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TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonin (5-hydroxytryptamine, 5-HT) is a unique neurotransmitter which can regulate various biological processes by activating thirteen different receptors. These serotonin receptors are divided into seven different classes based on their structure and functions. Since these receptors co-express in various tissue and cell types and share the same ligand (5-HT), it has been a challenge for the researchers to define specific pathway and separate physiological role for each of these serotonin receptors. Though the evidence of operational diversity of these receptors is continuously emerging, much work remains to be done. 5-HTR1E is a member of 5-HT1 receptor family which belongs to G-protein coupled receptors (GPCRs). Even after three decades since its discovery, 5-HTR1E remains the least explored serotonin receptor. Very high similarity with another family member (5-HTR1F) and its non-existence in mice or rats makes 5-HTR1E a difficult target to study. Despite these challenges, recent findings on the role of 5-HTR1E in neuroprotection and diseases such as cancer, have excited many researchers to explore this receptor in detail. Here, we provide the first review of 5-HTR1E, since its discovery in 1989 to 2023. We highlight the structural and functional characteristics of this important serotonin receptor in detail and propose future directions in developing 5-HTR1E as a drug target. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Alzheimer's Disease (AD) is a prevalent neurodegenerative disease characterized by tau hyperphosphorylation, Aβ1-42 aggregation and cognitive dysfunction. Therapeutic agents directed at mitigating tau aggregation and clearing Aβ1-42, and delivery of growth factor genes (BDNF, FGF2), have ameliorated cognitive deficits, but these approaches did not prevent or stop AD progression. Here we report that viral-(AAV) delivery of Neurotrophic Factor-α1/Carboxypeptidase E (NF-α1/CPE) gene in hippocampus at an early age prevented later development of cognitive deficits as assessed by Morris water maze and novel object recognition assays, neurodegeneration, and tau hyperphosphorylation in male 3xTg-AD mice. Additionally, amyloid precursor protein (APP) expression was reduced to near non-AD levels, and insoluble Aβ1-42 was reduced significantly. Pro-survival proteins: mitochondrial Bcl2 and Serpina3g were increased; and mitophagy inhibitor Plin4 and pro-inflammatory protein Card14 were decreased in AAV-NF-α1/CPE treated versus untreated AD mice. Thus NF-α1/CPE gene therapy targets many regulatory components to prevent cognitive deficits in 3xTg-AD mice and has implications as a new therapy to prevent AD progression by promoting cell survival, inhibiting APP overexpression and tau hyperphosphorylation., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

5-Hydroxytryptamine receptor 1E (5-HTR1E) is reported to activate cyclic AMP (cAMP) and extracellular-signal related kinases (ERK) pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin-induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin-induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gβγ and Gq were not associated with 5-HTR1E activation, while blocking protein kinase A (PKA) inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, β-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockdown studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 (RAMP1), Nuclear receptor 1 (NR4A1) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Carboxypeptidase E (CPE) is a multifunctional protein with many nonenzymatic functions in various systems. Previous studies using CPE knock-out mice have shown that CPE has neuroprotective effects against stress and is involved in learning and memory. However, the functions of CPE in neurons are still largely unknown. Here we used a Camk2a-Cre system to conditionally knockout CPE in neurons. The wild-type, CPE flox/- , and CPE flox/flox mice were weaned, ear-tagged, and tail clipped for genotyping at 3 weeks old, and they underwent open field, object recognition, Y-maze, and fear conditioning tests at 8 weeks old. The CPE flox/flox mice had normal body weight and glucose metabolism. The behavioral tests showed that CPE flox/flox mice had impaired learning and memory compared with wild-type and CPE flox/- mice. Surprisingly, the subiculum (Sub) region of CPE flox/flox mice was completely degenerated, unlike the CPE full knockout mice, which exhibit CA3 region neurodegeneration. In addition, doublecortin immunostaining suggested that neurogenesis in the dentate gyrus of the hippocampus was significantly reduced in CPE flox/flox mice. Interestingly, TrkB phosphorylation in the hippocampus was downregulated in CPE flox/flox mice, but brain-derived neurotrophic factor levels were not. In both the hippocampus and dorsal medial prefrontal cortex, we observed reduced MAP2 and GFAP expression in CPE flox/flox mice. Taken together, the results of this study demonstrate that specific neuronal CPE knockout leads to central nervous system dysfunction in mice, including learning and memory deficits, hippocampal Sub degeneration and impaired neurogenesis., (© 2023. The Author(s).)

It is well known that peptide hormones and neurotrophic factors are intercellular messengers that are packaged into secretory vesicles in endocrine cells and neurons and released by exocytosis upon the stimulation of the cells in a calcium-dependent manner. These secreted molecules bind to membrane receptors, which then activate signal transduction pathways to mediate various endocrine/trophic functions. Recently, there is evidence that these molecules are also in extracellular vesicles, including small extracellular vesicles (sEVs), which appear to be taken up by recipient cells. This finding raised the hypothesis that they may have functions differentiated from their classical secretory hormone/neurotrophic factor actions. In this article, the historical perspective and updated mechanisms for the sorting and packaging of hormones and neurotrophic factors into secretory vesicles and their transport in these organelles for release at the plasma membrane are reviewed. In contrast, little is known about the packaging of hormones and neurotrophic factors into extracellular vesicles. One proposal is that these molecules could be sorted at the trans-Golgi network, which then buds to form Golgi-derived vesicles that can fuse to endosomes and subsequently form intraluminal vesicles. They are then taken up by multivesicular bodies to form extracellular vesicles, which are subsequently released. Other possible mechanisms for packaging RSP proteins into sEVs are discussed. We highlight some studies in the literature that suggest the dual vesicular pathways for the release of hormones and neurotrophic factors from the cell may have some physiological significance in intercellular communication., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest.

