Circular Rims2 Deficiency Causes Retinal Degeneration

Advanced biology

2021 Nov 05

Sun, LF;Ma, Y;Ji, YY;Wu, Z;Wang, YH;Mou, H;Jin, ZB;
PMID: 34738746 | DOI: 10.1002/adbi.202100906

Circular RNAs (circRNAs) refer to a newly recognized family of non-coding RNA with single-stranded RNAs. Despite emerging evidence indicating that circRNAs are abundantly expressed in various tissues, especially in the brain and retina, the role of circRNAs in retinal function and diseases is still largely unknown. Circular Rims2 (circRims2) is highly expressed and conserved in both the human and mouse brains. However, little is known about the expression and function of circRims2 in the retina. In the current study, the high-throughput RNA-seq analysis reveals a high expression of circRims2 in the retina. In addition, it is found that circRims2 is mainly located in plexiform layers that contain synapses between retinal neurons. Knocking down circRims2 with short hairpin RNA through subretinal adeno-associated viral (AAV) delivery in the mice leads to the decrease of the thickness of the outer and inner segment (OS/IS) layers and outer nuclear layer (ONL), and cessation of scotopic and photopic electroretinogram responses. Furthermore, the current study finds that circRims2 deficiency evokes retinal inflammation and activates the tumor necrosis factor (TNF) signaling pathway. Therefore, circRims2 may play an important role in the maintenance of retinal structure and function, and circRims2 deficiency may lead to pathogenic changes in the retina.

Epigenetic targeting of SLC30A3 by HDAC1 is related to the malignant phenotype of glioblastoma

IUBMB life

2021 Mar 14

Zhang, L;Liu, Z;Dong, Y;Kong, L;
PMID: 33715270 | DOI: 10.1002/iub.2463

The epigenetic abnormality is believed as a major driver for cancer initiation. Histone modification plays a vital role in tumor formation and progression. Particularly, alteration in histone acetylation has been highly associated with gene expression, cell cycle, as well as carcinogenesis. By analyzing glioblastoma (GBM)-related microarray from the GEO database and conducting chromatin immunoprecipitation-sequencing (ChIP-seq), we discovered that solute carrier family 30 member 3 (SLC30A3), a super enhancer (SE)-regulated factor, was significantly reduced in GBM tissues. Furthermore, histone deacetylase 1 (HDAC1), overexpressed in GBM tissues, could inhibit SLC30A3 expression by promoting histone H3K27ac deacetylation modification of the SE region of SLC30A3. Our functional validation revealed that SLC30A3 can inhibit the growth and metastatic spread of GBM cells in vitro and in vivo, and can activate the MAPK signaling pathway to promote apoptosis of GBM cells. Moreover, overexpression of HDAC1 resulted in a significant increase in DNA replication activity, a significant decline in apoptosis and cell cycle arrest in GBM cells. In a word, these findings indicate that combined epigenetic targeting of SLC30A3 by HDAC1 and SE is potentially therapeutically feasible in GBM.

PP 2.15- 00169 Macrophages are the primary source of virus in semen and male genital tract organs in acutely and chronically infected rhesus macaques

Journal of Virus Eradication

2022 Dec 01

Deleage, C;Fennessey, C;Harper, J;Florea, S;Lipkey, L;Fast, R;Paiardini, M;Lifson, J;Keele, B;
| DOI: 10.1016/j.jve.2022.100170

