulum stresses in ALG3-CDG.

ALG3-CDG is a rare congenital disorder of glycosylation (CDG) with a clinical phenotype that includes neurological manifestations, transaminitis, and frequent infections. The ALG3 enzyme catalyzes the first step of endoplasmic reticulum (ER) luminal glycan extension by adding mannose from Dol-P-Man to Dol-PP-Man5GlcNAc2 (Man5) forming Dol-PP-Man6. Such glycan extension is the first and fastest cellular response to ER stress, which is deficient in ALG3-CDG. In this study, we provide evidence that the unfolded protein response (UPR) and ER-associated degradation activities are increased in ALG3-CDG patient-derived cultured skin fibroblasts and there is constitutive activation of UPR mediated by the IRE1-α pathway. In addition, we show that N-linked Man3-4 glycans are increased in cellular glycoproteins and secreted plasma glycoproteins with hepatic or non-hepatic origin. We found that like other CDGs such as ALG1- or PMM2-CDG, in transferrin, the assembling intermediate Man5 in ALG3-CDG, are likely further processed into a distinct glycan, NeuAc1Gal1GlcNAc1Man3GlcNAc2, probably by Golgi mannosidases and glycosyltransferases. We predict it to be a mono-antennary glycan with the same molecular weight as the truncated glycan described in MGAT2-CDG. In summary, this study elucidates multiple previously unrecognized biochemical consequences of the glycan extension deficiency in ALG3-CDG which will have important implications in the pathogenesis of CDG.