Mechanisms driving tumor growth and metastasis are complex, and involve the recruitment of many genes working in concert with each other. The tumor is characterized by the expression of specific sets of genes depending on its environment. Here we review the role of the carboxypeptidase E (CPE) gene which has been shown to be important in driving growth, survival and metastasis in many cancer types. CPE was first discovered as a prohormone processing enzyme, enriched in endocrine tumors, and later found to be expressed and secreted from many epithelial-derived tumors and cancer cell lines. Numerous studies have shown that besides wild-type CPE, a N-terminal truncated splice variant form of CPE (CPE-ΔN) has been cloned and found to be highly expressed in malignant tumors and cell lines derived from prostate, breast, liver and lung cancers and gliomas. The mechanisms of action of CPE and the splice variant in promoting tumor growth and metastasis in different cancer types are discussed. Mechanistically, secreted CPE activates the Erk/wnt pathways, while CPE-ΔN interacts with HDACs in a protein complex in the nucleus, to recruit various cell cycle genes and metastatic genes, respectively. Clinical studies suggest that CPE and CPE-ΔN mRNA and protein are potential diagnostic and prognostic biomarkers for multiple cancer types, assayed using solid tumors and secreted serum exosomes. CPE has been shown to be a therapeutic target for multiple cancer types. CPE/CPE-ΔN siRNA transported via exosomes and taken up by recipient high metastatic cancer cells, suppressed growth and proliferation of these cells. Thus future studies, delivering CPE/CPE-ΔN siRNA, perhaps via exosomes, to the tumor could be a novel treatment approach to suppress tumor growth and metastasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Depression is a major psychiatric disease affecting all ages and is often co-morbid with neurodegeneration in the elderly. Depression and neurodegeneration are associated with decreased neurotrophic factors. In this mini-review the functions and potential therapeutic use of a newly discovered trophic factor, Neurotrophic factor-α1 (NF-α1), also known as Carboxypeptidase E (CPE), in depression and neuroprotection are discussed. NF-α1/CPE expression is enriched in CA3 neurons of the hippocampus. Families carrying null and homozygous non-sense mutations of the NF-α1/CPE gene share common clinical features including childhood onset obesity, type 2 diabetes, impaired intellectual abilities and hypogonadotrophic hypogonadism. Studies in animal models such as CPE knockout (KO) mice and CPE fat/fat mutant mice exhibit similar phenotypes. Analysis of CPE-KO mouse brain revealed that hippocampal CA3 was completely degenerated after weaning stress, along with deficits in hippocampal long-term potentiation. Carbamazepine effectively blocked weaning stress-induced hippocampal CA3 degeneration, suggesting the stress induced epileptic-like neuronal firing led to the degeneration. Analysis of possible mechanisms underlying NF-α1/CPE -mediated neuroprotection revealed that it interacts with the serotonin receptor, 5-HTR1E, and via β arrestin activation, subsequently upregulates ERK1/2 signaling and pro-survival protein, BCL2, levels. Furthermore, the NF-α1/CPE promoter contains a peroxisome proliferator-activated receptor (PPARγ) binding site which can be activated by rosiglitazone, a PPARγ agonist, to up-regulate expression of NF-α1/CPE and neurogenesis, resulting in anti-depression in animal models. Rosiglitazone, an anti-diabetic drug administered to diabetic patients resulted in decline of depression. Thus, NF-α1/CPE is a potential therapeutic agent or drug target for treating depression and neurodegenerative disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xiao and Loh.)

Background: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model., Methods: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays., Results: Exosomes released from cancer cells contain CPE mRNA and protein. CPE mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc , a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells., Conclusions: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.

Background: Effective biomarkers for prediction of recurrence of lung adenocarcinoma cancer (LADC) patients are needed to determine treatment strategies post-surgery to improve outcome., Objective: This study evaluates the efficacy of carboxypeptidase E (CPE) mRNA including its splice isoforms, CPE-ΔN, as a biomarker for predicting recurrence in adenocarcinoma patients., Methods: RNA was extracted from resected tumors from 86 patients with different stages of non-small cell LADC. cDNA was synthesized and qRT-PCR carried out to determine the copy numbers of CPE/CPE-ΔN mRNA. Patients were followed for 7 years post-tumor resection to determine recurrence and death., Results: ROC curve analysis showed the overall AUC for CPE/CPE-ΔN copy number was 0.563 in predicting recurrence and 0.562 in predicting death. Kaplan-Meier survival analysis showed statistical difference (p= 0.018), indicating that patients with high CPE/CPE-ΔN copy numbers had a shorter time of disease-free survival and also shorter time to death (p= 0.035). Subgroup analyses showed that association of disease-free survival time with CPE/CPE-ΔN copy number was stronger among stage I and II LADC patients (p= 0.047)., Conclusions: CPE/CPE-ΔN mRNA is a potentially useful biomarker for predicting recurrence and death in LADC patients, especially in identifying patients at high risk of recurrence at early stages I and II.

Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125 I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H 2 O 2 -induced oxidative stress. Cell survival assay in β-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was β-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited β-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of β-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the β-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Chromatin interactions play important roles in regulating gene expression. However, the availability of genome-wide chromatin interaction data is limited. We develop a computational method, chromatin interaction neural network (ChINN), to predict chromatin interactions between open chromatin regions using only DNA sequences. ChINN predicts CTCF- and RNA polymerase II-associated and Hi-C chromatin interactions. ChINN shows good across-sample performances and captures various sequence features for chromatin interaction prediction. We apply ChINN to 6 chronic lymphocytic leukemia (CLL) patient samples and a published cohort of 84 CLL open chromatin samples. Our results demonstrate extensive heterogeneity in chromatin interactions among CLL patient samples., (© 2021. The Author(s).)

