TRP channels in thermosensation

The ability to sense external temperature is assumed by somatosensory neurons, in which temperature information is converted to neural activity by afferent input to the central nervous system. Somatosensory neurons consist of various populations with specialized gene expression, including thermosensitive transient receptor potential ion channels (thermo-TRPs). Thermo-TRPs are responsible for thermal transduction at the peripheral ends of somatosensory neurons and over a wide range of temperatures. In this review, we focus on several thermo-TRPs expressed in sensory neurons: TRPV1, TRPV4, TRPM2, TRPM3, TRPM8, TRPC5, and TRPA1. TRPV3, TRPV4, and TRPC5 expressed in non-neuronal cells that are also involved in somatosensation are also discussed, whereas TRPM2 and TRPM8 are involved in thermosensation in the brain.     

Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina

Phosphatidic acid is a key signaling molecule heavily implicated in exocytosis due to its protein-binding partners and propensity to induce negative membrane curvature. One phosphatidic acid-producing enzyme, phospholipase D (PLD), has also been implicated in neurotransmission. Unfortunately, due to the unreliability of reagents, there has been confusion in the literature regarding the expression of PLD isoforms in the mammalian brain which has hampered our understanding of their functional roles in neurons. To address this, we generated epitope-tagged PLD1 and PLD2 knockin mice using CRISPR/Cas9. Using these mice, we show that PLD1 and PLD2 are both localized at synapses by adulthood, with PLD2 expression being considerably higher in glial cells and PLD1 expression predominating in neurons. Interestingly, we observed that only PLD1 is expressed in the mouse retina, where it is found in the synaptic plexiform layers. These data provide critical information regarding the localization and potential role of PLDs in the central nervous system.     

Impaired neurite development and mitochondrial dysfunction associated with calcium accumulation in dopaminergic neurons differentiated from the dental pulp stem cells of a patient with metatropic dysplasia

Transient receptor potential vanilloid member 4 (TRPV4) is a Ca²⁺ permeable nonselective cation channel, and mutations in the TRPV4 gene cause congenital skeletal dysplasias and peripheral neuropathies. Although TRPV4 is widely expressed in the brain, few studies have assessed the pathogenesis of TRPV4 mutations in the brain. We aimed to elucidate the pathological associations between a specific TRPV4 mutation and neurodevelopmental defects using dopaminergic neurons (DNs) differentiated from dental pulp stem cells (DPSCs). DPSCs were isolated from a patient with metatropic dysplasia and multiple neuropsychiatric symptoms caused by a gain-of-function TRPV4 mutation, c.1855C>T (p.L619F). The mutation was corrected by CRISPR/Cas9 to obtain isogenic control DPSCs. Mutant DPSCs differentiated into DNs without undergoing apoptosis; however, neurite development was significantly impaired in mutant vs. control DNs. Mutant DNs also showed accumulation of mitochondrial Ca²⁺ and reactive oxygen species, low adenosine triphosphate levels despite a high mitochondrial membrane potential, and lower peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression and mitochondrial content. These results suggested that the persistent Ca²⁺ entry through the constitutively activated TRPV4 might perturb the adaptive coordination of multiple mitochondrial functions, including oxidative phosphorylation, redox control, and biogenesis, required for dopaminergic circuit development in the brain. Thus, certain mutations in TRPV4 that are associated with skeletal dysplasia might have pathogenic effects on brain development, and mitochondria might be a potential therapeutic target to alleviate the neuropsychiatric symptoms of TRPV4-related diseases.     

