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Probes for INS

ACD can configure probes for the various manual and automated assays for INS for RNAscope Assay, or for Basescope Assay compatible for your species of interest.

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Prolonged Posttreatment Virologic Control and Complete Seroreversion After Advanced Human Immunodeficiency Virus-1 Infection

Open forum infectious diseases

2021 Jan 01

Uruena, A;Cassetti, I;Kashyap, N;Deleage, C;Estes, JD;Trindade, C;Hammoud, DA;Burbelo, PD;Natarajan, V;Dewar, R;Imamichi, H;Ward, AJ;Poole, A;Ober, A;Rehm, C;Jones, S;Liang, CJ;Chun, TW;Nath, A;Lane, HC;Smith, BR;Connors, M;Migueles, SA;
PMID: 33511235 | DOI: 10.1093/ofid/ofaa613

Possible human immunodeficiency virus (HIV)-1 clearance has rarely been reported. In this study, we describe a unique case of an HIV-positive, combination antiretroviral therapy (cART)-experienced woman with prior acquired immunodeficiency syndrome (AIDS) who has not experienced viral rebound for over 12 years since discontinuing cART. Leukapheresis, colonoscopy, and lymph node excision were performed for detailed examination of virologic (including HIV reservoir) and immunologic features. Comparisons were made with chronically infected patients and healthy controls. No HIV-specific antibodies were detected in serum. Plasma HIV ribonucleic acid (RNA) levels were <0.2 copies/mL, and, except for low-frequency HIV deoxyribonucleic acid (DNA)+ cells in lymph node tissue (1 copy/3 × 106 cells), HIV antigen could not be detected by quantitative virus outgrowth (<0.0025 infectious units/106 CD4+ T cells) or by most measurements of HIV RNA or DNA in blood, lymph node, or gut-associated mononuclear cells. Human immunodeficiency virus-specific T-cell responses were detectable but low. Brain imaging revealed a prior biopsy site and persistent white matter disease since 1996. Human immunodeficiency virus DNA+ cells in the 1996 brain biopsy specimen confirmed her identity and initial HIV diagnosis. This represents the first report of complete seroreversion, prolonged posttreatment virus suppression, a profoundly small HIV reservoir, and persistent HIV-specific T cells in an adult with prior AIDS.
Susceptibility of Domestic Swine to Experimental Infection with Severe Acute Respiratory Syndrome Coronavirus 2

Emerging infectious diseases

2021 Jan 01

Pickering, BS;Smith, G;Pinette, MM;Embury-Hyatt, C;Moffat, E;Marszal, P;Lewis, CE;
PMID: 33350904 | DOI: 10.3201/eid2701.203399

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent that causes coronavirus disease, has been shown to infect several species. The role of domestic livestock and associated risks for humans in close contact with food production animals remains unknown for many species. Determining the susceptibility of pigs to SARS-CoV-2 is critical to a One Health approach to manage potential risk for zoonotic transmission. We found that pigs are susceptible to SARS-CoV-2 after oronasal inoculation. Among 16 animals, we detected viral RNA in group oral fluids and in nasal wash from 2 pigs, but live virus was isolated from only 1 pig. Antibodies also were detected in only 2 animals at 11 and 13 days postinoculation but were detected in oral fluid samples at 6 days postinoculation, indicating antibody secretion. These data highlight the need for additional livestock assessment to determine the potential role of domestic animals in the SARS-CoV-2 pandemic.
Safety and efficacy of rogaratinib in combination with atezolizumab in cisplatin-ineligible patients (pts) with locally advanced or metastatic urothelial cancer (UC) and FGFR mRNA overexpression in the phase Ib/II FORT-2 study.