Background: Pancreatic cancer is one of the leading cause of cancer-related death globally. The molecular basis of this disease is complex and not fully understood. Previous studies have indicated that carboxypeptidase E (CPE) plays a role in promoting tumorigenesis in many cancer types. Here we have investigated the effect of carboxypeptidase E (CPE), including its isoform, in regulating the proliferation, migration and invasion of Panc-1 cells, a pancreatic cell line. Methods: Panc-1 cells were transfected with CPE siRNA which targets both CPE-wild type and its isoform, or scrambled siRNA, for 24 h and then assayed for proliferation by the MTT and colony formation assays, and migration and invasion by wound healing and matrigel assays, respectively. Results: CPE siRNA treatment of Panc-1 cells down-regulated the expression of CPE mRNA by 94.8%. Silencing of CPE mRNA expression resulted in a significant decrease in proliferation as revealed by the MTT assay and a 62.8% decrease in colony formation. Western blot analysis of expression of Cyclin D1 in Panc-1 cells treated with CPE siRNA showed a decrease of 32.5% compared to scr siRNA treated cells, indicating that CPE regulates proliferation through modulating this cell cycle protein.  Additionally, suppression of CPE expression in Panc-1 cells significantly decreased migration and invasion. Conclusions: Our findings indicate that CPE may play an important role in regulating cell proliferation, migration and invasion to promote pancreatic cancer tumorigenesis., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Lou H and Loh YP.)

The mechanisms underlying gene repression and silencers are poorly understood. Here we investigate the hypothesis that H3K27me3-rich regions of the genome, defined from clusters of H3K27me3 peaks, may be used to identify silencers that can regulate gene expression via proximity or looping. We find that H3K27me3-rich regions are associated with chromatin interactions and interact preferentially with each other. H3K27me3-rich regions component removal at interaction anchors by CRISPR leads to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. Cells with H3K27me3-rich regions knockout also show changes in phenotype associated with cell identity, and altered xenograft tumor growth. Finally, we observe that H3K27me3-rich regions-associated genes and long-range chromatin interactions are susceptible to H3K27me3 depletion. Our results characterize H3K27me3-rich regions and their mechanisms of functioning via looping.

Stress leads to brain pathology including hippocampal degeneration, cognitive dysfunction, and potential mood disorders. Hippocampal CA3, a most stress-vulnerable region, consists of pyramidal neurons that regulate cognitive functions e.g. learning and memory. These CA3 neurons express high levels of the neuroprotective protein, neurotrophic factor-α1 (NF-α1), also known as carboxypeptidase E (CPE), and receive contacts from granule cell projections that release BDNF which has neuroprotective activity. Whether NF-α1-CPE and/or BDNF are critical in protecting these CA3 neurons against severe stress-induced cell death is unknown. Here we show that social combined with the physical stress of maternal separation, ear tagging, and tail snipping at weaning in 3-week-old mice lacking NF-α1-CPE, led to complete hippocampal CA3 degeneration, despite having BDNF and active phosphorylated TrkB receptor levels similar to WT animals. Mice administered TrkB inhibitor, ANA12 which blocked TrkB phosphorylation showed no degeneration of the CA3 neurons after the weaning stress paradigm. Furthermore, transgenic knock-in mice expressing CPE-E342Q, an enzymatically inactive form, replacing NF-α1-CPE, showed no CA3 degeneration and exhibited normal learning and memory after the weaning stress, unlike NF-α1-CPE-KO mice. Mechanistically, we showed that radio-labeled NF-α1-CPE bound HT22 hippocampal cells in a saturable manner and with high affinity (Kd = 4.37 nM). Subsequently, treatment of the HT22 cpe-/- cells with NF-α1-CPE or CPE-E342Q equivalently activated ERK signaling and increased BCL2 expression to protect these neurons against H 2 O 2 -or glutamate-induced cytotoxicity. Our findings show that NF-α1-CPE is more critical compared to BDNF in protecting CA3 pyramidal neurons against stress-induced cell death and cognitive dysfunction, independent of its enzymatic activity.

Exosomes are a subtype of extracellular vesicles released from different cell types including those in the nervous system, and are enriched in a variety of bioactive molecules such as RNAs, proteins and lipids. Numerous studies have indicated that exosomes play a critical role in many physiological and pathological activities by facilitating intercellular communication and modulating cells' responses to external environments. Particularly in the central nervous system, exosomes have been implicated to play a role in many neurological disorders such as abnormal neuronal development, neurodegenerative diseases, epilepsy, mental disorders, stroke, brain injury and brain cancer. Since exosomes recapitulate the characteristics of the parental cells and have the capacity to cross the blood-brain barrier, their cargo can serve as potential biomarkers for early diagnosis and clinical assessment of disease treatment. In this review, we describe the latest findings and current knowledge of the roles exosomes play in various neurological disorders and brain cancer, as well as their application as promising biomarkers. The potential use of exosomes to deliver therapeutic molecules to treat diseases of the central nervous system is also discussed., Competing Interests: Conflicts of interest The authors declare no conflict of interest.