Background: Most new HIV infections result from sexual interactions with infected but untreated individuals. Semen is the main vector for viral transmission globally, however, little is known regarding the anatomic origin and form of virus in semen. Methods: In this study, we were able to combine numerous new technologies to characterize the virus present in the semen during SIV infection. Six rhesus macaques (RM) were challenged intravenously with barcoded virus SIVmac239M. Semen and blood samples were collected longitudinally for 17 days post-infection with all male genital tract (MGT) and multiple lymphoid tissues collected at necropsy and subjected to quantitative PCR, next generation sequencing of the viral barcode, and tissue analysis (RNAscope, DNAscope and immunophenotyping). Semen was also collected from 6 animals chronically infected with SIVmac251 and in five CD4 depleted animals in acute phase and 2 weeks post ART initiation. Results: Extremely high levels of viral RNA (vRNA) were detected in seminal plasma (up to 10^9cp/ml) as well as comparable levels of cell associated vRNA and vDNA in seminal cells with detection starting as early as 4 days post-infection. RNAscope and immunophenotyping of seminal cells and MGT tissues revealed myeloid cells as the main source of virus (Fig. 1), while CD4+T cells were harboring vRNA in lymphoid tissues. Sequences show evidence of an early compartment between seminal and blood plasma and no difference in the env gene of virus present in semen/MGT and in Lymph Nodes. Finally, multinuclear giant cells harboring vRNA were the only source of virus in semen in chronically infected and in CD4 depleted RM. Moreover, vRNA + myeloid cells were highly present in semen after 2 weeks on ART.

An Unbiased Approach to Identifying Changes in the Gene Expression Profile of Sensory Neurons in Painful Diabetic Neuropathy

The Journal of Pain

2021 May 01

George, D;Jayaraj, N;Ren, D;Miller, R;Menichella, D;
| DOI: 10.1016/j.jpain.2021.03.014

Painful diabetic neuropathy (PDN) is an intractable and debilitating disease characterized by neuropathic pain and small-fiber degeneration. Given the prevalence of the disease, there is a dire need to identify new targets for the development of disease-modifying therapeutics for PDN. In patients with PDN, dorsal root ganglion (DRG) nociceptors become hyperexcitable and eventually degenerate, but the molecular mechanism underlying the phenomenon is unknown. We aim to identify the gene expression profile of the DRG neurons in PDN pathology to facilitate the discovery of novel druggable targets. Using a well-established mouse model of PDN, mice were either fed a regular diet (RD) or a high-fat diet (HFD) for 10 weeks. We used a single-cell RNA (scRNA-seq) sequencing approach to capture the changes in the DRG in an unbiased fashion. As expected, analysis of the scRNA-seq identified both neuronal and non-neuronal clusters and several differentially expressed genes. Interestingly, we saw two closely related non-peptidergic clusters expressing a Mas-related G protein-coupled receptor (Mrgprd). While there were no differences in the expression of Mrgprd in one of the clusters (NP1 Type1), there seemed to be a significant increase in the expression of Mrgprd in a cluster we refer to as the NP1 Type2. To determine the functional relevance of the overexpression of Mrgprd, we used in vivo 2-photon calcium imaging with Nav1.8 Cre-GCaMP6 animals fed an RD or HFD and examined whether administration of β-alanine (a known agonist of Mrgprd) in the hind paw would directly activate Mrgprd positive DRG neurons. We observed an increase in the number, as well as an increase in the magnitude of response in the HFD indicating the hyperexcitability of neurons expressing Mrgprd. Taken together, our data highlights an important role of the Mrgprd receptor in the generation and maintenance of hyperexcitability in a mouse model of PDN. NS104295-01.

Long-lasting analgesia via targeted in situ repression of NaV1.7 in mice

Science translational medicine

2021 Mar 10

Moreno, AM;Alemán, F;Catroli, GF;Hunt, M;Hu, M;Dailamy, A;Pla, A;Woller, SA;Palmer, N;Parekh, U;McDonald, D;Roberts, AJ;Goodwill, V;Dryden, I;Hevner, RF;Delay, L;Gonçalves Dos Santos, G;Yaksh, TL;Mali, P;
PMID: 33692134 | DOI: 10.1126/scitranslmed.aay9056