Journal of Clinical Oncology

2021 May 20

Rosenberg, J;Gajate, P;Morales-Barrera, R;Lee, J;Necchi, A;Penel, N;Zagonel, V;Sierecki, M;Bao, W;Zhou, Y;Ellinghaus, P;Sweis, R;
| DOI: 10.1200/JCO.2021.39.15_suppl.4521

4521 Background: Rogaratinib (R) is a novel pan-FGFR inhibitor that showed promising efficacy and safety in a Phase I trial in pts with advanced solid tumors, including UC, with FGFR1-3 mRNA overexpression. The Phase Ib/II FORT-2 study (NCT03473756) of R plus atezolizumab (A) in pts with first-line cisplatin-ineligible, FGFR-positive, advanced/metastatic UC previously identified a maximum tolerated dose of R 600 mg twice daily (BID) plus A (1200 mg every 3 weeks) . We report updated safety, efficacy, and the recommended Phase II dose (RP2D) for combination therapy from the Phase Ib study. Methods: Pts with cisplatin-ineligible, locally advanced/metastatic UC with FGFR1/3 mRNA overexpression detected by RNA in situ hybridization of archival tissue (RNAscope) received oral R 600 mg BID plus A 1200 mg on day 1 of a 21-day cycle. Archival tissue was examined for programmed cell-death ligand 1 (PD-L1) protein expression levels, FGFR3-activating mutations via a targeted Illumina NGS panel, and FGFR fusions via an Archer fusion plex assay. Primary objectives were safety, tolerability, and determination of the RP2D. Results: 26 pts (enrolled May 25, 2018 to Nov 25, 2020) were treated; 89% were male, median age was 76 years (range 47-85), 58% had an ECOG performance status of 1, and 77% displayed low or absent (negative or non-detectable) PD-L1 expression (combined positive score < 10%). Common treatment-emergent adverse events (TEAEs) included diarrhea (n = 17, 65%; 1 grade [G] 3), hyperphosphatemia (n = 15, 58%; all G1 or 2), and nausea (n = 11, 42%; 1 G3). The most common G3/4 TEAEs were elevated lipase without pancreatitis (n = 5, 19%), elevated amylase (n = 3, 12%), and rash and syncope (n = 2, 8% each). TEAEs led to interruption/reduction/discontinuation of R in 69%/46%/19% of pts. R-related unique TEAEs were hyperphosphatemia in 15 pts (58%) and retinal pigment epithelium detachment in 1 pt (4%). G5 events occurred in 3 pts (12%), unrelated to treatment. 13 of 24 evaluable pts (54%) had an objective response (OR) per RECIST v1.1. The disease control rate was 83%, including 3 pts (13%) with a complete response (CR), 10 (42%) with a partial response (PR), and 7 (29%) with stable disease. Median duration of response was not reached. OR rate was 56% (2 CRs and 7 PRs) in the 16 pts with tumors having low/negative PD-L1 protein and FGFR3 mRNA overexpression without mutation. The RP2D for R+A was 600 mg BID. Conclusions: First-line treatment with the RP2D of R+A achieved favorable clinical efficacy and tolerability in pts with cisplatin-ineligible, metastatic UC characterized by high FGFR1/3 mRNA expression and generally low/negative PD-L1 expression. Encouraging efficacy was observed regardless of PD-L1 expression or FGFR3 mutation status, warranting future investigation. Clinical trial information: NCT03473756.
1384P DKN-01 in combination with tislelizumab and chemotherapy as a first-line therapy in unselected patients with advanced gastroesophageal adenocarcinoma (GEA): DisTinGuish trial

Annals of Oncology

2021 Sep 01

Klempner, S;Sirard, C;Chao, J;Chiu, V;Mahalingam, D;Uronis, H;Kagey, M;Baum, J;Dayyani, F;Song, J;Wang, J;Iqbal, S;Tejani, M;Sonbol, M;Scott, A;Wainberg, Z;Ajani, J;
| DOI: 10.1016/j.annonc.2021.08.1493