Bone and energy metabolism are integrated by common regulatory mechanisms. Carboxypeptidase E (CPE), also known as obesity susceptibility protein or neurotrophic factor-α1, is recognized for its function in processing prohormones, including proinsulin and pro-opiomelanocortin polypeptide. Independent of its enzymatic activity, CPE may also act as a secreted factor with divergent roles in neuroprotection and cancer growth; however, its role in the regulation of bone mass and skeletal cell differentiation is unknown. Male mice with global deficiency in CPE are characterized with profound visceral obesity, low bone mass in both appendicular and axial skeleton, and high volume of marrow fat. Interestingly, although metabolic deficit of CPE KO mice develops early in life, bone deficit develops in older age, suggesting that CPE bone-specific activities differ from its enzymatic activities. Indeed, mutated CPE knockin (mCPE KI) mice ectopically expressing CPE-E342Q, a mutated protein lacking enzymatic activity, develop the same obese phenotype and accumulate the same volume of marrow fat as CPE KO mice, but their bone mass is normal. In addition, differentiation of marrow hematopoietic cells toward tartrate-resistant acid phosphatase-positive multinucleated osteoclasts is highly increased in CPE KO mice, but normal in mCPE KI mice. Moreover, in murine skeletal stem cells, nonenzymatic trophic CPE has activated ERK signaling, increased cell proliferation and increased mitochondrial activity. Treatment of preosteoblastic cells with intact or mutated recombinant CPE led to a transient accumulation of small lipid droplets, increased oxidative phosphorylation, and increased cellular dependence on fatty acids as fuel for energy production. In human marrow aspirates, CPE expression increases up to 30-fold in osteogenic conditions. These findings suggest that nonenzymatic and trophic activities of CPE regulate bone mass, whereas marrow adiposity is controlled by CPE enzymatic activity. Thus, CPE can be positioned as a factor regulating simultaneously bone and energy metabolism through a combination of shared and distinct mechanisms. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research., (© 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.)

The Editor and the Publisher have retracted this article [1] following an investigation by the National Institutes of Health (NIH) into the primary data used to construct Table 2. This investigation concluded that the data in Table 2 are unreliable.

Astrocytes are the most abundant type of glial cells in the brain, and they play a key role in Alzheimer's disease (AD). Fibroblast Growth Factor-2 (FGF-2) has been implicated as a potential therapeutic agent for treating AD. In the present study, we investigated the protective effects of low molecular weight (LMW; 17 KDa) and high molecular weight (HMW; 23 KDa) forms of FGF-2 on Aβ 1-42 -induced toxicity, and proliferation in astrocytes. We show that both isoforms of FGF-2 have similar protective effects against Aβ 1-42 -induced cytotoxicity in primary cultured cortical astrocytes as measured by Lactate Dehydrogenase (LDH) release assay. Additionally, 17 KDa FGF-2 significantly promoted astrocyte proliferation as measured by Trypan Blue, DRAQ5 and 5-ethynyl-2'-deoxyuridine (EdU) staining, but not 23 kDa FGF-2. Furthermore, our results demonstrated that AKT signaling pathway was required for the protective and proliferative effects of FGF-2. Downstream effector studies indicated that 17 kDa FGF-2 promoted astrocyte proliferation by enhanced expression of c-Myc, Cyclin D1, Cyclin E. Furthermore, our data suggested that Cyclin D1 was required for the proliferative effect of LMW FGF2 in astrocytes. Taken together, our findings provide important information for the similarities and differences between 23 kDa and17 kDa isoforms of FGF-2 on astrocyte survival and proliferation., (Copyright © 2020 Chen, Li, Cheng, Kardami and Loh.)

Pancreatic cancer is one of the leading causes of cancer-related mortality worldwide. The molecular basis for the pathogenesis of this disease remains elusive. In this study, we have investigated the role of wild-type Carboxypeptidase E (CPE-WT) and a 40 kDa N-terminal truncated isoform, CPE-ΔN in promoting proliferation and invasion of Panc-1 cells, a pancreatic cancer cell line. Both CPE-WT and CPE-ΔN were expressed in Panc-1 and BXPC-3 pancreatic cancer cells. Immunocytochemical studies revealed that in CPE transfected Panc-1 cells, CPE-ΔN was found primarily in the nucleus, whereas CPE-WT was present exclusively in the cytoplasm as puncta, characteristic of secretory vesicles. Endogenous CPE-WT was secreted into the media. Overexpression of CPE-ΔN in Panc-1 cells resulted in enhancement of proliferation and invasion of these cells, as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay and Matrigel invasion assay, respectively. In contrast, the expression of CPE-WT protein at comparable levels to CPE-ΔN in Panc-1 cells resulted in promotion of proliferation but not invasion. Importantly, there was an upregulation of the expression of CXCR2 mRNA and protein in Panc-1 cells overexpressing CPE-ΔN, and these cells exhibited significant increase in proliferation in a CXCR2-dependent manner. Thus, CPE-ΔN may play an important role in promoting pancreatic cancer growth and malignancy through upregulating the expression of the metastasis-related gene, CXCR2 .

Carboxypeptidase E, also known as neurotrophic factor-α1 (CPE-NFα1), was first discovered as an exopeptidase and is known to work by cleaving C-terminal basic amino acids from prohormone intermediates to produce mature peptide hormones and neuropeptides in the endocrine and central nervous systems, respectively. CPE-NFα1 also plays a critical role in prohormone sorting and secretory vesicle transportation. Recently, emerging studies have indicated that CPE-NFα1 exerts multiple non-enzymatic physiological roles in maintaining normal central nervous system function and in neurodevelopment. This includes potent neuroprotective and anti-depressant activities, as well as stem cell differentiation functions. In addition, N-terminal truncated variants of CPE-NFα1 have been identified to regulate expression of important neurodevelopmental genes. This mini-review summarizes recent advances in understanding the mechanisms underlying CPE-NFα1's function in neuroprotection during stress and aspects of neurodevelopment.

