Nucleoredoxin is a thioredoxin-like redoxin that has been recognized as redox modulator of WNT signaling. Using a Yeast-2-Hybrid screen, we identified calcium calmodulin kinase 2a, Camk2a, as a prominent prey in a brain library. Camk2a is crucial for nitric oxide dependent processes of neuronal plasticity of learning and memory. Therefore, the present study assessed functions of NXN in neuronal Nestin-NXN^-/- deficient mice. The NXN-Camk2a interaction was confirmed by coimmunoprecipitation, and by colocalization in neuropil and dendritic spines. Functionally, Camk2a activity was reduced in NXN deficient neurons and restored with recombinant NXN. Proteomics revealed reduced oxidation in the hippocampus of Nestin-NXN^-/- deficient mice, including Camk2a, further synaptic and mitochondrial proteins, and was associated with a reduction of mitochondrial respiration. Nestin-NXN^-/- mice were healthy and behaved normally in behavioral tests of anxiety, activity and sociability. They had no cognitive deficits in touchscreen based learning & memory tasks, but omitted more trials showing a lower interest in the reward. They also engaged less in rewarding voluntary wheel running, and in exploratory behavior in IntelliCages. Accuracy was enhanced owing to the loss of exploration. The data suggested that NXN maintained the oxidative state of Camk2a and thereby its activity. In addition, it supported oxidation of other synaptic and mitochondrial proteins, and mitochondrial respiration. The loss of NXN-dependent pro-oxidative functions manifested in a loss of exploratory drive and reduced interest in reward in behaving mice.

Keywords: Behavior; Calcium calmodulin kinase; IntelliCage; Play; Pleasure; Proteomics; Redoxin.

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase II α (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C-terminus with phenylalanine compromising the αkap-α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap-α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

Keywords: AChR clusters; Muscle-specific anchoring protein; Phosphorylation of alpha dystrobrevin; nicotinic acetylcholine receptors (nAChR); proteasome inhbitor; protein complex; protein degradation; protein phosphorylation; protein stability; stability of αkap.

Adipogenesis and osteoblastogenesis (adipo-osteoblastogenesis) are closely related processes involving with the phosphorylation of numerous cytoplasmic proteins and key transcription factors. Despite the recognition of the importance of protein phosphorylation in adipo-osteoblastocyte biology, relatively little is known about the specific kinases for adipo-osteoblastogenesis. Here, we constructed the comprehensive gene transcriptional landscapes of kinases at 3, 5, and 7 days during adipo-osteoblastogenesis from human bone marrow mesenchymal stem cells (hMSCs). We identified forty-four and eight significant DEGs (differentially expressed genes) separately for adipo-osteoblastogenesis. Five significant DEGs, namely CAMK2A, NEK10, PAK3, PRKG2, and PTK2B, were simultaneously shared by adipo-osteoblastogenic anecdotes. Using a lentivirus system, we confirmed that PTK2B (non-receptor protein tyrosine kinase 2 beta) simultaneously inhibited adipo-osteoblastogenesis through RNAi assays, and PRKG2 (protein kinase cGMP-dependent 2) facilitated adipogenesis and weakened osteoblastogenesis. The only certainty was that the identified candidate significant DEGs encoding kinases responsible for protein phosphorylation, especially PTK2B and PRKG2, were the potential molecular switches of cell fate determination for hMSCs. This study would provide novel study targets for hMSC differentiation and potential clues for the therapy of the adipo-osteoblastogenic balance-derived disorders.

The hippocampus of cases with NFT pathology classified as stages I-II, III-IV, and V-VI without comorbidities, and middle-aged (MA) individuals with no NFT pathology, were examined to learn about the composition of granulovacuolar degeneration (GVD). Our results confirm the presence of CK1-δ, p38-P Thr180/Tyr182, SAPK/JNK-P Thr183/Thr185, GSK-3α/β-P Tyr279/Tyr216, and GSK-3β Ser9 in the cytoplasmic granules in a subset of neurons of the CA1 and CA2 subfields of the hippocampus. Also, we identify the presence of PKA α/β-P Thr197, SRC-P Tyr416, PAK1-P Ser199/Ser204, CAMK2A-P Tyr197, and PKCG-P Thr655 in cytoplasmic granules in cases with NFT pathology, but not in MA cases. Our results also confirm the presence of β-catenin-P Ser45/Thr41, IREα-P Ser274, eIF2α-P Ser51, TDP-43-P Ser403-404 (but absent TDP-43), and ubiquitin in cytoplasmic granules. Other components of the cytoplasmic granules are MAP2-P Thr1620/1623, MAP1B-P Thr1265, ADD1-P Ser726, and ADD1/ADD1-P Ser726/Ser713, in addition to several tau species including 3Rtau, 4Rtau, and tau-P Ser262. The analysis of GVD at progressive stages of NFT pathology reveals the early appearance of phosphorylated kinases and proteins in cytoplasmic granules at stages I-II, before the appearance of pre-tangles and NFTs. Most of these granules are not surrounded by LAMP1-positive membranes. Markers of impaired ubiquitin-protesome system, abnormal reticulum stress response, and altered endocytic and autophagic pathways occur in a subpopulation of neurons containing cytoplasmic granules, and they appear later. These observations suggest early phosphorylation of kinases leading to their activation, and resulting in the abnormal phosphorylation of various substrates, including tau, as a main alteration at the first stages of GVD.