Background Dickkopf-1 (DKK1) modulates Wnt signaling and contributes to an immune suppressive tumor microenvironment. DKN-01 (D), a neutralizing DKK1 antibody, in combination with an anti-PD1 antibody, has demonstrated safety and clinical activity in advanced previously treated DKK1-high GEA. We report response and survival outcomes in GEA patients (pts) treated with D + tislelizumab (T) + capecitabine/oxaliplatin (CAPOX) as a first line therapy. Methods We enrolled advanced GEA pts in a phase IIa study of D + T + CAPOX (NCT04363801). Tumoral DKK1 mRNA expression was assessed by a chromogenic in situ hybridization RNAscope assay and assigned an H-score (0-300). Objective response rate (ORR) [primary efficacy objective], duration of response (DoR), disease control rate (DCR), and progression free survival (PFS) were evaluated in a modified intent to treat (mITT) population (completed ≥ 1 cycle) as well as compared between DKK1 high (H-score ≥35) and low groups. Results Twenty-five GEA pts were enrolled. Median age was 61 (22, 80); 19 males, 6 females. 17 pts (68%) had gastroesophageal junction (GEJ) adenocarcinoma; 8 pts (32%) had gastric cancer (GC). 18 GEA pts had RNAscope DKK1 expression available; 9 pts DKK1-high [5 GEJ, 4 GC) and 9 pts DKK1-low [7 GEJ, 2 GC]. Mean duration of treatment 3 mos, longest duration to date on study 7 mos, 19 pts remain on therapy. Most common D + T + CAPOX regimen related TEAEs were G1/2: anemia, thrombocytopenia, fatigue, diarrhea, nausea each in 3 pts. No related G3/4 toxicities; overall four G5 events; 1 related event pulmonary embolism. mITT analysis included 22 pts. Preliminary ORR in response evaluable (RE) mITT was 68% (13 PR, 6 SD, 1 NE, 2 pending first scan) and DCR 100%. In RE DKK1 high pts (n=7) there was an ORR of 100% (6 PR, 1 NE) compared with DKK1 low pts (n=9) ORR of 56% (5 PR, 4 SD). Median DoR and PFS were not reached. Conclusions D + T + CAPOX was well tolerated and has encouraging early activity as first line treatment for advanced GEA (unselected for PD-L1), with a preliminary ORR of 68% and DCR of 100%. Higher ORR in biomarker RE population: DKK1 high compared with DKK1 low (ORR 100% vs 56%). Updated ORR, DoR, PFS and PD-L1 expression will be reported.
A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization

Scientific reports

2021 Jan 28

Uddin, MI;Kilburn, TC;Jamal, SZ;Duvall, CL;Penn, JS;
PMID: 33510218 | DOI: 10.1038/s41598-021-81367-5

Diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA-lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA-lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA-lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.
DKN-01 and tislelizumab ± chemotherapy as a first-line (1L) and second-line (2L) investigational therapy in advanced gastroesophageal adenocarcinoma (GEA): DisTinGuish Trial.

Journal of Clinical Oncology

2022 Feb 01

Klempner, S;Chao, J;Uronis, H;Sirard, C;Kagey, M;Baum, J;Song, J;Wang, J;Kim, I;Lee, K;Oh, D;Sonbol, B;Wainberg, Z;Ajani, J;
| DOI: 10.1200/jco.2022.40.4_suppl.292

292 Background: Despite recent approval of anti-PD-1 antibodies as 1L therapy in HER2(-) advanced GEA, benefit remains modest and limited largely to PD-L1(+) patients (pts), primarily those with combined positive scores (CPS) ≥5. Thus novel therapeutic approaches are needed for this pt population. DKN-01 is a targeted anti-DKK1 mAb which has demonstrated improved clinical outcomes in pts with elevated tumoral DKK1 expression, a subset of pts with more aggressive disease and shorter overall survival. Methods: DisTinGuish (NCT04363801) is a Phase 2a single arm 2-part trial; Part A investigated DKN-01 (D) + tislelizumab (TS) + CAPOX as 1L therapy for pts with advanced HER2(-) GEA regardless of DKK1 status; Part B investigated two dosing cohorts of D (300 mg and 600 mg) + TS as 2L therapy for DKK1-high advanced GEA pts. Primary objective was to examine safety and tolerability and secondary objectives evaluated multiple efficacy endpoints including overall response rate (ORR) in a modified intent to treat (mITT) population (>1 dose D). Results: Forty-nine pts enrolled between 01 Sept 2020 and 15 Sept 2021; 25 pts in Part A and 24 pts in Part B (D-300 mg). Key clinicopathologic features and efficacy outcomes are shown in Table. The most common D-related AEs were low grade (G1/2) fatigue, nausea, and diarrhea. Nine pts had D-related ≥G3 toxicities, elevated AST/ALT, elevated alkaline phosphatase, hypophosphatemia, hyponatremia, lymphopenia, neutropenia, diarrhea, vomiting, fatigue all occurring in 1 pt and pulmonary embolism in 2 pts (one G5 event). No new safety signals were observed in Part A or B1. Duration of response (DoR), median PFS and median OS have not been reached for Part A. Last pt enrolled in Part B1 on 15 Sept 2021. Conclusions: The combination of D/TS + CAPOX represents a well-tolerated, active 1L combination, particularly for DKK1-high patients consistent with the proposed mechanism of action. Activity appears to be independent of PD-L1 status. Part B1 is aligned with biomarker enrichment and efficacy and biomarker data will be presented along with updated Part A efficacy data. Clinical trial information: NCT04363801. [Table: see text]
Spatiotemporal control of noradrenaline-dependent synaptic transmission in mouse dorsal raphe serotonin neurons