Current treatments for chronic pain rely largely on opioids despite their substantial side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing. In particular, a hereditary loss-of-function mutation in NaV1.7, a sodium channel protein associated with signaling in nociceptive sensory afferents, leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence and structural similarity between NaV subtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 in primary afferents via epigenome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins at the spinal level as a potential treatment for chronic pain. Toward this end, we first optimized the efficiency of NaV1.7 repression in vitro in Neuro2A cells and then, by the lumbar intrathecal route, delivered both epigenome engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain, and BzATP-induced pain. Our results show effective repression of NaV1.7 in lumbar dorsal root ganglia, reduced thermal hyperalgesia in the inflammatory state, decreased tactile allodynia in the neuropathic state, and no changes in normal motor function in mice. We anticipate that this long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7 methodology we dub pain LATER, might have therapeutic potential in management of persistent pain states.

Adenosine A3 And Kappa Opioid Receptors In Satellite Glial Cells Of Human Dorsal Root Ganglion

The Journal of Pain

2023 Apr 01

Iadarola, M;Staedtler, E;Sapio, M;Maric, D;Ghetti, A;Mannes, A;
| DOI: 10.1016/j.jpain.2023.02.066

Controlling primary afferent nociceptive input is a known route to analgesia. This path is exemplified by agonists of the mu opioid receptors that are expressed in nociceptive DRG neurons and can be activated by intrathecal or systemic opioid agonists. For other peripheral analgesic targets, the hypothesis is that similar neuronal expression would validate their development as analgesic agents. In this study, two potential candidates, the kappa opioid and adenosine A3 receptors, are evaluated in human DRG for transcript levels, across species expression, and cell type localization using RNA Scope multiplex fluorescence microscopy. The Kappa receptor shows strong species variation in expression. No expression is detectable in dog DRG, low expression in mouse and rat, and relatively high expression in human DRG. This prompted an anatomical localization study to determine which cell types in the human DRG express the Kappa receptor. In situ hybridization disclosed Kappa receptor expression in satellite glial cells (SGCs) and most neurons were surrounded by fluorescent signal. This result was verified using a second independent probe to a distinct region of the OPRK1 transcript. Essentially no signal was seen in DRG neurons. The adenosine A3 receptor (ADORA3) is another GPCR suggested as a peripheral analgesic drug target. ADORA3 shows strong species expression variation, being absent in mouse, and expressed at low levels in rat and dog DRG. In contrast, ADORA3 was abundant in human DRG, but restricted to SGCs. These data suggest peripherally targeted agonists for either receptor may not be effective analgesic agents. Intramural Research Program, Clinical Center, NIH.

Sleep restriction during opioid abstinence affects the hypothalamic-pituitary-adrenal (HPA) axis in male and female rats

Stress (Amsterdam, Netherlands)

2023 Jan 01

Raff, H;Glaeser, BL;Szabo, A;Olsen, CM;Everson, CA;
PMID: 36856367 | DOI: 10.1080/10253890.2023.2185864

Hypothalamic-pituitary-adrenal (HPA) axis dynamics are disrupted by opioids and may be involved in substance abuse; this persists during withdrawal and abstinence and is associated with co-morbid sleep disruption leading to vulnerability to relapse. We hypothesized that chronic sleep restriction (SR) alters the HPA axis diurnal rhythm and the sexually dimorphic response to acute stressor during opioid abstinence. We developed a rat model to evaluate the effect of persistent sleep loss during opioid abstinence on HPA axis dynamics in male and female rats. Plasma ACTH and corticosterone were measured diurnally and in response to acute restraint stress in rats Before (control) compared to During subsequent opioid abstinence without or with SR. Abstinence, regardless of sleep state, led to an increase in plasma ACTH and corticosterone in the morning in males. There was a tendency for higher PM plasma ACTH during abstinence in SR males (p = 0.076). ACTH and corticosterone responses to restraint were reduced in male SR rats whereas there was a failure to achieve the post-restraint nadir in female SR rats. There was no effect of the treatments or interventions on adrenal weight normalized to body weight. SR resulted in a dramatic increase in hypothalamic PVN AVP mRNA and plasma copeptin in male but not female rats. This corresponded to the attenuation of the HPA axis stress response in SR males during opioid abstinence. We have identified a potentially unique, sexually dimorphic role for magnocellular vasopressin in the control of the HPA axis during opioid abstinence and sleep restriction.