Neurod1 mediates the reprogramming of NG2 glial into neurons in vitro

Neuronal defect and loss are the main pathological processes of many central nervous system diseases. Cellular reprogramming is a promising method to supplement lost neurons. However, study on cellular reprogramming is still limited and its mechanism remains unclear. Herein, the effect of Neurod1 expression on differentiation of NG2 glia into neurons was investigated. In this study, we successfully isolated NG2 glial cells from mice prior to identification with immunofluorescence. Afterwards, AAV-Neurod1 virus was used to construct Neurod1 overexpression vectors in NG2 glia. Later, we detected neuronal markers expression with immunofluorescence and real time quantitative polymerase-chain reaction (qRT-PCR). Besides, expression of MAPK-signaling-pathway-related proteins were detected by western blotting technique. Through immunofluorescence and qRT-PCR techniques, we observed that Neurod1 overexpression contributed to NG2 cells differentiated into neurons. Further experiments also showed that Neurod1 overexpression induced the activation of MAPK pathway, but PD98059 (a selective inhibitor of MAPK pathway) partly inhibited the neuronal differentiation induced by Neurod1 overexpression. These findings suggest that Neurod1 could promote NG2 glia cells differentiating into neurons, wherein the mechanism under the differentiation is related to activation of MAPK pathway.     

Mass Spectrometry–Based Proteomics Analysis of Human Substantia Nigra From Parkinson's Disease Patients Identifies Multiple Pathways Potentially Involved in the Disease

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) of the brain. Despite decades of studies, the precise pathogenic mechanism of PD is still elusive. An unbiased proteomic analysis of PD patient’s brain allows the identification of critical proteins and molecular pathways that lead to dopamine cell death and α-synuclein deposition and the resulting devastating clinical symptoms. In this study, we conducted an in-depth proteome analysis of human SN tissues from 15 PD patients and 15 healthy control individuals combining Orbitrap mass spectrometry with the isobaric tandem mass tag–based multiplexing technology. We identified 10,040 proteins with 1140 differentially expressed proteins in the SN of PD patients. Pathway analysis showed that the ribosome pathway was the most enriched one, followed by gamma-aminobutyric acidergic synapse, retrograde endocannabinoid signaling, cell adhesion molecules, morphine addiction, Prion disease, and PD pathways. Strikingly, the majority of the proteins enriched in the ribosome pathway were mitochondrial ribosomal proteins (mitoribosomes). The subsequent protein–protein interaction analysis and the weighted gene coexpression network analysis confirmed that the mitoribosome is the most enriched protein cluster. Furthermore, the mitoribosome was also identified in our analysis of a replication set of ten PD and nine healthy control SN tissues. This study provides potential disease pathways involved in PD and paves the way to study further the pathogenic mechanism of PD.     

Dhh signaling pathway regulates reconstruction of seminiferous tubule-like structure

The reconstruction of a tubule-like structure in vitro has provided a promising system to analyze factors that drive morphogenesis and the underlying mechanisms. In this study, we took advantage of the inhibitor cyclopamine and a smoothened agonist to detect the role of the Dhh signaling pathway in the reconstructed tubule-like structure. Sertoli cells did not show polarity, rounded peritubular myoid cells and scattered Leydig cells were observed, combined with less laminin and lower proliferation of Leydig, peritubular myoid, germ, and Sertoli cells. However, in the presence of SAG, elongated peritubular myoid cells gathered at the bottom of polarized Sertoli cells, and most of the Leydig cells gathered at the outer part of the elongated peritubular myoid cells. Moreover, SAG promoted the secretion of laminin, assisting in the formation of the basal membrane and promoting the proliferation of Leydig, peritubular myoid, and germ cells. The level of Gli1 was significantly downregulated when treated with cyclopamine, whereas it was significantly upregulated when treated with SAG. These results indicate that the Dhh signaling pathway regulates the reconstruction of tubule-like structures by regulating the expression of Gli1.     