The Journal of neuroscience : the official journal of the Society for Neuroscience

2021 Dec 13

Khamma, JK;Copeland, DS;Hake, HS;Gantz, SC;
PMID: 34921047 | DOI: 10.1523/JNEUROSCI.1176-21.2021

Activity of dorsal raphe neurons is controlled by noradrenaline afferents. In this brain region, noradrenaline activates Gαq-coupled α1-adrenergic receptors (α1-AR), causing action potential firing and serotonin release. In vitro, electrical stimulation elicits vesicular noradrenaline release and subsequent activation of α1-AR to produce an excitatory postsynaptic current (α1-AR-EPSC). The duration of the α1-AR-EPSC (∼27 s) is much longer than that of most other synaptic currents, but the factors that govern the spatiotemporal dynamics of α1-AR are poorly understood. Using an acute brain slice preparation from adult male and female mice and electrophysiological recordings from dorsal raphe neurons, we found that the time-course of the α1-AR-EPSC was slow, but highly consistent within individual serotonin neurons. The amount of noradrenaline released influenced the amplitude of the α1-AR-EPSC without altering the time constant of decay suggesting that once released, extracellular noradrenaline was cleared efficiently. Reuptake of noradrenaline via noradrenaline transporters was a primary means of terminating the α1-AR-EPSC, with little evidence for extrasynaptic diffusion of noradrenaline unless transporter-dependent reuptake was impaired. Taken together, the results demonstrate that despite slow intrinsic signaling kinetics, noradrenaline-dependent synaptic transmission in the dorsal raphe is temporally and spatially controlled and noradrenaline transporters are critical regulators of serotonin neuron excitability. Given the functionally distinct types of neurons intermingled in the dorsal raphe nucleus and the unique roles of these neural circuits in physiological responses, transporters may preserve independence of each synapse to encode a long-lasting but discrete signal.SIGNIFICANCE STATEMENTThe dorsal raphe nucleus is the predominant source of serotonin in the brain and is controlled by another monoamine, noradrenaline. In this brain region, noradrenaline activates G protein-coupled α1-adrenergic receptors causing action potential firing and serotonin release. In spite of high interest in pharmacotherapies to enhance serotonin signaling, the factors that govern noradrenaline α1-AR signaling have received little attention. Here we show using mouse brain slices that the time-course of α1-AR signaling is slow, persisting for tens of seconds. Despite slow intrinsic signaling kinetics, noradrenaline-dependent synaptic transmission in the dorsal raphe is controlled temporally and spatially by efficient noradrenaline transporter-dependent clearance extracellular noradrenaline. Thus, noradrenaline transporters are critical regulators of serotonin neuron excitability.
Atypical Ebola Virus Disease in a Nonhuman Primate following Monoclonal Antibody Treatment Is Associated with Glycoprotein Mutations within the Fusion Loop

mBio

2021 Jan 12

Banadyga, L;Zhu, W;Kailasan, S;Howell, KA;Franaszek, K;He, S;Siragam, V;Cheng, K;Yan, F;Moffat, E;Cao, W;Leung, A;Embury-Hyatt, C;Aman, MJ;Qiu, X;
PMID: 33436428 | DOI: 10.1128/mBio.01438-20