Carboxypeptidase E (CPE), an exopeptidase involved in proneuropeptide processing, is also a neurotrophic factor, named neurotrophic factor-α1 (NF-α1) and has important roles in neuroprotection, stem cell differentiation, and neurite outgrowth, independent of enzymatic activity. Additionally, an N-terminal-truncated CPE/NF-α1 variant, (CPE/NF-α1)-ΔN, proposed from bioinformatic analysis of GenBank (National Center for Biotechnology Information, Bethesda, MD, USA) DNA sequences and encoding a 40-kDa protein, has been found to be exclusively expressed in embryonic neurons. To investigate the function of (CPE/NF-α1)-ΔN in neurodevelopment, we first cloned (CPE/NF-α1)-ΔN transcripts from an embryonic mouse brain. A rapid amplification of cDNA ends assay, DNA sequencing, and Northern blot revealed 1.9- and 1.73-kb transcripts, which encoded 47- and 40-kDa (CPE/NF-α1)-ΔN proteins, respectively. Those proteins were expressed in embryonic mouse brain. Expression of the 2 (CPE/NF-α1)-ΔN mRNAs surged at embryonic d 10.5, correlating with the time of neurogenesis in the developing brain and also at postnatal d 1. HT22 cells, a mouse hippocampal cell line, transduced with 40 kDa (CPE/NF-α1)-ΔN up-regulated expression of genes involved in embryonic neurodevelopment: insulin-like growth factor binding protein 2 ( IGFBP2), death-associated protein 1, and ephrin A1, which regulate proliferation, programmed cell death, and neuronal migration, respectively. HT22 cells and embryonic cortical neurons overexpressing 40 kDa (CPE/NF-α1)-ΔN exhibited enhanced proliferation, which was inhibited by IGFBP2 short interfering RNA treatment. Thus, 40 kDa (CPE/NF-α1)-ΔN has an important, enzymatically independent role in the regulation of genes critical for neurodevelopment.-Xiao, L., Yang, X., Sharma, V. K., Loh, Y. P. Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain.

Carboxypeptidase E (CPE), a prohormone processing enzyme, is a 476- amino acid protein with a signal peptide in its N-terminus and is expressed in the nervous and the endocrine systems. Recent evidence indicate CPE plays various non-enzymatic roles in the endocrine and nervous systems and in various cancers. Besides wild type (WT) CPE, a 40-kDa CPE protein that localizes in the nucleus and cytoplasm has been described in embryonic mouse brain. In this study we have cloned this CPE variant encoding the 40kDa CPE-ΔN protein from human cancer cells. RACE assay and sequence analysis confirmed existence of this CPE variant mRNA, which has 198 nucleotides removed within the first exon and 589 nucleotides from the 3'-UTR, respectively, compared to WT-CPE mRNA. Bioinformatic analysis revealed that this CPE variant mRNA has a shortened open reading frame, which starts coding from the 3rd ATG relative to WT-CPE mRNA and encodes a 40kDa N-terminus truncated CPE protein. RT-PCR and Western blot analysis showed that 40kDa CPE-ΔN is expressed in multiple cancer cell lines and tumor tissues. Overexpression of this 40kDa CPE-ΔN variant up-regulated expression of multiple metastatic genes encompassing different signaling pathways, suggesting potentially an important role of CPE-ΔN in tumor metastasis., Competing Interests: COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests., (Copyright: © 2019 Yang et al.)

The repression of telomerase activity during cellular differentiation promotes replicative aging and functions as a physiological barrier for tumorigenesis in long-lived mammals, including humans. However, the underlying mechanisms remain largely unclear. Here we describe how miR-615-3p represses hTERT expression. mir-615-3p is located in an intron of the HOXC5 gene, a member of the highly conserved homeobox family of transcription factors controlling embryogenesis and development. Unexpectedly, we found that HoxC5 also represses hTERT expression by disrupting the long-range interaction between hTERT promoter and its distal enhancer. The 3'UTR of hTERT and its upstream enhancer region are well conserved in long-lived primates. Both mir-615-3p and HOXC5 are activated upon differentiation, which constitute a feed-forward loop that coordinates transcriptional and post-transcriptional repression of hTERT during cellular differentiation. Deregulation of HOXC5 and mir-615-3p expression may contribute to the activation of hTERT in human cancers.

