Cytomembrane Trafficking Pathways of Connexin 26, 30, and 43

International journal of molecular sciences

2023 Jun 19

Zong, YJ;Liu, XZ;Tu, L;Sun, Y;
PMID: 37373495 | DOI: 10.3390/ijms241210349

The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6, respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1, appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2, GJB6, and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.

Age-dependent immune and lymphatic responses after spinal cord injury

Neuron

2023 Apr 28

Salvador, AFM;Dykstra, T;Rustenhoven, J;Gao, W;Blackburn, SM;Bhasiin, K;Dong, MQ;Guimarães, RM;Gonuguntla, S;Smirnov, I;Kipnis, J;Herz, J;
PMID: 37148871 | DOI: 10.1016/j.neuron.2023.04.011

Spinal cord injury (SCI) causes lifelong debilitating conditions. Previous works demonstrated the essential role of the immune system in recovery after SCI. Here, we explored the temporal changes of the response after SCI in young and aged mice in order to characterize multiple immune populations within the mammalian spinal cord. We revealed substantial infiltration of myeloid cells to the spinal cord in young animals, accompanied by changes in the activation state of microglia. In contrast, both processes were blunted in aged mice. Interestingly, we discovered the formation of meningeal lymphatic structures above the lesion site, and their role has not been examined after contusive injury. Our transcriptomic data predicted lymphangiogenic signaling between myeloid cells in the spinal cord and lymphatic endothelial cells (LECs) in the meninges after SCI. Together, our findings delineate how aging affects the immune response following SCI and highlight the participation of the spinal cord meninges in supporting vascular repair.

Advanced analysis and applications of single-cell transcriptome sequencing

All Life

2023 Dec 31

Ruohan, Z;Yicheng, B;Jingying, Z;Mei, H;Xinyan, Z;Min, Y;Tengfei, D;Junjing, J;
| DOI: 10.1080/26895293.2023.2199140

In summary, with the continuous improvement of technology and methods, scRNA-seq is becoming an indispensable tool in many biomedical fields. It is predicted that single-cell multiplex technology will play a more powerful role in single-cell research of complex organs and tissues in the future. It is expected that the demand and application of scRNA-seq technology will increase greatly in the future, and the technology will become more refined, high-throughput, affordable, and easier to use in scientific research laboratories and clinical laboratories. Especially in the new era of precision medicine, the study of the characteristics of high intercellular heterogeneity and clonal evolution in the occurrence, development, and treatment of diseases brings hope for the accurate diagnosis and treatment of diseases. In particular, it can be used to monitor the progress, efficacy, and prognosis of hematological tumors, and is likely to find potential therapeutic targets, providing a basis for accurate diagnosis, dynamic monitoring, and individualized treatment of the disease. More importantly, innovative single-cell technology is expected to greatly promote the effective control of diseases in IVF and early pregnancy screening and diagnosis of chromosomal and genetic diseases by improving the efficiency and detection quality. Thus, scRNA-seq is of great significance to improve human genetic health.

Tbx2 and Tbx3 regulate cell fate progression of the otic vesicle for inner ear development

Developmental biology

2022 Dec 12

Song, H;Morrow, BE;
PMID: 36521641 | DOI: 10.1016/j.ydbio.2022.12.003

The morphogenesis of the otic vesicle (OV) to form inner ear organs serves as an excellent model system to understand cell fate acquisition on a single cell level. Tbx2 and Tbx3 (Tbx2/3) encode closely related T-box transcription factors that are expressed widely in the mammalian OV. Inactivation of both genes in the OV (Tbx2/3cKO) results in failed morphogenesis into inner ear organs. To understand the basis of these defects, single cell RNA-sequencing (scRNA-seq) was performed on the OV lineage, in controls versus Tbx2/3cKO embryos. We identified a multipotent population termed otic progenitors in controls that are marked by expression of the known otic placode markers Eya1, Sox2, and Sox3 as well as new markers Fgf18, Cxcl12, and Pou3f3. The otic progenitor population was increased three-fold in Tbx2/3cKO embryos, concomitant with dysregulation of genes in these cells as well as reduced progression to more differentiated states of prosensory and nonsensory cells. An ectopic neural population of cells was detected in the posterior OV of Tbx2/3cKO embryos but had reduced maturation to delaminated neural cells. As all three cell fates were affected in Tbx2/3cKO embryos, we suggest that Tbx2/3 promotes progression of multipotent otic progenitors to more differentiated cell types in the OV.