Ebola virus (EBOV) is responsible for numerous devastating outbreaks throughout Africa, including the 2013-2016 West African outbreak as well as the two recent outbreaks in the Democratic Republic of the Congo (DRC), one of which is ongoing. Although EBOV disease (EVD) has typically been considered a highly lethal acute infection, increasing evidence suggests that the virus can persist in certain immune-privileged sites and occasionally lead to EVD recrudescence. Little is understood about the processes that contribute to EBOV persistence and recrudescence, in part because of the rarity of these phenomena but also because of the absence of an animal model that recapitulates them. Here, we describe a case of EBOV persistence associated with atypical EVD in a nonhuman primate (NHP) following inoculation with EBOV and treatment with an experimental monoclonal antibody cocktail. Although this animal exhibited only mild signs of acute EVD, it developed severe disease 2 weeks later and succumbed shortly thereafter. Viremia was undetectable at the time of death, despite abundant levels of viral RNA in most tissues, each of which appeared to harbor a distinct viral quasispecies. Remarkably, sequence analysis identified a single mutation in glycoprotein (GP) that not only resisted antibody-mediated neutralization but also increased viral growth kinetics and virulence. Overall, this report represents the most thoroughly characterized case of atypical EVD in an NHP described thus far, and it provides valuable insight into factors that may contribute to EBOV persistence and recrudescent disease.IMPORTANCE Ebola virus remains a global threat to public health and biosecurity, yet we still know relatively little about its pathogenesis and the complications that arise following recovery. With nearly 20,000 survivors from the 2013-2016 West African outbreak, as well as over 1,000 survivors of the recent outbreak in the DRC, we must consider the consequences of virus persistence and recrudescent disease, even if they are rare. In this study, we describe a case of atypical Ebola virus disease in a nonhuman primate after treatment with a monoclonal antibody. Not only does this study underscore the potential for atypical disease presentations, but it also emphasizes the importance of considering how medical countermeasures might relate to these phenomena, especially as antibodies are incorporated into the standard of care. The results presented herein provide a foundation from which we can continue to investigate these facets of Ebola virus disease.
Signaling of Phoenixin via GPR173 and the Paradox of Increased Vasopressin Secretion in Preeclampsia

The FASEB Journal

2021 Jan 01

Yosten, G;Kolar, G;Stein, L;Haddock, C;
| DOI: 10.1096/fasebj.2021.35.S1.04204

Preterm birth affects over 15 million pregnancies annually worldwide, and is associated with severe health risks for both mother and child. The leading cause of clinically indicated, non-spontaneous preterm delivery is preeclampsia, which can lead to maternal and fetal morbidity and mortality if not appropriately and promptly managed. At this time, the only “cure” for preeclampsia is delivery of the fetus and associated products of conception, which presents the clinical challenge of weighing the risks versus benefits of delivery if the pregnancy is preterm. The etiology of preeclampsia is multifactorial and incompletely understood. However, several carefully conducted studies have implicated the paradoxical secretion of the posterior pituitary hormone, arginine vasopressin (AVP) as a potential causative factor. Women with preeclampsia exhibit elevated plasma levels of AVP even in the face of the ongoing hypertension and hyponatremia, and infusion of AVP into pregnant rodents recapitulated multiple features of preeclampsia. The reason for the inappropriately elevated AVP in preeclampsia is unknown. Using a bioinformatic algorithm, our group identified a novel peptide that is conserved across multiple species, and named it phoenixin (PNX) (PMID 22963497). Our published data demonstrate a reproductive role of the peptide, and indicate that hypothalamic and pituitary PNX expression is modulated by sex hormones (PMID 27440717). Likewise, we recently have demonstrated that cerebroventrcular administration of PNX elevated plasma AVP levels in rats (PMID 29364701). Using the RNAscope approach we have now demonstrated that the receptor for PNX, identified by our group as GPR173 (PMID 27440717), is expressed by AVP neurons. Importantly expression of GPR173 increased across pregnancy in the rat hypothalamus coincident with increased AVP production and release (PMID 27330717). These data strongly suggest that PNX, acting through GPR173, could act as a molecular link between sex hormones and AVP secretion. In addition, we now demonstrate that central injection of PNX increases blood pressure, likely through PNX-induced elevation in AVP secretion. We hypothesize that the paradox of increased AVP secretion in the hyponatremic and hypervolemic state of preeclampsia is due to exaggerated PNX signaling in hypothalamus via GPR173 on AVP neurons and possibly on GPR173 expressing neurons in autonomic centers in hindbrain.
SARS‐CoV‐2‐associated neuropathology in non‐human primates