Ablation of Growth Hormone Receptor in GABAergic Neurons Leads to Increased Pulsatile Growth Hormone Secretion

Endocrinology

2022 Aug 01

Dos Santos, WO;Wasinski, F;Tavares, MR;Campos, AMP;Elias, CF;List, EO;Kopchick, JJ;Szawka, RE;Donato, J;
PMID: 35803590 | DOI: 10.1210/endocr/bqac103

Growth hormone (GH) acts in several hypothalamic neuronal populations to modulate metabolism and the autoregulation of GH secretion via negative-feedback loops. However, few studies have investigated whether GH receptor (GHR) expression in specific neuronal populations is required for the homeostatic control of GH secretion and energy homeostasis. In the present study, we investigated the consequences of the specific GHR ablation in GABAergic (VGAT-expressing) or glutamatergic (VGLUT2-expressing) cells. GHR ablation in GABAergic neurons led to increased GH secretion, lean mass, and body growth in male and female mice. VGAT-specific GHR knockout (KO) male mice also showed increased serum insulin-like growth factor-1, hypothalamic Ghrh, and hepatic Igf1 messenger RNA levels. In contrast, normal GH secretion, but reduced lean body mass, was observed in mice carrying GHR ablation in glutamatergic neurons. GHR ablation in GABAergic cells increased weight loss and led to decreased blood glucose levels during food restriction, whereas VGLUT2-specific GHR KO mice showed blunted feeding response to 2-deoxy-D-glucose both in males and females, and increased relative food intake, oxygen consumption, and serum leptin levels in male mice. Of note, VGLUT2-cre female mice, independently of GHR ablation, exhibited a previously unreported phenotype of mild reduction in body weight without further metabolic alterations. The autoregulation of GH secretion via negative-feedback loops requires GHR expression in GABAergic cells. Furthermore, GHR ablation in GABAergic and glutamatergic neuronal populations leads to distinct metabolic alterations. These findings contribute to the understanding of the neuronal populations responsible for mediating the neuroendocrine and metabolic effects of GH.

TIPE2 Promotes Tumor Initiation But Inhibits Tumor Progression in Murine Colitis-Associated Colon Cancer

Inflammatory bowel diseases

2021 Dec 11

Etwebi, Z;Goldsmith, JR;Bou-Dargham, M;Tian, Y;Hood, R;Spitofsky, N;Li, M;Sun, H;Lou, Y;Liu, S;Lengner, C;Chen, YH;
PMID: 34894222 | DOI: 10.1093/ibd/izab306

Colorectal cancer (CRC) is the third leading cause of cancer in the United States, and inflammatory bowel disease patients have an increased risk of developing CRC due to chronic intestinal inflammation with it being the cause of death in 10% to 15% of inflammatory bowel disease patients. TIPE2 (TNF-alpha-induced protein 8-like 2) is a phospholipid transporter that is highly expressed in immune cells and is an important regulator of immune cell function.The azoxymethane/dextran sulfate sodium murine model of colitis-associated colon cancer (CAC) was employed in Tipe2 -/- and wild-type mice, along with colonoid studies, to determine the role of TIPE2 in CAC.Early on, loss of TIPE2 led to significantly less numbers of visible tumors, which was in line with its previously described role in myeloid-derived suppressor cells. However, as time went on, loss of TIPE2 promoted tumor progression, with larger tumors appearing in Tipe2 -/- mice. This was associated with increased interleukin-22/STAT3 phosphorylation signaling. Similar effects were also observed in primary colonoid cultures, together demonstrating that TIPE2 also directly regulated colonocytes in addition to immune cells.This work demonstrates that TIPE2 has dual effects in CAC. In the colonocytes, it works as a tumor suppressor. However, in the immune system, TIPE2 may promote tumorigenesis through suppressor cells or inhibit it through IL-22 secretion. Going forward, this work suggests that targeting TIPE2 for CRC therapy requires cell- and pathway-specific approaches and serves as a cautionary tale for immunotherapy approaches in general in terms of colon cancer, as intestinal inflammation can both promote and inhibit cancer.