Keywords: Alzheimer’s disease; brain aging; granulovacuolar degeneration; kinases; protein phosphorylation; tau.

Bipolar disorder (BPD) is a severe mental illness characterized by fluctuations in mood states, behaviors and energy levels. Growing evidence suggests that genes associated with specific illnesses tend to interact together and encode a tight protein-protein interaction (PPI) network, providing valuable information for understanding their pathogenesis. To gain insights into the genetic and physiological foundation of BPD, we conduct the physical PPI analysis of 184 BPD risk genes distilled from genome-wide association studies and exome sequencing studies. We have identified several hub genes (CAMK2A, HSP90AA1 and PLCG1) among those risk genes, and observed significant enrichment of the BPD risk genes in certain pathways such as calcium signaling, oxytocin signaling and circadian entrainment. Furthermore, while none of the 184 genetic risk genes are "well established" BPD drug targets, our PPI analysis showed that αCaMKII (encoded by CAMK2A) had direct physical PPIs with targets (HRH1, SCN5A and CACNA1E) of clinically used anti-manic BPD drugs, such as carbamazepine. We thus speculated that αCaMKII might be involved in the cellular pharmacological actions of those drugs. Using cultured rat primary cortical neurons, we found that carbamazepine treatment induced phosphorylation of αCaMKII in dose-dependent manners. Intriguingly, previous study showed that CAMK2A heterozygous knockout (CAMK2A^+/-) mice exhibited infradian oscillation of locomotor activities that can be rescued by carbamazepine. Our data, in combination with previous studies, provide convergent evidence for the involvement of CAMK2A in the risk of BPD.

It has become increasingly evident that the methylation of DNA, known as an epigenetic marker, affects behavior in animals. In our previous study, a methyl-donors (folate, methionine, and choline)-deficient (FMCD) diet during the juvenile period could be shown to affect anxiety-like behavior and fear memory, accompanied by alteration in some gene expression and their methylations in the hippocampus. One question is whether the fear memory of a parent affects the fear responses of offspring. To explore this question in the present study, C57BL/6 J male (F0) mice were given a FMCD diet from 3 to 12 weeks of age. After confirming the effect of the FMCD diet on the behavior and gene expression of F0 mice, their male offspring (F1-FMCD mice) were examined using the same behavioral batteries and genetic analysis. F0 diet-based differences in F1 behavior were observed, accompanied by the differences in the expression of memory-related genes (Camk2α and PP1) and promoter methylation of the PP1 gene in the hippocampus. Our results add evidence that behavior and gene expression of the F1 generation could be altered due to differences in the father's intake of methyl-donor nutrients.

The aim of this study was to investigate the effects of ethyl acetate extract from Coreopsis tinctoria (EACC) on learning and memory impairment in d-galactose-induced aging mice and the underlying molecular mechanism. The composition of EACC was analyzed by UPLC-MS, and the targets and pathways of EACC to improve learning and memory impairment were predicted and analyzed by the network pharmacology method. A mouse aging model was established by subcutaneous injection of d-galactose in mice, and EACC and piracetam were given to the model mice by gavage to observe their behavioral changes and changes in their SOD and GSH-Px activities in MDA contents in their peripheral blood serum and in the contents of Glu and GABA in their brain tissues. Then the hippocampus of the three mice selected from each of the MOD group and EACC-H group was separated for RT-qPCR assay. The results of the animal experiments showed that EACC could improve the learning and memory impairment of model mice by affecting the level of oxidative stress enzymes in serum and the content of neurotransmitters in the brain tissue. The results of network pharmacology analysis showed that the EACC components corresponded to 74 learning and memory-related targets, of which 13 were enriched in the long-term potentiation pathway. The results of RT-qPCR showed that 12 of the 13 detected targets were consistent with the predicted targets, and 9 of them were located in the NMDA receptor-related pathway of the long-term potentiation process and the pathway played an important regulatory role. It is believed that EACC could improve the learning and memory impairment of d-galactose-induced aging mice by acting on the nine targets Grin1, Grin2a, Camk2a, Camk2b, Kras, Raf1, Mapk1, Mapk3 and Creb to affect the NMDA receptor-related pathway of long-term potentiation.