AGR3 promotes the stemness of colorectal cancer via modulating Wnt/β-catenin signalling

Cancer cells with stem cell properties have been acknowledged to be responsible for cancer initiation and progression. Wnt/β-catenin signalling is a major signal pathway promoting the stemness of cancer cells. Anterior gradient 3 (AGR3), a member of the protein disulfide isomerase (PDI) family, was found to be overexpressed in several cancers. However, the roles and mechanisms of AGR3 in colorectal cancer (CRC) have not been previously described. In our study, we find that AGR3 is highly expressed in CRC and associated with poor prognosis. Functional studies show that AGR3 promotes the stemness of CRC cells. Mechanically, AGR3 activates Wnt/β-catenin signalling and promotes the nuclear translocation of β-catenin to upregulate stemness related genes. Wnt/β-catenin signalling inhibition counteracts the promoting effect of AGR3 on cancer stemness. Moreover, the effect of AGR3 on Wnt/β-catenin signalling and cancer stemness depends on the presence of frizzled 4 (FZD4). Thus, our study first uncovers the stemness-promoting role and the oncogenic mechanism of AGR3 in CRC, which might provide a novel target for designing anti-CRC strategies.     

Impact on S. aureus and E. coli Membranes of Treatment with Chlorhexidine and Alcohol Solutions: Insights from Molecular Simulations and Nuclear Magnetic Resonance

Membranes form the first line of defence of bacteria against potentially harmful molecules in the surrounding environment. Understanding the protective properties of these membranes represents an important step towards development of targeted anti-bacterial agents such as sanitizers. Use of propanol, isopropanol and chlorhexidine can significantly decrease the threat imposed by bacteria in the face of growing anti-bacterial resistance via mechanisms that include membrane disruption. Here we have employed molecular dynamics simulations and nuclear magnetic resonance to explore the impact of chlorhexidine and alcohol on the S. aureus cell membrane, as well as the E. coli inner and outer membranes. We identify how sanitizer components partition into these bacterial membranes, and show that chlorhexidine is instrumental in this process.     

Identification of prognostic biomarkers for breast cancer brain metastases based on the bioinformatics analysis

PurposeThe prognosis of breast cancer (BC) patients who develop into brain metastases (BMs) is very poor. Thus, it is of great significance to explore the etiology of BMs in BC and identify the key genes involved in this process to improve the survival of BC patients with BMs.Patients and methodsThe gene expression data and the clinical information of BC patients were downloaded from TCGA and GEO database. Differentially expressed genes (DEGs) in TCGA-BRCA and GSE12276 were overlapped to find differentially expressed metastatic genes (DEMGs). The protein-protein interaction (PPI) network of DEMGs was constructed via STRING database. ClusterProfiler R package was applied to perform the gene ontology (GO) enrichment analysis of DEMGs. The univariate Cox regression analysis and the Kaplan-Meier (K-M) curves were plotted to screen DEMGs associated with the overall survival and the metastatic recurrence survival, which were identified as the key genes associated with the BMs in BC. The immune infiltration and the expressions of immune checkpoints for BC patients with brain relapses and BC patients with other relapses were analyzed respectively. The correlations among the expressions of key genes and the differently infiltrated immune cells or the differentially expressed immune checkpoints were calculated. The gene set enrichment analysis (GSEA) of each key gene was conducted to investigate the potential mechanisms of key genes involved in BC patients with BMs. Moreover, CTD database was used to predict the drug-gene interaction network of key genes.ResultsA total of 154 DEGs were identified in BC patients at M0 and M1 in TCGA database. A total of 667 DEGs were identified in BC patients with brain relapses and with other relapses. By overlapping these DEGs, 17 DEMGs were identified, which were enriched in the cell proliferation related biological processes and the immune related molecular functions. The univariate Cox regression analysis and the Kaplan-Meier curves revealed that CXCL9 and GPR171 were closely associated with the overall survival and the metastatic recurrence survival and were identified as key genes associated with BMs in BC. The analyses of immune infiltration and immune checkpoint expressions showed that there was a significant difference of the immune microenvironment between brain relapses and other relapses in BC. GSEA indicated that CXCL9 and GPR171 may regulate BMs in BC via the immune-related pathways.ConclusionOur study identified the key genes associated with BMs in BC patients and explore the underlying mechanisms involved in the etiology of BMs in BC. These findings may provide a promising approach for the treatments of BC patients with BMs.     