LncRNA expression and SDHB mutations in pheochromocytomas and paragangliomas

Annals of diagnostic pathology

2021 Jul 31

Li, H;Hardin, H;Zaeem, M;Huang, W;Hu, R;Lloyd, RV;
PMID: 34461576 | DOI: 10.1016/j.anndiagpath.2021.151801

Although pheochromocytomas and paragangliomas (PPGLs) are usual low-grade neoplasms, the metastatic forms of these lesions are associated with high morbidity and mortality. Recent studies have discovered multiple aberrantly expressed long non-coding RNAs (lncRNAs) in cancers that may have regulatory roles in tumor pathogenesis and metastasis; however, the roles of some lncRNAs in PPGLs are still unknown. The expression levels of lncRNAs including metastasis-associated lung adenocarcinoma transcript (MALAT1), prostate cancer antigen 3 (PCA3), and HOX transcript antisense intergenic RNA (HOTAIR) in PPGLs were analyzed by in situ hybridization, using two tissue microarrays (TMAs). The pheochromocytoma (PCC) TMA consisted of normal adrenal medulla (N = 25), non-metastatic PCCs (N = 76) and metastatic PCCs (N = 5) while the paraganglioma (PGL) TMA had 73 non-metastatic PGLs and 5 metastatic PGLs. Immunohistochemical staining was performed on all samples with an anti-SDHB antibody. The correlations between lncRNA expression, loss of SDHB expression and clinical characteristics including tumor progression and disease prognosis were investigated. The expression levels of MALAT1 and PCA3 were significantly elevated (2.5-3.9 folds) in both non-metastatic and metastatic PCCs compared to normal adrenal medulla, although there were no significant differences between the non-metastatic and metastatic neoplasms. In contrast to non-metastatic PGLs, metastatic PGLs had significantly upregulated expression of MALAT1, PCA3, and HOTAIR. SDHB loss was more frequently observed in PGLs (25 of 78), especially in metastatic PGLs (5 of 5), compared to PCCs (2 of 81) and in 0 of 5 metastatic PCCs. Patients with SDHB loss, in contrast to SDHB retained, were younger at diagnosis, had higher rates of tumor recurrence, metastatic disease, and mortality. In addition, PGLs with SDHB loss had significantly increased expression of PCA3 compared to tumors with intact SDHB expression. Our findings suggest that specific lncRNAs may be involved in the SDHx signaling pathways in the tumorigenesis and in the development of PPGL.

Comprehensive Search for Novel Circulating miRNAs and Axon Guidance Pathway Proteins Associated with Risk of End Stage Kidney Disease in Diabetes

Journal of the American Society of Nephrology : JASN

2021 Jun 17

Satake, E;Saulnier, PJ;Kobayashi, H;Gupta, M;Looker, H;Wilson, J;Md Dom, Z;Ihara, K;O'Neil, K;Krolewski, B;Pipino, C;Pavkov, M;Nair, V;Bitzer, M;Niewczas, M;Kretzler, M;Mauer, M;Doria, A;Najafian, B;Kulkarni, R;Duffin, K;Pezzolesi, M;Kahn, CR;Nelson, R;Krolewski, A;
PMID: 34140396 | DOI: 10.1681/ASN.2021010105