The FASEB Journal

2021 Jan 01

Rutkai, I;Mayer, M;Hellmers, L;Ning, B;Huang, Z;
| DOI: 10.1096/fasebj.2021.35.S1.03000

SARS-CoV-2 infection impacts multiple organ systems, including the central nervous system (CNS). Multiple reports have described a variety of neurological manifestations associated with infection that may contribute to worsening COVID-19. The neuropathology of SARS-CoV-2 is not well understood, necessitating the development of relevant animal models for investigation. Here, we report marked neuropathology but with limited virus in the CNS of two non-human primate models (NHPs) of SARS-CoV-2 infection. Adult male and female purpose-bred Rhesus macaques (RMs; n = 4) and wild-caught African green monkeys (AGMs; n = 4) were inoculated with the 2019-nCoV/USA-WA1/2020 strain of SARS-CoV-2 via multi-route mucosal or aerosol challenge. SARS-CoV-2 nucleocapsid (SARS-N) mRNA was detected in nasal swabs within the first week of inoculation, demonstrating infection of all study animals. All animals were euthanized at the study endpoint of 4 weeks post-inoculation, with the exception of two AGMs that reached humane endpoints at 8- and 22-days post-challenge. Seven regions of the CNS were investigated for pathology and virus infection. Archival brain tissues from two non-infected adult female RMs were used as aged-matched controls. Mild, but chronic, hypoxemia with impaired gas exchange were suggested by SpO2 values that stayed at or below 95% and elevated blood CO2 in the majority of the study animals. Neuroinflammation was seen throughout the brain and brainstem but with limited virus detection by immunohistochemistry and RNAscope of fixed tissues and viral RNA detection using a highly sensitive CRISPR-fluorescent detection system on RNA extracted from sectioned brain lysates. In addition, neuronal injury and death were suggested by pyknotic and karyolytic nuclei and cellular blebbing. Limited myelin vacuolation was revealed in two infected animals through Luxol Fast Blue staining. Neuronal cleaved caspase 3 positivity was seen at a greater frequency in infected animals compared to controls, suggesting increased apoptosis in infection. Microhemorrhages were larger and more frequent among infected NHPs, as compared to controls. Neuroinflammation, neuronal injury and death, and microhemorrhages were seen in animals that did not develop severe respiratory disease and may suggest neuropathology contributes to on-going symptoms of convalesced patients. Our findings in NHPs are in agreement with human autopsy and neuroimaging studies and demonstrate this is a relevant animal model for investigating neuropathological changes associated with COVID-19. Our results also suggest that hypoxic-ischemic events leading to energy failure and neuronal injury, contribute to the neuropathological consequences of COVID-19. Further studies are warranted to elucidate the mechanisms of SARS-CoV-2 neuropathogenesis.
TREM2 splice isoforms generate soluble TREM2 species that disrupt long-term potentiation

Genome medicine

2023 Feb 20

Moutinho, M;Coronel, I;Tsai, AP;Di Prisco, GV;Pennington, T;Atwood, BK;Puntambekar, SS;Smith, DC;Martinez, P;Han, S;Lee, Y;Lasagna-Reeves, CA;Lamb, BT;Bissel, SJ;Nho, K;Landreth, GE;
PMID: 36805764 | DOI: 10.1186/s13073-023-01160-z