Characteristic fragmentation of polyunsaturated fatty acids with allylic vicinal diols in positive-ion LC/ESI-MS/MS

A characteristic fragmentation was observed for PUFAs that contain allylic vicinal diol groups (resolvin D1, D2, D4, E3, lipoxin A4, B4, and maresin 2), which were derivatized with N,N-dimethylethylenediamine (DMED), in positive-ion ESI-MS/MS. The findings indicate that when these compounds contain an allylic hydroxyl group that is located distal to the terminal DMED moiety in the case of resolvin D1, D4, and lipoxin A4, an aldehyde (-CH=O) is predominately formed, which arises from the breakdown in between vicinal diols, whereas, in the case of an allylic hydroxyl group that is located proximal to the DMED moiety, as in resolvin D2, E3, lipoxin B4, and maresin 2, an allylic carbene (-CH=CH-CH:) is formed. These specific fragmentations could be used as diagnostic ions for characterizing the above seven PUFAs. As a result, it was possible to detect resolvin D1, D2, E3, lipoxin A4, and B4 in sera (20 μl) obtained from healthy volunteers by multiple-reaction monitoring using LC/ESI-MS/MS.     

Consequences of excessive glucosylsphingosine in glucocerebrosidase-deficient zebrafish.

In Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson’s disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency, we studied zebrafish that have two orthologs of acid ceramidase, Asah1a and Asah1b. Only the latter is involved in the formation of GlcSph in glucocerebrosidase-deficient zebrafish as revealed by knockouts of Asah1a or Asah1b with glucocerebrosidase deficiency (either pharmacologically induced or genetic). Comparison of zebrafish with excessive GlcSph (gba1^-/- fish) and without GlcSph (gba1^-/-:asah1b^-/- fish) allowed us to study the consequences of chronic high levels of GlcSph. Prevention of excessive GlcSph in gba1^-/-:asah1b^-/- fish did not restrict storage cells, GlcCer accumulation, or neuroinflammation. However, GD fish lacking excessive GlcSph show an ameliorated course of disease reflected by significantly increased lifespan, delayed locomotor abnormality, and delayed development of an abnormal curved back posture. The loss of tyrosine hydroxylase 1 (th1) mRNA, a marker of dopaminergic neurons, is slowed down in brain of GD fish lacking excessive GlcSph. In conclusion, in the zebrafish GD model, excess GlcSph has little impact on (neuro)inflammation or the presence of GlcCer-laden macrophages but rather seems harmful to th1-positive dopaminergic neurons.     

Epigenetic regulator KDM4A activates Notch1-NICD-dependent signaling to drive tumorigenesis and metastasis in breast cancer

BackgroundAltered epigenetic reprogramming and events contribute to breast cancer (Bca) progression and metastasis. How the epigenetic histone demethylases modulate breast cancer progression remains poorly defined. We aimed to elucidate the biological roles of KDM4A in driving Notch1 activation and Bca progression.MethodsThe KDM4A expression in Bca specimens was analyzed using quantitative PCR and immunohistochemical assays. The biological roles of KDM4A were evaluated using wound-healing assays and an in vivo metastasis model. The Chromatin Immunoprecipitation (ChIP)-qPCR assay was used to determine the role of KDM4A in Notch1 regulation.ResultsHere, we screened that targeting KDM4A could induce notable cell growth suppression. KDM4A is required for the growth and progression of Bca cells. High KDM4A enhances tumor migration abilities and in vivo lung metastasis. Bioinformatic analysis suggested that KDM4A was highly expressed in tumors and high KDM4A correlates with poor survival outcomes. KDM4A activates Notch1 expressions via directly binding to the promoters and demethylating H3K9me3 modifications. KDM4A inhibition reduces expressions of a list of Notch1 downstream targets, and ectopic expressions of ICN1 could restore the corresponding levels. KDM4A relies on Notch1 signaling to maintain cell growth, migration and self-renewal capacities. Lastly, we divided a panel of cell lines into KDM4A^high and KDM4A^low groups. Targeting Notch1 using specific LY3039478 could efficiently suppress cell growth and colony formation abilities of KDM4A^high Bca.ConclusionTaken together, KDM4A could drive Bca progression via triggering the activation of Notch1 pathway by decreasing H3K9me3 levels, highlighting a promising therapeutic target for Bca.     