Aim: To investigate biomarkers for predicting papillary thyroid cancer outcomes., Materials & Methods: The expression of biomarkers ( ITGA2, SYT12 and CDH3 ) was studied in a prospective cohort of patients with papillary thyroid cancer. Three outcomes of initial metastases, baseline status and longitudinal status were analyzed and correlated with the biomarkers., Results: SYT12 provided the best prediction of initial metastasis (sensitivity: 72%; specificity: 54%). SYT12 had the highest accuracy for predicting longitudinal status (sensitivity: 100%; specificity: 47%). The best performance for longitudinal status resulted from combining SYT12 with American Thyroid Association risk stratification, with sensitivity and specificity of 88 and 73%, respectively., Conclusion: SYT12 has some prognostic significance in papillary thyroid cancer. Further validation studies in larger populations are warranted., Competing Interests: Financial & competing interests disclosure The research of SRK Murthy, D Liu and YP Loh is supported by the Intramural Research Program of the National Institutes of Health (NIH), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Neurotrophins particularly brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are crucial modulators in the neurodevelopment and maintenance of central and peripheral nervous systems. Neurotrophin hypothesis of schizophrenia (SCZ) postulated that the changes in the brains of SCZ patients are the result of disturbances of developing processes involving neurotrophic factors. This hypothesis was mainly supported by the abnormal regulation of BDNF in SCZ, especially the decreased peripheral blood BDNF levels in SCZ patients validated by several meta-analyses. However, the regulation of NGF in SCZ remains unclear because of the inconsistent findings from the clinical studies. Therefore, we undertook, to the best of our knowledge, the first systematic review with a meta-analysis to quantitatively summarize the peripheral blood NGF data in SCZ patients compared with healthy control (HC) subjects. A systematic search of Pubmed, PsycINFO and Web of Science identified 13 articles encompassing a sample of 1693 individuals for the meta-analysis. Random-effects meta-analysis showed that patients with SCZ had significantly decreased peripheral blood levels of NGF when compared with the HC subjects (Hedges's g=-0.633, 95% confidence interval (CI)=-0.948 to -0.318, P<0.001). Subgroup analyses revealed reduced NGF levels both in serum (Hedges's g=-0.671, 95% CI=-1.259 to -0.084, P=0.025) and plasma (Hedges's g=-0.621, 95% CI=-0.980 to -0.261, P<0.001) of the patients, and in drug-free (Hedges's g=-0.670, 95% CI=-1.118 to -0.222, P=0.003) and medicated (Hedges's g=-0.357, 95% CI=-0.592 to -0.123, P=0.003) patients with SCZ. Furthermore, meta-regression analyses showed that age, gender and sample size had no moderating effects on the outcome of the meta-analysis, whereas disease severity might be a confounding factor for the meta-analysis. These results demonstrated that patients with SCZ are accompanied by the decreased peripheral blood NGF levels, strengthening the clinical evidence of an abnormal neurotrophin profile in the patients with SCZ.

Embryonic neurodevelopment involves inhibition of proliferation of multipotent neural stem cells (NSCs) followed by differentiation into neurons, astrocytes and oligodendrocytes to form the brain. We have identified a new neurotrophic factor, NF-α1, which inhibits proliferation and promotes differentiation of NSC/progenitors derived from E13.5 mouse cortex. Inhibition of proliferation of these cells was mediated through negatively regulating the Wnt pathway and decreasing β-catenin. NF-α1 induced differentiation of NSCs to astrocytes by enhancing Glial Fibrillary Acidic Protein (GFAP) expression through activating the ERK1/2-Sox9 signaling pathway. Cultured E13.5 cortical stem cells from NF-α1-knockout mice showed decreased astrocyte numbers compared to wild-type mice, which was rescued by treatment with NF-α1. In vivo, immunocytochemistry of brain sections and Western blot analysis of neocortex of mice showed a gradual increase of NF-α1 expression from E14.5 to P1 and a surge of GFAP expression at P1, the time of increase in astrogenesis. Importantly, NF-α1-Knockout mice showed ∼49% fewer GFAP positive astrocytes in the neocortex compared to WT mice at P1. Thus, NF-α1 is critical for regulating antiproliferation and cell fate determination, through differentiating embryonic stem cells to GFAP-positive astrocytes for normal neurodevelopment. Stem Cells 2017;35:557-571., (© 2016 AlphaMed Press.)

Studies suggest that dysfunction of brain-derived neurotrophic factor (BDNF) is a possible contributor to the pathology and symptoms of Alzheimer's disease (AD). Several studies report reduced peripheral blood levels of BDNF in AD, but findings are inconsistent. This study sought to quantitatively summarize the clinical BDNF data in patients with AD and mild cognitive impairment (MCI, a prodromal stage of AD) with a meta-analytical technique. A systematic search of Pubmed, PsycINFO and the Cochrane Library identified 29 articles for inclusion in the meta-analysis. Random-effects meta-analysis showed that patients with AD had significantly decreased baseline peripheral blood levels of BDNF compared with healthy control (HC) subjects (24 studies, Hedges' g=-0.339, 95% confidence interval (CI)=-0.572 to -0.106, P=0.004). MCI subjects showed a trend for decreased BDNF levels compared with HC subjects (14 studies, Hedges' g=-0.201, 95% CI=-0.413 to 0.010, P=0.062). No differences were found between AD and MCI subjects in BDNF levels (11 studies, Hedges' g=0.058, 95% CI=-0.120 to 0.236, P=0.522). Interestingly, the effective sizes and statistical significance improved after excluding studies with reported medication in patients (between AD and HC: 18 studies, Hedges' g=-0.492, P<0.001; between MCI and HC: 11 studies, Hedges' g=-0.339, P=0.003). These results strengthen the clinical evidence that AD or MCI is accompanied by reduced peripheral blood BDNF levels, supporting an association between the decreasing levels of BDNF and the progression of AD.

Report of a human with a homozygous truncating null mutation of the Carboxypeptidase E (CPE) gene with endocrinological and neurological deficits prompted us to search for other mutations in the human CPE gene that might be linked to disease. We searched an EST database and identified from a small population of patients, a novel T to C single nucleotide polymorphism (SNP) in the CPE gene at bp980 of exon 4, herein called TC-CPE. This introduces a tryptophan to arginine (W235R) mutation in the catalytic domain of human CPE protein. Over-expression of TC-CPE in N2A cells, a neuroendocrine cell line, showed that it was synthesized, but was found in lesser amounts compared to over-expressed WT-CPE in these cells. Furthermore, TC-CPE was secreted poorly from these N2A cells. The levels of TC-CPE were significantly increased after the N2A cells were treated with MG132 (a proteasome inhibitor), suggesting that TC-CPE was targeted to proteasomes for degradation in N2A cells. In addition, TC-CPE induced ER stress as demonstrated by the increased expression of CHOP in N2A cells. Double labeling of CPE and calnexin (and ER marker) suggested the accumulation of TC-CPE in the ER, and the accumulation appears to be enhanced by the treatment of MG132 in the cells. Moreover, the secreted levels of TC-CPE were not affected by the treatment of MG132 in the cells. Over-expression studies revealed that while N2A cells transfected with WT-CPE showed reduced cytotoxicity when challenged with H2O2 compared to cells expressing an empty vector, cells transfected with TC-CPE had no effect. Furthermore, WT-CPE condition medium showed protective effect against oxidative stress, but not TC-CPE condition medium. Although co-expression of WT-CPE and TC-CPE in N2A cells resulted in the reduction in secretion of WT-CPE, co-expression of WT-CPE and TC-CPE did not significantly affect the protective effect of WT-CPE. Taken together, we have identified a novel SNP in the CPE gene which results in the loss of its neuroprotective function in cells and may confer neurological disorders in humans., Competing Interests: The authors have declared that no competing interests exist.