Daylength regulates the development of spring migratory and subsequent reproductive phenotypes in avian migrants. This study used molecular approaches, and compared mRNA and proteome-wide expressions in captive redheaded buntings that were photostimulated under long days (LD) for 4 days (early stimulated, LD-eS) or for ∼3 weeks until each bird had shown successive 4 nights of Zugunruhe (stimulated, LD-S); controls were maintained under short-days. After ∼3 weeks of LD, photostimulated indices of the migratory preparedness (fattening, weight gain and Zugunruhe) were paralleled with upregulated expression of acc, dgat2 and apoa1 genes in the liver, and of cd36, fabp3 and cpt1 genes in the flight muscle, suggesting an enhanced fatty acids (FAs) synthesis and transport in the LD-S state. Concurrently elevated expression of genes involved in the calcium-ion signaling and transport (camk1 and atp2a2; camk2a in LD-eS instead), cellular stress (hspa8 and sod1, not nos2) and metabolic pathways (apoa1 and sirt1), but not of the genes associated with migratory behaviour (adcyap1 and vps13a), were found in the mediobasal hypothalamus (MBH). Further, the MBH-specific quantitative proteomics revealed that out of 503 annotated proteins, 28 were differentially expressed (LD-eS vs. LD-S: 21 up- and 7 down-regulated) and they enriched five physiological pathways that are associated with fatty acids transport and metabolism. These first comprehensive results on gene and protein expressions suggest that changes in molecular correlates of fatty acids transport and metabolism may aid the decision for migratory departure from wintering areas in obligate songbird migrants.

Keywords: Bunting; Gene expression; Migration; Proteome; Songbird.

Day length regulates the development of spring migratory and subsequent reproductive phenotypes in avian migrants. This study used molecular approaches, and compared mRNA and proteome-wide expression in captive redheaded buntings that were photostimulated under long-day (LD) conditions for 4 days (early stimulated, LD-eS) or for ∼3 weeks until each bird had shown 4 successive nights of Zugunruhe (stimulated, LD-S); controls were maintained under short days. After ∼3 weeks of LD, photostimulated indices of the migratory preparedness (fattening, weight gain and Zugunruhe) were paralleled with upregulated expression of acc, dgat2 and apoa1 genes in the liver, and of cd36, fabp3 and cpt1 genes in the flight muscle, suggesting enhanced fatty acid (FA) synthesis and transport in the LD-S state. Concurrently, elevated expression of genes involved in the calcium ion signalling and transport (camk1 and atp2a2; camk2a in LD-eS), cellular stress (hspa8 and sod1, not nos2) and metabolic pathways (apoa1 and sirt1), but not of genes associated with migratory behaviour (adcyap1 and vps13a), were found in the mediobasal hypothalamus (MBH). Further, MBH-specific quantitative proteomics revealed that out of 503 annotated proteins, 28 were differentially expressed (LD-eS versus LD-S: 21 up-regulated and 7 down-regulated) and they enriched five physiological pathways that are associated with FA transport and metabolism. These first comprehensive results on gene and protein expression suggest that changes in molecular correlates of FA transport and metabolism may aid the decision for migratory departure from wintering areas in obligate songbird migrants.

Keywords: Bunting; Gene expression; Migration; Proteome; Songbird.

Investigating novel genetic variants involved in intellectual disability (ID) development is essential. X-linked intellectual disability (XLID) accounts for over 10% of all cases of ID in males. XLID genes are involved in many cellular pathways and processes. Some of them are not specific to the development and functioning of the neural system. The implementation of exome sequencing simplifies the search for novel variants, especially those less expected. Here, we describe a nonsense variant of the XLID gene, WDR13. The mutation c.757C>T (p.Arg253Ter) was uncovered by X-chromosome exome sequencing in males with a familial form of intellectual disability. Quantitative PCR (qPCR) analysis showed that variant c.757C>T caused a significant decrease in WDR13 expression in the patient's fibroblast. Moreover, it dysregulated other genes linked to intellectual disability, such as FMR1, SYN1, CAMK2A, and THOC2. The obtained results indicate the pathogenic nature of the detected variant and suggest that the WDR13 gene interacts with other genes essential for the functioning of the nervous system, especially the synaptic plasticity process.

Keywords: WD-40 protein family; X-chromosome exome sequencing; X-linked intellectual disability.

Growth mechanism of economically important aquaculture species has aroused widespread interest among scholars. Rice flower carp (Cyprinus carpio), commonly cultured in rice-fish farming systems, shows wide variation in body mass at the same age, which limits the development of commercial aquaculture. In this study, muscle tissues from 20-month-old fish of different sizes were used for transcriptome analysis and muscle histological studies. The muscle histological analysis showed the muscle growth in rice flower carp main depends on the hypertrophic growth of muscle fibers. A total of 30,590 unigenes were generated by muscle trancriptome analysis, including 403 differentially expressed genes (DEGs). Of these, 157 DEGs were upregulated and 246 DEGs were downregulated. Nine unigenes related to the ubiquitin-proteasome pathway were identified using differential expression analysis. This study initially revealed that the differences in growth of rice flower carp could be due to hypertrophic growth of muscle fibers caused by higher protein deposition, and the ubiquitin-proteasome pathway was an important factor affecting the growth rate of rice flower carp. E3 ubiquitin-protein ligase ari7, g2e3, Neurl1 and rnf144ab were upregulated in the slow-growing fish, indicating the binding of ubiquitin to target protein was enhanced. Foxo3 was upregulated in the slow-growing fish, which could promote the muscle loss. Eif4a2 was upregulated in the fast-growing fish, increasing protein translation efficiency. Some genes related to active muscle contraction such as actb, actg, camk2a, and camk2b were upregulated in the fast-growing rice flower carp muscle. In summary, these results provide valuable information about the key genes for use as biomarkers of growth in selective breeding programs for rice flower carp and provide novel insights into the regulatory mechanisms of muscle growth.