Immunohistochemical Characterization of the Nervous System of Culex pipiens (Diptera, Culicidae)

Biology

2022 Jan 01

Gregor, KM;Becker, SC;Hellhammer, F;Baumgärtner, W;Puff, C;
PMID: 35053056 | DOI: 10.3390/biology11010057

Arthropod-borne diseases represent one of the greatest infection-related threats as a result of climate change and globalization. Repeatedly, arbovirus-infected mosquitoes show behavioral changes whose underlying mechanisms are still largely unknown, but might help to develop control strategies. However, in contrast to well-characterized insects such as fruit flies, little is known about neuroanatomy and neurotransmission in mosquitoes. To overcome this limitation, the study focuses on the immunohistochemical characterization of the nervous system of Culex pipiens biotype molestus in comparison to Drosophila melanogaster using 13 antibodies labeling nervous tissue, neurotransmitters or neurotransmitter-related enzymes. Antibodies directed against γ-aminobutyric acid, serotonin, tyrosine-hydroxylase and glutamine synthetase were suitable for investigations in Culex pipiens and Drosophila melanogaster, albeit species-specific spatial differences were observed. Likewise, similar staining results were achieved for neuronal glycoproteins, axons, dendrites and synaptic zones in both species. Interestingly, anti-phosphosynapsin and anti-gephyrin appear to represent novel markers for synapses and glial cells, respectively. In contrast, antibodies directed against acetylcholine, choline acetyltransferase, elav and repo failed to produce a signal in Culex pipiens comparable to that in Drosophila melanogaster. In summary, present results enable a detailed investigation of the nervous system of mosquitoes, facilitating further studies of behavioral mechanisms associated with arboviruses in the course of vector research.

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency

Med

2021 Nov 01

O’Connor, M;Kallenberg, D;Camilli, C;Pilotti, C;Dritsoula, A;Jackstadt, R;Bowers, C;Watson, H;Alatsatianos, M;Ohme, J;Dowsett, L;George, J;Blackburn, J;Wang, X;Singhal, M;Augustin, H;Ager, A;Sansom, O;Moss, S;Greenwood, J;
| DOI: 10.1016/j.medj.2021.10.002

Background A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. Methods Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. Findings In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. Conclusions LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics.

LINC01348 suppresses hepatocellular carcinoma metastasis through inhibition of SF3B3-mediated EZH2 pre-mRNA splicing

Oncogene

2021 Jun 17

Lin, YH;Wu, MH;Liu, YC;Lyu, PC;Yeh, CT;Lin, KH;
PMID: 34140643 | DOI: 10.1038/s41388-021-01905-3

Long non-coding RNAs (lncRNA) play crucial roles in hepatocellular carcinoma (HCC) progression. However, the specific functions of lncRNAs in alternative splicing (AS) and the metastatic cascade in liver cancer remain largely unclear. In this study, we identified a novel lncRNA, LINC01348, which was significantly downregulated in HCC and correlated with survival functions in HCC patients. Ectopic expression of LINC01348 induced marked inhibition of cell growth, and metastasis in vitro and in vivo. Conversely, these phenotypes were reversed upon knockdown of LINC01348. Mechanistically, LINC01348 complexed with splicing factor 3b subunit 3 (SF3B3) acted as a modulator of EZH2 pre-mRNA AS, and induced alterations in JNK/c-Jun activity and expression of Snail. Notably, C-terminal truncated HBx (Ct-HBx) negatively regulated LINC01348 through c-Jun signaling. Our data collectively highlight those novel regulatory associations involving LINC01348/SF3B3/EZH2/JNK/c-Jun/Snail are an important determinant of metastasis in HCC cells and support the potential utility of targeting LINC01348 as a therapeutic strategy for HCC.