Common Mechanisms Underlying α-Synuclein-Induced Mitochondrial Dysfunction in Parkinson’s Disease

Parkinson’s disease (PD) is the most common neurological movement disorder characterized by the selective and irreversible loss of dopaminergic neurons in substantia nigra pars compacta resulting in dopamine deficiency in the striatum. While most cases are sporadic or environmental, about 10% of patients have a positive family history with a genetic cause. The misfolding and aggregation of α-synuclein (α-syn) as a casual factor in the pathogenesis of PD has been supported by a great deal of literature. Extensive studies of mechanisms underpinning degeneration of the dopaminergic neurons induced by α-syn dysfunction suggest a complex process that involves multiple pathways, including mitochondrial dysfunction and increased oxidative stress, impaired calcium homeostasis through membrane permeabilization, synaptic dysfunction, impairment of quality control systems, disruption of microtubule dynamics and axonal transport, endoplasmic reticulum/Golgi dysfunction, nucleus malfunction, and microglia activation leading to neuroinflammation. Among them mitochondrial dysfunction has been considered as the most primary target of α-syn-induced toxicity, leading to neuronal cell death in both sporadic and familial forms of PD. Despite reviewing many aspects of PD pathogenesis related to mitochondrial dysfunction, a systemic study on how α-syn malfunction/aggregation damages mitochondrial functionality and leads to neurodegeneration is missing in the literature. In this review, we give a detailed molecular overview of the proposed mechanisms by which α-syn, directly or indirectly, contributes to mitochondrial dysfunction. This may provide valuable insights for development of new therapeutic approaches in relation to PD. Antioxidant-based therapy as a potential strategy to protect mitochondria against oxidative damage, its challenges, and recent developments in the field are discussed.     

What does engram encode?: Heterogeneous memory engrams for different aspects of experience

Long-lasting synaptic changes within the neuronal network mediate memory. Neurons bearing such physical traces of memory (memory engram cells) are often equated with neurons expressing immediate early genes (IEGs) during a specific experience. However, past studies observed the expression of different IEGs in non-overlapping neurons or synaptic plasticity in neurons that do not express a particular IEG. Importantly, recent studies revealed that distinct subsets of neurons expressing different IEGs or even IEG negative-(yet active) neurons support different aspects of memory or computation, suggesting a more complex nature of memory engram cells than previously thought. In this short review, we introduce studies revealing such heterogeneous composition of the memory engram and discuss how the memory system benefits from it.     

Deep generative models for 3D molecular structure

Deep generative models have gained recent popularity for chemical design. Many of these models have historically operated in 2D space; however, more recently explicit 3D molecular generative models have become of interest, which are the topic of this article. Dozens of published models have been developed in the last few years to generate molecules directly in 3D, outputting both the atom types and coordinates, either in one-shot or adding atoms or fragments step-by-step. These 3D generative models can also be guided by structural information such as a binding pocket representation to successfully generate molecules with docking score ranges similar to known actives, but still showing lower computational efficiency and generation throughput than 1D/2D generative models and sometimes producing unrealistic conformations. We advocate for a unified benchmark of metrics to evaluate generation and propose perspectives to be addressed in next implementations.     