Keywords: Cyprinus carpio; Hypertrophic growth; Protein deposition; Ubiquitin-proteasome pathway.

Fructose 1,6-bisphosphatase 2 (Fbp2) is a gluconeogenic enzyme and multifunctional protein modulating mitochondrial function and synaptic plasticity via protein-protein interactions. The ability of Fbp2 to bind to its cellular partners depends on a quaternary arrangement of the protein. NAD⁺ and AMP stabilize an inactive T-state of Fbp2 and thus, affect these interactions. However, more subtle structural changes evoked by the binding of catalytic cations may also change the affinity of Fbp2 to its cellular partners. In this report, we demonstrate that Fbp2 interacts with Co²⁺, a cation which in excessive concentrations, causes pathologies of the central nervous system and which has been shown to provoke the octal-like events in hippocampal slices. We describe for the first time the kinetics of Fbp2 in the presence of Co²⁺, and we provide a line of evidence that Co²⁺ blocks the AMP-induced transition of Fbp2 to the canonical T-state triggering instead of a new, non-canonical T-state. In such a state, Fbp2 is still partially active and may interact with its binding partners e.g., Ca²⁺/calmodulin-dependent protein kinase 2α (Camk2α). The Fbp2-Camk2α complex seems to be restricted to mitochondria membrane and it facilitates the Camk2α autoactivation and thus, synaptic plasticity.

Keywords: Fbp2; mitochondria; moonlighting protein; protein-protein interaction.

Background: The pathogenesis of glioma-related seizures (GRS) is poorly understood. Here in, we aim to identify putative molecular pathways that lead to the development of GRS.

Methods: We determined brain transcriptome from intraoperative human brain tissue of patients with either GRS, glioma without seizures (non-GRS), or with idiopathic temporal lobe epilepsy (iTLE). We performed transcriptome-wide comparisons between disease groups tissue from non-epileptic controls (non-EC) to identify differentially-expressed genes (DEG). We compared DEGs to identify those that are specific or common to the groups. Through a gene ontology analysis, we identified molecular pathways enriched for genes with a Log-fold change ≥1.5 or ≤-1.5 and p-value <0.05 compared to non-EC.

Results: We identified 110 DEGs that are associated with GRS vs. non-GRS: 80 genes showed high and 30 low expression in GRS. There was significant overexpression of genes involved in cell-to-cell and glutamatergic signaling (CELF4, SLC17A7, and CAMK2A) and down-regulation of genes involved immune-trafficking (CXCL8, H19, and VEGFA). In the iTLE vs GRS analysis, there were 1098 DEGs: 786 genes were overexpressed and 312 genes were underexpressed in the GRS samples. There was significant enrichment for genes considered markers of oncogenesis (GSC, MYBL2, and TOP2A). Further, there was down-regulation of genes involved in the glutamatergic neurotransmission (vesicular glutamate transporter-2) in the GRS vs. iTLE samples.

Conclusions: We identified a number of altered processes such as cell-to-cell signaling and interaction, inflammation-related, and glutamatergic neurotransmission in the pathogenesis of GRS. Our findings offer a new landscape of targets to further study in the fields of brain tumors and seizures.

Keywords: Brain tumor-related epilepsy; Cytokines; Epileptogenesis; Glioma-related seizures; Glutamatergic signaling; Immune-trafficking; Vesicular glutamate transporter.

Camk2a-Cre mice have been widely used to study the postnatal function of several genes in forebrain projection neurons, including cortical projection neurons (CPNs) and striatal medium-sized spiny neurons (MSNs). We linked heterozygous deletion of TSHZ3/Tshz3 gene to autism spectrum disorder (ASD) and used Camk2a-Cre mice to investigate the postnatal function of Tshz3, which is expressed by CPNs but not MSNs. Recently, single-cell transcriptomics of the adult mouse striatum revealed the expression of Camk2a in interneurons and showed Tshz3 expression in striatal cholinergic interneurons (SCINs), which are attracting increasing interest in the field of ASD. These data and the phenotypic similarity between the mice with Tshz3 haploinsufficiency and Camk2a-Cre-dependent conditional deletion of Tshz3 (Camk2a-cKO) prompted us to better characterize the expression of Tshz3 and the activity of Camk2a-Cre transgene in the striatum. Here, we show that the great majority of Tshz3-expressing cells are SCINs and that all SCINs express Tshz3. Using lineage tracing, we demonstrate that the Camk2a-Cre transgene is expressed in the SCIN lineage where it can efficiently elicit the deletion of the Tshz3-floxed allele. Moreover, transcriptomic and bioinformatic analysis in Camk2a-cKO mice showed dysregulated striatal expression of a number of genes, including genes whose human orthologues are associated with ASD and synaptic signaling. These findings identifying the expression of the Camk2a-Cre transgene in SCINs lineage lead to a reappraisal of the interpretation of experiments using Camk2a-Cre-dependent gene manipulations. They are also useful to decipher the cellular and molecular substrates of the ASD-related behavioral abnormalities observed in Tshz3 mouse models.