Chromogranin A (CgA) is an acidic protein co-stored with catecholamines, hormones and neuropeptides in the secretory granules of endocrine, neuronal and other cell types (including cardiomyocytes). Proteolytic cleavage in the C terminus of CgA generates a 2.9kDa peptide named serpinin (Serp; Ala26Leu) that can be modified at its N terminus to form a pyroglutamate residue (pGlu-Serp). In the rat heart, both peptides increase contractility and relaxation through a β-adrenergic-like action mechanism. Accordingly, Serp and pGlu-Serp were proposed as novel myocardial sympatho-adrenergic modulators in mammals. On a comparative basis, here we report the actions of Serp and pGlu-Serp on myocardial contractility in three poikilotherm vertebrate species: the eel (Anguilla anguilla), the goldfish (Carassius auratus) and the frog (Rana esculenta). Using isolated working heart preparations, we show that pGlu-Serp reduces stroke volume in all species tested, while Serp reduces contractility in the frog heart, but is uneffective in eel and goldfish hearts. In the goldfish and frog hearts, pGlu-Serp activates the Nitric Oxide/cGMP pathway involving Endothelin-1 B receptors (frog) and β 3 adrenergic receptors (goldfish). pGlu-Serp-treated hearts from goldfish and frog show increased cGMP content. Moreover, the exposure of the frog heart to pGlu-Serp is accompanied by an increased expression of activated eNOS and Akt. In conclusion, this first report showing that pGlu-Serp inhibits mechanical cardiac performance in teleost and amphibians supports an evolutionary role of the CgA system, and particularly its serpinin component, in the sympatho-adrenergic control of the vertebrate heart., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Oxidative stress plays a critical role in neuronal injury and is associated with various neurological diseases. Here, we explored the potential protective effect of neuroserpin against oxidative stress in primary cultured hippocampal neurons. Our results show that neuroserpin inhibits H 2 O 2 -induced neurotoxicity in hippocampal cultures as measured by WST, LDH release, and TUNEL assays. We found that neuroserpin enhanced the activation of AKT in cultures subjected to oxidative stress and that the AKT inhibitor Ly294002 blocked this neuroprotective effect. Neuroserpin increased the expression of the anti-apoptotic protein BCL-2 and blocked the activation of caspase-3. Neuroserpin did not increase the level of neuroprotection over levels seen in neurons transduced with a BCL-2 expression vector, and an inhibitor of Trk receptors, K252a, did not block neuroserpin's effect. Taken together, our study demonstrates that neuroserpin protects against oxidative stress-induced dysfunction and death of primary cultured hippocampal neurons through the AKT-BCL-2 signaling pathway through a mechanism that does not involve the Trk receptors and leads to inhibition of caspase-3 activation.

The Wnt pathway has essential roles in cell proliferation, cell fate determination and tumorigenesis by regulating the expression of a wide range of target genes. As a core signaling cascade, the canonical Wnt pathway is regulated at different levels by numerous proteins. We have previously shown that carboxypeptidase E (CPE) is a novel regulator of the canonical Wnt signaling pathway. Here, we show that CPE and the Wnt3a ligand are co-secreted from cells. We show that although the C'-terminal Lys residue of Wnt3a is critical for its activity and is important for the effect of CPE on the Wnt pathway, CPE does not execute its effect by removing this Wnt3a residue. Interestingly, CPE through its N'-terminal sequence, forms aggregates with Wnt3a and possible endoplasmic reticulum (ER) stress leading to its loss of function. Together, our current results provide a mechanistic insight into the way CPE regulates the canonical Wnt signaling pathway.

Patients with Alzheimer's disease (AD), a common dementia among the aging population, often also suffer from depression. This comorbidity is poorly understood. Although most forms of AD are not genetically inherited, we have identified a new human mutation in the carboxypeptidase E (CPE)/neurotrophic factor-α1 (NF-α1) gene from an AD patient that caused memory deficit and depressive-like behavior in transgenic mice. This mutation consists of three adenosine inserts, introducing nine amino acids, including two glutamines into the mutant protein, herein called CPE-QQ. Expression of CPE-QQ in Neuro2a cells demonstrated that it was not secreted, but accumulated in the endoplasmic reticulum and was subsequently degraded by proteasomes. Expression of CPE-QQ in rat hippocampal neurons resulted in cell death, through increased ER stress and decreased expression of pro-survival protein, BCL-2. Transgenic mice expressing CPE-QQ did not show any difference in the processing enzyme activity of CPE compared with wild-type mice. However, the transgenic mice exhibited poor memory, depressive-like behavior, severely decreased dendrites in the hippocampal CA3 region and medial prefrontal cortex indicative of neurodegeneration, hyperphosphorylation of tau at Ser 396 , and diminished neurogenesis in the dentate gyrus at 50 weeks old. All these pathologies are associated with AD and the latter with depression and were observed in 50-week-old mice. Interestingly, the younger CPE-QQ mice (11 weeks old) did not show deficits in dendrite outgrowth and neurogenesis. This study has uncovered a human CPE/NF-α1 gene mutation that could lead to comorbidity of dementia and depression, emphasizing the importance of this gene in cognitive function.