Differentiation of Sensory Neuron Lineage During the Late First and Early Second Trimesters of Human Foetal Development

Neuroscience

2021 May 24

Quinn, RK;Drury, HR;Lim, R;Callister, RJ;Tadros, MA;
PMID: 34033872 | DOI: 10.1016/j.neuroscience.2021.05.018

Sensory neurons within DRGs are broadly divided into three types that transmit nociceptive, mechanical, and proprioceptive signals. These subtypes are established during in utero development when sensory neurons differentiate into distinct categories according to a complex developmental plan. Most of what we know about this developmental plan comes from studies in rodents and little is known about this process in humans. The present study documents the expression of key genes involved in human sensory neuron development during the late first and early second trimesters (9-16WG). We observed a decrease in the expression of SOX10 and BRN3A, factors associated with migration and proliferation of sensory neurons, towards the end of the first trimester. Small and large sensory neuron populations also emerged at the end of the first trimester, as well as the transcription factors responsible for defining distinct sensory neuron types. NTRK1, which is expressed in nociceptive neurons, emerged first at ~11 WG followed by NTRK2 in mechanoreceptors at ~12 WG, with NTRK3 for proprioceptors peaking at ~14 WG. These peaks were followed by increased expression of their respective neurotrophic factors. Our results show significant differences in the expression of key signalling molecules for human DRG development versus that of rodents, most notably the expression of neurotrophins that promote the survival of sensory neuron types. This highlights the importance of examining molecular changes in humans to better inform the application of data collected in pre-clinical models.

The melanocortin-3 receptor is a pharmacological target for the regulation of anorexia

Science translational medicine

2021 Apr 21

Sweeney, P;Bedenbaugh, MN;Maldonado, J;Pan, P;Fowler, K;Williams, SY;Gimenez, LE;Ghamari-Langroudi, M;Downing, G;Gui, Y;Hadley, CK;Joy, ST;Mapp, AK;Simerly, RB;Cone, RD;
PMID: 33883274 | DOI: 10.1126/scitranslmed.abd6434

Ablation of hypothalamic AgRP (Agouti-related protein) neurons is known to lead to fatal anorexia, whereas their activation stimulates voracious feeding and suppresses other motivational states including fear and anxiety. Despite the critical role of AgRP neurons in bidirectionally controlling feeding, there are currently no therapeutics available specifically targeting this circuitry. The melanocortin-3 receptor (MC3R) is expressed in multiple brain regions and exhibits sexual dimorphism of expression in some of those regions in both mice and humans. MC3R deletion produced multiple forms of sexually dimorphic anorexia that resembled aspects of human anorexia nervosa. However, there was no sexual dimorphism in the expression of MC3R in AgRP neurons, 97% of which expressed MC3R. Chemogenetic manipulation of arcuate MC3R neurons and pharmacologic manipulation of MC3R each exerted potent bidirectional regulation over feeding behavior in male and female mice, whereas global ablation of MC3R-expressing cells produced fatal anorexia. Pharmacological effects of MC3R compounds on feeding were dependent on intact AgRP circuitry in the mice. Thus, the dominant effect of MC3R appears to be the regulation of the AgRP circuitry in both male and female mice, with sexually dimorphic sites playing specialized and subordinate roles in feeding behavior. Therefore, MC3R is a potential therapeutic target for disorders characterized by anorexia, as well as a potential target for weight loss therapeutics.

The effect of nerve growth factor on supporting spatial memory depends upon hippocampal cholinergic innervation

Translational psychiatry

2021 Mar 15

Eu, WZ;Chen, YJ;Chen, WT;Wu, KY;Tsai, CY;Cheng, SJ;Carter, RN;Huang, GJ;
PMID: 33723225 | DOI: 10.1038/s41398-021-01280-3