Keywords: Camk2a-Cre; Mus musculus; TSHZ3; autism spectrum disorder; striatal cholinergic interneurons.

Background: The development of functional neural circuits requires the precise formation of synaptic connections between diverse neuronal populations. The molecular pathways that allow GABAergic interneuron subtypes in the mammalian brain to initially recognize their postsynaptic partners remain largely unknown. The transmembrane glycoprotein Dystroglycan is localized to inhibitory synapses in pyramidal neurons, where it is required for the proper function of CCK+ interneurons. However, the precise temporal requirement for Dystroglycan during inhibitory synapse development has not been examined.

Methods: In this study, we use NEX^Cre or Camk2a^CreERT2 to conditionally delete Dystroglycan from newly-born or adult pyramidal neurons, respectively. We then analyze forebrain development from postnatal day 3 through adulthood, with a particular focus on CCK+ interneurons.

Results: In the absence of postsynaptic Dystroglycan in developing pyramidal neurons, presynaptic CCK+ interneurons fail to elaborate their axons and largely disappear from the cortex, hippocampus, amygdala, and olfactory bulb during the first two postnatal weeks. Other interneuron subtypes are unaffected, indicating that CCK+ interneurons are unique in their requirement for postsynaptic Dystroglycan. Dystroglycan does not appear to be required in adult pyramidal neurons to maintain CCK+ interneurons. Bax deletion did not rescue CCK+ interneurons in Dystroglycan mutants during development, suggesting that they are not eliminated by canonical apoptosis. Rather, we observed increased innervation of the striatum, suggesting that the few remaining CCK+ interneurons re-directed their axons to neighboring areas where Dystroglycan expression remained intact.

Conclusion: Together these findings show that Dystroglycan functions as part of a synaptic partner recognition complex that is required early for CCK+ interneuron development in the forebrain.

Keywords: Apoptosis; Cannabinoid receptor; Cholecystokinin; Dystroglycan; Interneuron; Synapse.

Objective To study the stemness characteristics of uterine corpus endometrial carcinoma(UCEC)and its potential regulatory mechanism.Methods Transcriptome sequencing data of UCEC was obtained from The Cancer Genome Atlas.Gene expression profile was normalized by edgeR package in R3.5.1.A one-class logistic regression machine learning algorithm was employed to calculated the mRNA stemness index(mRNAsi)of each UCEC sample.Then,the prognostic significance of mRNAsi and candidate genes was evaluated by survminer and survival packages.The high-frequency sub-pathways mining approach(HiFreSP)was used to identify the prognosis-related sub-pathways enriched with differentially expressed genes(DEGs).Subsequently,a gene co-expression network was constructed using WGCNA package,and the key gene modules were analyzed.The clusterProfiler package was adopted to the function annotation of the modules highly correlated with mRNAsi.Finally,the Human Protein Atlas(HPA)was retrieved for immunohistochemical validation.Results The mRNAsi of UCEC samples was significantly higher than that of normal tissues(t=25.095,P<0.001),and the lower degree of differentiation corresponded to higher mRNAsi in tumor tissues.The mRNAsi of UCEC increased gradually with tumor staging.The prognostic analysis showed that high mRNAsi was correlated with short overall survival in patients with UCEC(χ²=6.864,P=0.0088).There were 570 DEGs between the high-and low-mRNAsi groups.By using the HiFreSP algorithm,we identified that the oocyte meiosis sub-pathway(Oocyte meiosis_1)and cell cycle sub-pathway(Cell cycle_3)had significant prognostic significance.These pathways contained 11 DEGs(MAD2L1,CAMK2A,PTTG1,PLK1,CCNE1,CCNE2,ESPL1,CDC20,CCNB1,CCNB2,and SMC1B),which were significantly associated with the prognosis of UCEC patients.Gene co-expression network showed that mRNAsi,as well as MAD2L1,CAMK2A,and PTTG1,was associated with three gene modules.The immunohistochemical analysis demonstrated that MAD2L1 and PTTG1 showed up-regulated expression while CAMK2A showed down-regulated expression in UCEC,which was consistent with the results of transcriptome sequencing.Conclusions On the basis of machine learning,this study characterizes the stemness characteristics of UCEC.We identify the key sub-pathways related to prognosis and demonstrate that MAD2L1,CAMK2A,PTTG1 are closely related to the stemness of UCEC,which provides insight into the regulatory mechanism of cancer stemness and reveals the potential therapeutic targets of UCEC.