TREM2 is a transmembrane receptor expressed by myeloid cells and acts to regulate their immune response. TREM2 governs the response of microglia to amyloid and tau pathologies in the Alzheimer's disease (AD) brain. TREM2 is also present in a soluble form (sTREM2), and its CSF levels fluctuate as a function of AD progression. Analysis of stroke and AD mouse models revealed that sTREM2 proteins bind to neurons, which suggests sTREM2 may act in a non-cell autonomous manner to influence neuronal function. sTREM2 arises from the proteolytic cleavage of the membrane-associated receptor. However, alternatively spliced TREM2 species lacking a transmembrane domain have been postulated to contribute to the pool of sTREM2. Thus, both the source of sTREM2 species and its actions in the brain remain unclear.The expression of TREM2 isoforms in the AD brain was assessed through the analysis of the Accelerating Medicines Partnership for Alzheimer's Disease Consortium transcriptomics data, as well as qPCR analysis using post-mortem samples of AD patients and of the AD mouse model 5xFAD. TREM2 cleavage and secretion were studied in vitro using HEK-293T and HMC3 cell lines. Synaptic plasticity, as evaluated by induction of LTP in hippocampal brain slices, was employed as a measure of sTREM2 actions.Three distinct TREM2 transcripts, namely ENST00000373113 (TREM2230), which encodes the full-length transmembrane receptor, and the alternatively spliced isoforms ENST00000373122 (TREM2222) and ENST00000338469 (TREM2219), are moderately increased in specific brain regions of patients with AD. We provide experimental evidence that TREM2 alternatively spliced isoforms are translated and secreted as sTREM2. Furthermore, our functional analysis reveals that all sTREM2 species inhibit LTP induction, and this effect is abolished by the GABAA receptor antagonist picrotoxin.TREM2 transcripts can give rise to a heterogeneous pool of sTREM2 which acts to inhibit LTP. These results provide novel insight into the generation, regulation, and function of sTREM2 which fits into the complex biology of TREM2 and its role in human health and disease. Given that sTREM2 levels are linked to AD pathogenesis and progression, our finding that sTREM2 species interfere with LTP furthers our understanding about the role of TREM2 in AD.
Functionally clustered mRNAs are distinctly enriched at cortical astroglial processes and are preferentially affected by FMRP deficiency

The Journal of neuroscience : the official journal of the Society for Neuroscience

2022 Jun 13

Men, Y;Higashimori, H;Reynolds, K;Tu, L;Jarvis, R;Yang, Y;
PMID: 35701158 | DOI: 10.1523/JNEUROSCI.0274-22.2022

Mature protoplasmic astroglia in the mammalian central nervous system (CNS) uniquely possess a large number of fine processes that have been considered primary sites to mediate astroglia to neuron synaptic signaling. However, localized mechanisms for regulating interactions between astroglial processes and synapses, especially for regulating the expression of functional surface proteins at these fine processes, are largely unknown. Previously, we showed that the loss of the RNA binding protein FMRP in astroglia disrupts astroglial mGluR5 signaling and reduces expression of the major astroglial glutamate transporter GLT1 and glutamate uptake in the cortex of Fmr1 conditional deletion mice. In the current study, by examining ribosome localization using electron microscopy and identifying mRNAs enriched at cortical astroglial processes using SNS/TRAP and RNA-Seq in wild type and FMRP-deficient male mice, our results reveal interesting localization-dependent functional clusters of mRNAs at astroglial processes. We further showed that the lack of FMRP preferentially alters the subcellular localization and expression of process-localized mRNAs. Taken together, we defined the role of FMRP in altering mRNA localization and expression at astroglial processes at the postnatal development (P30-40) and provided new candidate mRNAs that are potentially regulated by FMRP in cortical astroglia.SIGNIFICANCE STATEMENTLocalized mechanisms for regulating interactions between astroglial processes and synapses, especially for regulating the expression of functional surface proteins at these fine processes, are largely unknown. Previously, we showed that the loss of the RNA binding protein FMRP in astroglia disrupts expression of several astroglial surface proteins such as mGluR5 and major astroglial glutamate transporter GLT1 in the cortex of FMRP-deficient mice. Our current study examined ribosome localization using electron microscopy and identified mRNAs enriched at cortical astroglial processes in wild type and FMRP-deficient mice. These results reveal interesting localization-dependent functional clusters of mRNAs at astroglial processes and demonstrate that the lack of FMRP preferentially alters the subcellular localization and expression of process-localized mRNAs.

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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
EnEmProbe targets exons n and m
En-EmProbe 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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