Importance: The association of nonmotor features and Parkinson disease (PD) is increasingly recognized. Evidence suggests that inflammation may play a role in PD pathologic features and symptoms., Objective: To quantitatively summarize the peripheral inflammatory cytokine data available for patients with PD., Data Source: A systematic search of peer-reviewed English-language articles from PubMed, PsycINFO, and the Cochrane Library without year limitation was performed from December 7, 2015, to March 23, 2016. The search terms included inflammation or cytokine or chemokine or tumor necrosis factor or interleukin or interferon or C-reactive protein AND Parkinson disease., Study Selection: Studies were included if they provided data on peripheral blood cytokine concentrations in patients with PD and a healthy control group. Studies were excluded if they contained in vitro analysis of stimulated or unstimulated levels of cytokines, samples that overlapped with other studies, patients not diagnosed with PD at blood sampling, or if the cytokine analyzed was assessed in fewer than 3 studies., Data Extraction and Synthesis: Data were extracted from the 25 included studies encompassing 1547 unique patients with PD and 1107 unique controls by 2 independent investigators. Data were pooled using a random-effects model with the Comprehensive Meta-analysis software. Effect sizes were generated as standardized mean differences of cytokine concentrations between patients with PD and healthy controls and converted to the Hedges g statistic., Main Outcomes and Measures: Blood cytokine concentrations in patients with PD compared with controls. Aberrations in peripheral cytokine levels were hypothesized to be related to PD., Results: Among the 2654 study participants, concentrations of interleukin 6 (IL-6) (Hedges g, 0.325; 95% CI, 0.007-0.643; P = .045) in 13 studies, tumor necrosis factor (Hedges g, 0.354; 95% CI, 0.144-0.563; P = .001) in 9 studies, IL-1β (Hedges g, 0.382; 95% CI, 0.142-0.621; P = .002) in 6 studies, C-reactive protein (Hedges g, 0.323; 95% CI, 0.052-0.593; P = .02) in 6 studies, IL-10 (Hedges g, 0.329; 95% CI, 0.051-0.607; P = .02) in 5 studies, RANTES (regulated on activation, normal T-expressed, and presumably secreted) (Hedges g, 0.605; 95% CI, 0.111-1.099; P = .02) in 5 studies, and IL-2 (Hedges g, 0.789; 95% CI, 0.105-1.472; P = .02) in 3 studies were significantly higher in patients with PD compared with healthy controls. No differences were found between patients with PD and healthy controls for concentrations of interferon-γ (Hedges g, 0.745; 95% CI, -0.192 to 1.682; P = .12) in 5 studies, IL-4 (Hedges g, 0.031; 95% CI, -0.191 to 0.253; P = .79) in 3 studies, and IL-8 (Hedges g, 0.072; 95% CI, -0.136 to 0.279; P = .50) in 3 studies., Conclusions and Relevance: The findings of the meta-analysis demonstrated higher peripheral concentrations of IL-6, tumor necrosis factor, IL-1β, IL-2, IL-10, C-reactive protein, and RANTES in patients with PD, strengthening the clinical evidence that PD is accompanied by an inflammatory response.

Importance: Accumulating evidence suggests that brain-derived neurotrophic factor (BDNF) may be implicated in the developmental outcomes of children with autism spectrum disorder (ASD)., Objective: To use meta-analysis to determine whether children with ASD have altered peripheral blood levels of BDNF., Data Source: A systematic search of PubMed, PsycINFO, and Web of Science was performed for English-language literature through February 7, 2016. The search terms included brain-derived neurotrophic factor or BDNF in combination with autism, without year restriction. Two additional records were retrieved after a review of the reference lists of selected articles., Study Selection: Studies were included if they provided data on peripheral blood levels of BDNF in children with ASD and healthy control children. Studies that included adults or with overlapping samples were excluded., Data Extraction and Synthesis: Data were extracted by 2 independent observers from 19 included studies. Data were pooled using a random-effects model with Comprehensive Meta-analysis software., Main Outcomes and Measures: Blood levels of BDNF in children with ASD compared with healthy controls. Altered levels of BDNF were hypothesized to be related to ASD., Results: This meta-analysis included 19 studies with 2896 unique participants. Random-effects meta-analysis of all 19 studies showed that children with ASD had significantly increased peripheral blood levels of BDNF compared with healthy controls (Hedges g, 0.490; 95% CI, 0.185-0.794; P = .002). Subgroup analyses in 4 studies revealed that neonates diagnosed with ASD later in life had no association with blood levels of BDNF (Hedges g, 0.384; 95% CI, -0.244 to 1.011; P = .23), whereas children in the nonneonate ASD group (15 studies) demonstrated significantly increased BDNF levels compared with healthy controls (Hedges g, 0.524; 95% CI, 0.206 to 0.842; P = .001). Further analysis showed that children in the nonneonate ASD group had increased BDNF levels in serum (10 studies) (Hedges g, 0.564; 95% CI, 0.168 to 0.960; P = .005) but not in plasma (5 studies) (Hedges g, 0.436; 95% CI, -0.176 to 1.048; P = .16). Meta-regression analyses revealed that sample size had a moderating effect on the outcome of the meta-analysis in the nonneonate group. In addition, no publication bias was found in the meta-analysis., Conclusions and Relevance: Children with ASD have increased peripheral blood levels of BDNF, strengthening the clinical evidence of an abnormal neurotrophic factor profile in this population.