Keywords: biomarker; cancer stem cell; co-expression network; stemness index; uterine corpus endometrial carcinoma.

Genomic alterations are crucial for the development and progression of human cancers. Copy number gains found in genes encoding metabolic enzymes may induce triple-negative breast cancer (TNBC) adaptation. However, little is known about how metabolic enzymes regulate TNBC metastasis. Using our previously constructed multiomic profiling of a TNBC cohort, we identified decaprenyl diphosphate synthase subunit 1 (PDSS1) as an essential gene for TNBC metastasis. PDSS1 expression was significantly upregulated in TNBC tissues compared to adjacent normal tissues and was positively associated with poor survival among TNBC patients. PDSS1 knockdown inhibited TNBC cell migration, invasion, and distant metastasis. Mechanistically, PDSS1, but not a catalytically inactive mutant, positively regulated the cellular level of coenzyme Q10 (CoQ10) and intracellular calcium levels, thereby inducing CAMK2A phosphorylation, which is essential for STAT3 phosphorylation in the cytoplasm. Phosphorylated STAT3 entered the nucleus, promoting oncogenic STAT3 signaling and TNBC metastasis. STAT3 phosphorylation inhibitors (e.g., Stattic) effectively blocked PDSS1-induced cell migration and invasion in vitro and tumor metastasis in vivo. Taken together, our study highlights the importance of targeting the previously uncharacterized PDSS1/CAMK2A/STAT3 oncogenic signaling axis, expanding the repertoire of precision medicine in TNBC.

Down syndrome (DS) is the leading genetic cause of intellectual disability and causes early-onset dementia and cerebellar hypoplasia. The prevalence of attention deficit hyperactivity disorder is elevated in children with DS. The aneuploid DS mouse model "Ts65Dn" shows prominent brain phenotypes, including learning and memory deficits, cerebellar hypoplasia, and locomotor hyperactivity. Previous studies indicate that impaired Sonic hedgehog (Shh) signaling contributes to neurological phenotypes associated with DS and neurodegenerative diseases. However, because of a lack of working inducible Shh knock-in mice, brain region-specific Shh overexpression and its effects on cognitive function have not been studied in vivo. Here, with Gli1-LacZ reporter mice, we demonstrated that Ts65Dn had reduced levels of Gli1, a sensitive readout of Shh signaling, in both hippocampus and cerebellum at postnatal day 6. Through site-specific transgenesis, we generated an inducible human Shh knock-in mouse, TRE-bi-hShh-Zsgreen1 (TRE-hShh), simultaneously expressing dually-lipidated Shh-Np and Zsgreen1 marker in the presence of transactivator (tTA). Double transgenic mice "Camk2a-tTA;TRE-hShh" and "Pcp2-tTA;TRE-hShh" induced Shh overexpression and activated Shh signaling in a forebrain and cerebellum, respectively, specific manner from the perinatal period. Camk2a-tTA;TRE-hShh normalized locomotor hyperactivity and improved learning and memory in 3-month-old Ts65Dn, mitigated early-onset severe cognitive impairment in 7-month-old Ts65Dn, and enhanced spatial cognition in euploid mice. Camk2a-tTA;TRE-hShh cohort maintained until 600days old showed that chronic overexpression of Shh in forebrain from the perinatal period had no effect on longevity of euploid or Ts65Dn. Pcp2-tTA;TRE-hShh did not affect cognition but mitigated the phenotype of cerebellar hypoplasia in Ts65Dn. Our study provides the first in vivo evidence that Shh overexpression from the perinatal period protects DS brain integrity and enhances learning and memory in normal mice, indicating the broad therapeutic potential of Shh ligand for other neurological conditions. Moreover, the first inducible hShh site-specific knock-in mouse could be widely used for spatiotemporal Shh signaling regulation.

Keywords: Cognitive function; Down Syndrome; Hyperactivity; Sonic hedgehog; TRE-hShh; Ts65Dn.

Aiming at a more comprehensive understanding of the molecular biomarkers and potential mechanisms of major depressive disorder (MDD), from the Gene Expression Omnibus (GEO) database, we first obtained mRNA expression profiles and identified 585 differentially expressed genes (DEGs) through the R software, including 263 upregulated genes and 322 downregulated genes. Then, through the Kyoto Encyclopedia of Genome and Genome (KEGG) pathway and biological process (BP) analysis, we found that the upregulated and downregulated DEGs were abundant in different pathways, respectively. It was noteworthy that upregulated DEGs were the most significantly enriched in the mTOR signaling pathway. Subsequently, through the protein-protein interaction (PPI) network, we identified seven hub genes, namely, EXOSC2, CAMK2A, PRIM1, SMC4, TYMS, CDK6, and RPA2. Finally, through gene set enrichment analysis (GSEA), we obtained that hypoxia, epithelial-mesenchymal transition, hedgehog signaling, and reactive oxygen species pathway were the enriched pathways for MDD patients. The above data results would provide a new direction for the treatment of MDD patients.