Background Mechanisms underlying the progression of diabetic kidney disease to end-stage kidney disease (ESKD) are not fully understood. Methods We performed global micro-RNA (miRNA) analysis in plasma in two cohorts encompassing 375 individuals with type 1 and type 2 diabetes with late diabetic kidney disease and targeted proteomics analysis in plasma in four cohorts encompassing 746 individuals with late and early diabetic kidney disease. We examined structural lesions in kidney biopsies from the 105 individuals with early diabetic kidney disease. Human umbilical vein endothelial cells were used to assess the effects of miRNA mimics or inhibitors on regulation of candidate proteins. Results In the late diabetic kidney disease cohorts, we identified 17 circulating miRNAs represented by four exemplars (miR-1287-5p, miR-197-5p, miR-339-5p, miR-328-3p), which were strongly associated with 10-year risk of ESKD. These miRNAs targeted proteins in the axon guidance pathway. Circulating levels of six of these proteins-most notably EFNA4 and EPHA2-were strongly associated with 10-year risk of ESKD in all cohorts. Furthermore, circulating levels of these proteins correlated with severity of structural lesions in kidney biopsies. In contrast, expression levels of genes encoding these proteins had no apparent effects on the lesions. In in vitro experiments, mimics of miR-1287-5p and miR-197-5p and inhibitors of miR-339-5p and miR328-3p upregulated concentrations of EPHA2 in either cell lysate, supernatant, or both. Conclusions This study reveals novel mechanisms involved in progression to ESKD and points to the importance of systemic factors in the development of diabetic kidney disease. Some circulating miRNAs and axon guidance pathway proteins represent potential targets for new therapies to prevent and treat this condition.

HIV DNA reservoir and elevated PD-1 expression of CD4 T-cell subsets particularly persist in the terminal ileum of HIV-positive patients despite cART

HIV medicine

2021 Jan 09

Horn, C;Augustin, M;Ercanoglu, MS;Heger, E;Knops, E;Bondet, V;Duffy, D;Chon, SH;Nierhoff, D;Oette, M;Schäfer, H;Vivaldi, C;Held, K;Anderson, J;Geldmacher, C;Suárez, I;Rybniker, J;Klein, F;Fätkenheuer, G;Müller-Trutwin, M;Lehmann, C;
PMID: 33421299 | DOI: 10.1111/hiv.13031

Despite its importance as an HIV anatomic sanctuary, little is known about the characteristics of the HIV reservoir in the terminal ileum (TI). In blood, the immune checkpoint inhibitor programmed-death-1 (PD-1) has been linked to the HIV reservoir and T-cell immune dysfunction. We thus evaluated PD-1 expression and cell-associated HIV DNA in memory CD4 T-cell subsets from TI, peripheral blood (PB) and rectum (RE) of untreated and treated HIV-positive patients to identify associations between PD-1 and HIV reservoir in other sites. Using mononuclear cells from PB, TI and RE of untreated HIV-positive (N = 6), treated (n = 18) HIV-positive and uninfected individuals (n = 16), we identified and sorted distinct memory CD4 T-cell subsets by flow cytometry, quantified their cell-associated HIV DNA using quantitative PCR and assessed PD-1 expression levels using geometric mean fluorescence intensity. Combined HIV-1 RNA in situ hybridization and immunohistochemistry was performed on ileal biopsy sections. Combined antiretroviral therapy (cART)-treated patients with undetectable HIV RNA and significantly lower levels of HIV DNA in PB showed particularly high PD-1 expression in PB and TI, and high HIV DNA levels in TI, irrespective of clinical characteristics. By contrast, in treatment-naïve patients HIV DNA levels in memory CD4 T-cell subsets were high in PB and TI. Elevated PD-1 expression on memory CD4 T-cells in PB and TI despite treatment points to continuous immune dysfunction and underlines the importance of evaluating immunotherapy in reversing HIV latency and T-cell reconstitution. As HIV DNA particularly persists in TI despite cART, investigating samples from TI is crucial in understanding HIV immunopathogenesis.

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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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