Nerve growth factor (NGF) gene therapy has been used in clinical trials of Alzheimer's disease. Understanding the underlying mechanisms of how NGF influences memory may help develop new strategies for treatment. Both NGF and the cholinergic system play important roles in learning and memory. NGF is essential for maintaining cholinergic innervation of the hippocampus, but it is unclear whether the supportive effect of NGF on learning and memory is specifically dependent upon intact hippocampal cholinergic innervation. Here we characterize the behavior and hippocampal measurements of volume, neurogenesis, long-term potentiation, and cholinergic innervation, in brain-specific Ngf-deficient mice. Our results show that knockout mice exhibit increased anxiety, impaired spatial learning and memory, decreased adult hippocampal volume, neurogenesis, short-term potentiation, and cholinergic innervation. Overexpression of Ngf in the hippocampus of Ngf gene knockout mice rescued spatial memory and partially restored cholinergic innervations, but not anxiety. Selective depletion of hippocampal cholinergic innervation resulted in impaired spatial memory. However, Ngf overexpression in the hippocampus failed to rescue spatial memory in mice with hippocampal-selective cholinergic fiber depletion. In conclusion, we demonstrate the impact of Ngf deficiency in the brain and provide evidence that the effect of NGF on spatial memory is reliant on intact cholinergic innervations in the hippocampus. These results suggest that adequate cholinergic targeting may be a critical requirement for successful use of NGF gene therapy of Alzheimer's disease.

The Perils of Navigating Activity-Dependent Alternative Splicing of Neurexins

Frontiers in molecular neuroscience

2021 Mar 09

Liakath-Ali, K;Südhof, TC;
PMID: 33767611 | DOI: 10.3389/fnmol.2021.659681

Neurexins are presynaptic cell-adhesion molecules essential for synaptic function that are expressed in thousands of alternatively spliced isoforms. Recent studies suggested that alternative splicing at splice site 4 (SS4) of Nrxn1 is tightly regulated by an activity-dependent mechanism. Given that Nrxn1 alternative splicing at SS4 controls NMDA-receptor-mediated synaptic responses, activity-dependent SS4 alternative splicing would suggest a new synaptic plasticity mechanism. However, conflicting results confound the assessment of neurexin alternative splicing, prompting us to re-evaluate this issue. We find that in cortical cultures, membrane depolarization by elevated extracellular K+-concentrations produced an apparent shift in Nrxn1-SS4 alternative splicing by inducing neuronal but not astroglial cell death, resulting in persistent astroglial Nrxn1-SS4+ expression and decreased neuronal Nrxn1-SS4- expression. in vivo, systemic kainate-induced activation of neurons in the hippocampus produced no changes in Nrxn1-SS4 alternative splicing. Moreover, focal kainate injections into the mouse cerebellum induced small changes in Nrxn1-SS4 alternative splicing that, however, were associated with large decreases in Nrxn1 expression and widespread DNA damage. Our results suggest that although Nrxn1-SS4 alternative splicing may represent a mechanism of activity-dependent synaptic plasticity, common procedures for testing this hypothesis are prone to artifacts, and more sophisticated approaches will be necessary to test this important question.

SOX9 is required for kidney fibrosis and activates NAV3 to drive renal myofibroblast function

Science signaling

2021 Mar 02

Raza, S;Jokl, E;Pritchett, J;Martin, K;Su, K;Simpson, K;Birchall, L;Mullan, AF;Athwal, VS;Doherty, DT;Zeef, L;Henderson, NC;Kalra, PA;Hanley, NA;Piper Hanley, K;
PMID: 33653921 | DOI: 10.1126/scisignal.abb4282

Renal fibrosis is a common end point for kidney injury and many chronic kidney diseases. Fibrogenesis depends on the sustained activation of myofibroblasts, which deposit the extracellular matrix that causes progressive scarring and organ failure. Here, we showed that the transcription factor SOX9 was associated with kidney fibrosis in humans and required for experimentally induced kidney fibrosis in mice. From genome-wide analysis, we identified Neuron navigator 3 (NAV3) as acting downstream of SOX9 in kidney fibrosis. NAV3 increased in abundance and colocalized with SOX9 after renal injury in mice, and both SOX9 and NAV3 were present in diseased human kidneys. In an in vitro model of renal pericyte transdifferentiation into myofibroblasts, we demonstrated that NAV3 was required for multiple aspects of fibrogenesis, including actin polymerization linked to cell migration and sustained activation of the mechanosensitive transcription factor YAP1. In summary, our work identifies a SOX9-NAV3-YAP1 axis involved in the progression of kidney fibrosis and points to NAV3 as a potential target for pharmacological intervention.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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