The molecular heterogeneity of gene expression profiles of glioblastoma multiforme (GBM) are the most important prognostic factors for tumor recurrence and drug resistance. Thus, the aim of this study was to identify potential target genes related to temozolomide (TMZ) resistance and GBM recurrence. The genomic data of patients with GBM from The Cancer Genome Atlas (TCGA; 154 primary and 13 recurrent tumors) and a local cohort (29 primary and 4 recurrent tumors), samples from different tumor regions from a local cohort (29 tumor and 25 peritumoral regions), and Gene Expression Omnibus data (GSE84465, single-cell RNA sequencing; 3589 cells) were included in this study. Critical gene signatures were identified based an analysis of differentially expressed genes (DEGs). DEGs were further used to evaluate gene enrichment levels among primary and recurrent GBMs and different tumor regions through gene set enrichment analysis. Protein-protein interactions (PPIs) were incorporated into gene regulatory networks to identify the affected metabolic pathways. The enrichment levels of 135 genes were identified in the peritumoral regions as being risk signatures for tumor recurrence. Fourteen genes (DVL1, PRKACB, ARRB1, APC, MAPK9, CAMK2A, PRKCB, CACNA1A, ERBB4, RASGRF1, NF1, RPS6KA2, MAPK8IP2, and PPM1A) derived from the PPI network of 135 genes were upregulated and involved in the regulation of cancer stem cell (CSC) development and relevant signaling pathways (Notch, Hedgehog, Wnt, and MAPK). The single-cell data analysis results indicated that 14 key genes were mainly expressed in oligodendrocyte progenitor cells, which could produce a CSC niche in the peritumoral region. The enrichment levels of 336 genes were identified as biomarkers for evaluating TMZ resistance in the solid tumor region. Eleven genes (ARID5A, CDC42EP3, CDKN1A, FLT3, JUNB, MAP2K3, MYBPC2, RGS14, RNASEK, TBC1D30, and TXNDC11) derived from the PPI network of 336 genes were upregulated and may be associated with a high risk of TMZ resistance; these genes were identified in both the TCGA and local cohorts. Furthermore, the expression patterns of ARID5A, CDKN1A, and MAP2K3 were identical to the gene signatures of TMZ-resistant cell lines. The identified enrichment levels of the two gene sets expressed in tumor and peritumoral regions are potentially helpful for evaluating TMZ resistance in GBM. Moreover, these key genes could be used as biomarkers, potentially providing new molecular strategies for GBM treatment.

Keywords: TMZ resistance; biomarker; glioblastoma; tumor recurrence.

The hippocampus is essential for consolidating transient experiences into long-lasting memories. Memory consolidation is facilitated by postlearning sleep, although the underlying cellular mechanisms are largely unknown. We took an unbiased approach to this question by using a mouse model of hippocampally mediated, sleep-dependent memory consolidation (contextual fear memory). Because synaptic plasticity is associated with changes to both neuronal cell membranes (e.g., receptors) and cytosol (e.g., cytoskeletal elements), we characterized how these cell compartments are affected by learning and subsequent sleep or sleep deprivation (SD). Translating ribosome affinity purification was used to profile ribosome-associated RNAs in different subcellular compartments (cytosol and membrane) and in different cell populations (whole hippocampus, Camk2a+ neurons, or highly active neurons with phosphorylated ribosomal subunit S6 [pS6+]). We examined how transcript profiles change as a function of sleep versus SD and prior learning (contextual fear conditioning; CFC). While sleep loss altered many cytosolic ribosomal transcripts, CFC altered almost none, and CFC-driven changes were occluded by subsequent SD. In striking contrast, SD altered few transcripts on membrane-bound (MB) ribosomes, while learning altered many more (including long non-coding RNAs [lncRNAs]). The cellular pathways most affected by CFC were involved in structural remodeling. Comparisons of post-CFC MB transcript profiles between sleeping and SD mice implicated changes in cellular metabolism in Camk2a+ neurons and protein synthesis in highly active pS6+ (putative "engram") neurons as biological processes disrupted by SD. These findings provide insights into how learning affects hippocampal neurons and suggest that the effects of SD on memory consolidation are cell type and subcellular compartment specific.

Keywords: bioinformatics; memory consolidation; ribosomes; synaptic plasticity; translation.