Functional analysis of thermo-sensitive TRPV1 in an aquatic vertebrate,masu salmon (Oncorhynchus masou ishikawae)

Transient receptor potential vanilloid 1 (TRPV1) is mainly expressed in nociceptive primary sensory neurons and acts as a sensor for heat and capsaicin. The functional properties of TRPV1 have been reported to vary among species and, in some cases, the species difference in its thermal sensitivity is likely to be associated with thermal habitat conditions. To clarify the functional properties and physiological roles of TRPV1 in aquatic vertebrates, we examined the temperature and chemical sensitivities of TRPV1 in masu salmon (Oncorhynchus masou ishikawae, Om) belonging to a family of salmonids that generally prefer cool environments. First, behavioral experiments were conducted using a video tracking system. Application of capsaicin, a TRPV1 agonist, induced locomotor activities in juvenile Om. Increasing the ambient temperature also elicited locomotor activity potentiated by capsaicin. RT-PCR revealed TRPV1 expression in gills as well as spinal cord. Next, electrophysiological analyses of OmTRPV1 were performed using a two-electrode voltage-clamp technique with a Xenopus oocyte expression system. Heat stimulation evoked an inward current in heterologously expressed OmTRPV1. In addition, capsaicin produced current responses in OmTRPV1-expressing oocytes, but higher concentrations were needed for its activation compared to the mammalian orthologues. These results indicate that Om senses environmental stimuli (heat and capsaicin) through the activation of TRPV1, and this channel may play important roles in avoiding environments disadvantageous for survival in aquatic vertebrates.     

Construction and validation of a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis and immune infiltration

BackgroundApoptosis played vital roles in the formation and progression of osteosarcoma. However, no studies elucidated the prognostic relationships between apoptosis-associated genes (AAGs) and osteosarcoma.MethodsThe differentially expressed genes associated with osteosarcoma metastasis and apoptosis were identified from GEO and MSigDB databases. The apoptosis-associated prognostic signature was established through univariate and multivariate cox regression analyses. The Kaplan–Meier (KM) survival curve, ROC curve and nomogram were constructed to investigate the predictive value of this signature. CIBERSORT algorithm and ssGSEA were used to explore the relationships between immune infiltration and AAG signature. The above results were validated in another GEO dataset and the expression of AAGs was also validated in osteosarcoma patient samples by immunohistochemistry.ResultsHSPB1 and IER3 were involved in AAG signature. In training and validation datasets, apoptosis-associated risk scores were negatively related to patient survival rates and the AAG signature was regarded as the independent prognostic factor. ROC and calibration curves demonstrated the signature and nomogram were reliable. GSEA revealed the signature related to immune-associated pathways. ssGSEA indicated that one immune cell and three immune functions were significantly dysregulated. The immunohistochemistry analyses of patients’ samples revealed that AAGs were significantly differently expressed between metastasis and non-metastasis osteosarcomas.ConclusionsThe present study identified and validated a novel apoptosis-associated prognostic signature related to osteosarcoma metastasis. It could serve as the potential biomarker and therapeutic targets for osteosarcoma in the future.     

Solution Structure of the C-terminal Domain of A20, the Missing Brick for the Characterization of the Interface between Vaccinia Virus DNA Polymerase and its Processivity Factor

Poxviruses are enveloped viruses with a linear, double-stranded DNA genome. Viral DNA synthesis is achieved by a functional DNA polymerase holoenzyme composed of three essential proteins. For vaccinia virus (VACV) these are E9, the catalytic subunit, a family B DNA polymerase, and the heterodimeric processivity factor formed by D4 and A20. The A20 protein links D4 to the catalytic subunit. High-resolution structures have been obtained for the VACV D4 protein in complex with an N-terminal fragment of A20 as well as for E9. In addition, biochemical studies provided evidence that a poxvirus-specific insertion (insert 3) in E9 interacts with the C-terminal residues of A20. Here, we provide solution structures of two different VACV A20 C-terminal constructs containing residues 304–426, fused at their C-terminus to either a BAP (Biotin Acceptor Peptide)-tag or a short peptide containing the helix of E9 insert 3. Together with results from titration studies, these structures shed light on the molecular interface between the catalytic subunit and the processivity factor component A20. The interface comprises hydrophobic residues conserved within the Chordopoxvirinae subfamily. Finally, we constructed a HADDOCK model of the VACV A20_304-426-E9 complex, which is in excellent accordance with previous experimental data.