Bipolar disorder (BD) is a severe psychiatric disorder characterized by repeated conflicting manic and depressive states. In addition to genetic factors, complex gene-environment interactions, which alter the epigenetic status in the brain, contribute to the etiology and pathophysiology of BD. Here, we performed a promoter-wide DNA methylation analysis of neurons and nonneurons derived from the frontal cortices of mutant Polg1 transgenic (n = 6) and wild-type mice (n = 6). The mutant mice expressed a proofreading-deficient mitochondrial DNA (mtDNA) polymerase under the neuron-specific CamK2a promoter and showed BD-like behavioral abnormalities, such as activity changes and altered circadian rhythms. We identified a total of 469 differentially methylated regions (DMRs), consisting of 267 neuronal and 202 nonneuronal DMRs. Gene ontology analysis of DMR-associated genes showed that cell cycle-, cell division-, and inhibition of peptide activity-related genes were enriched in neurons, whereas synapse- and GABA-related genes were enriched in nonneurons. Among the DMR-associated genes, Trim2 and Lrpprc showed an inverse relationship between DNA methylation and gene expression status. In addition, we observed that mutant Polg1 transgenic mice shared several features of DNA methylation changes in postmortem brains of patients with BD, such as dominant hypomethylation changes in neurons, which include hypomethylation of the molecular motor gene and altered DNA methylation of synapse-related genes in nonneurons. Taken together, the DMRs identified in this study will contribute to understanding the pathophysiology of BD from an epigenetic perspective.

Keywords: Bipolar disorder; DNA methylation; Mitochondrial dysfunction; Neuron; Nonneuron; Schizophrenia.

Objectives: Aging and common diseases alter the stiffness of bone tissue, causing changes to the microenvironment of the mechanosensitive bone cells. Osteoclasts, the sole bone-resorbing cells, play a vital role in bone remodeling. This study was performed to elucidate the mechanism through which osteoclasts sense and react to substrate stiffness signals.

Materials and methods: We fabricated polydimethylsiloxane (PDMS) substrates of different stiffness degrees for osteoclast formation progressed from osteoclast precursors including bone marrow-derived macrophages (BMMs) and RAW264.7 monocytes. Osteoclast differentiation in response to the stiffness signals was determined by examining the cell morphology, fusion/fission activities, transcriptional profile, and resorption function. Cytoskeletal changes and mechanosensitive adhesion molecules were also assessed.

Results: Stiffer PDMS substrates accelerated osteoclast differentiation, firstly observed by variations in their morphology and fusion/fission activities. Upregulation of canonical osteoclast markers (Nfatc1, Acp5, Ctsk, Camk2a, Mmp9, Rela, and Traf6) and the fusion master regulator DC-stamp were detected on stiffer substrates, with similar increases in their bone resorption functions. Additionally, the activation of cytoskeleton-associated adhesion molecules, including fibronectin and integrin αvβ3, followed by biochemical signaling cascades of paxillin, FAK, PKC, and RhoA, was detected on the stiffer substrates.

Conclusions: This is the first study to provide evidence proving that extracellular substrate stiffness is a strong determinant of osteoclast differentiation and functions. Higher stiffness upregulated the differentiation profile and activity of osteoclasts, revealing the mechanical regulation of osteoclast activity in bone homeostasis and diseases.

Keywords: PDMS substrates; cytoskeletons; osteoclast differentiation; osteoclasts; substrate stiffness.

Introduction: Prenatal stress (PS) can cause depression in offspring. However, the underlying biological mechanism of these influences is still unclear. This work was implemented to investigate the molecular mechanisms of depressive-like behavior of offspring rats insulted with PS.

Methods: Relative quantitative phosphoproteomics of the hippocampus of PS susceptibility (PS-S) and control (CON) rat offspring was performed using liquid chromatography-tandem mass spectrometry to confirm known pathways and to identify new mechanisms involved in depression.

Results: A total of 6790 phosphopeptides, 9817 phosphorylation sites, and 2978 phosphoproteins were detected. Among the 2978 phosphoproteins, 1760 (59.09%) had more than two phosphorylated sites, the ENSRNOP00000023460 protein had more than 117 phosphorylated sites, and the average distribution of modification sites per 100 amino acids was 2.97. There were 197 different phosphopeptides, including 140 increased phosphopeptides and 57 decreased phosphopeptides in the PS-S offspring rats, compared to the CON offspring rats. These differential phosphopeptides corresponded to 100 upregulated and 44 downregulated phosphoproteins, respectively. Gene ontology enrichment analysis revealed that these different phosphoproteins in the top five enriched terms in the cellular component, molecular function, and biological proces categories were involved in a total of 35 different phosphoproteins, and these phosphoproteins were mainly related to myelin-, microtubule- and synapse-associated proteins. The enrichment of Kyoto Encyclopedia of Genes and Genome pathways was found to be involved in many essential biological pathways, and the top five pathways included amphetamine addiction, insulin secretion, Cushing syndrome, and the circadian entrainment signaling pathway. These first five pathways were related to nine phosphoproteins, including Adcy9, Apc, Cacna1c, Camk2a, Camk2b, Camk2g, Ctnnd2, Grin2a, and Stx1a. The full data are available via ProteomeXchange with identifier PXD019117.

Conclusion: We preliminarily identified 144 different phosphoproteins involved in myelin, microtubule, and synapse formation and plasticity in the hippocampus of susceptible offspring rats exposed to PS.

Keywords: depressive-like behaviors; hippocampus; offspring; phosphoproteomics; prenatal stress.