Mu opioid receptors on hippocampal GABAergic interneurons are critical for the antidepressant effects of tianeptine

Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

2021 Sep 30

Han, J;Andreu, V;Langreck, C;Pekarskaya, EA;Grinnell, SG;Allain, F;Magalong, V;Pintar, J;Kieffer, BL;Harris, AZ;Javitch, JA;Hen, R;Nautiyal, KM;
PMID: 34593976 | DOI: 10.1038/s41386-021-01192-2

Tianeptine is an atypical antidepressant used in Europe to treat patients who respond poorly to selective serotonin reuptake inhibitors (SSRIs). The recent discovery that tianeptine is a mu opioid receptor (MOR) agonist has provided a potential avenue for expanding our understanding of antidepressant treatment beyond the monoamine hypothesis. Thus, our studies aim to understand the neural circuits underlying tianeptine's antidepressant effects. We show that tianeptine induces rapid antidepressant-like effects in mice after as little as one week of treatment. Critically, we also demonstrate that tianeptine's mechanism of action is distinct from fluoxetine in two important aspects: (1) tianeptine requires MORs for its chronic antidepressant-like effect, while fluoxetine does not, and (2) unlike fluoxetine, tianeptine does not promote hippocampal neurogenesis. Using cell-type specific MOR knockouts we further show that MOR expression on GABAergic cells-specifically somatostatin-positive neurons-is necessary for the acute and chronic antidepressant-like responses to tianeptine. Using central infusion of tianeptine, we also implicate the ventral hippocampus as a potential site of antidepressant action. Moreover, we show a dissociation between the antidepressant-like phenotype and other opioid-like phenotypes resulting from acute tianeptine administration such as analgesia, conditioned place preference, and hyperlocomotion. Taken together, these results suggest a novel entry point for understanding what circuit dysregulations may occur in depression, as well as possible targets for the development of new classes of antidepressant drugs.

The noncoding genome and hearing loss

Human genetics

2021 Sep 07

Avraham, KB;Khalaily, L;Noy, Y;Kamal, L;Koffler-Brill, T;Taiber, S;
PMID: 34491412 | DOI: 10.1007/s00439-021-02359-z

The age of sequencing has provided unprecedented insights into the human genome. The coding region of the genome comprises nearly 20,000 genes, of which approximately 4000 are associated with human disease. Beyond the protein-coding genome, which accounts for only 3% of the genome, lies a vast pool of regulatory elements in the form of promoters, enhancers, RNA species, and other intricate elements. These features undoubtably influence human health and disease, and as a result, a great deal of effort is currently being invested in deciphering their identity and mechanism. While a paucity of material has caused a lag in identifying these elements in the inner ear, the emergence of technologies for dealing with a minimal number of cells now has the field working overtime to catch up. Studies on microRNAs (miRNAs), long non-coding RNAs (lncRNAs), methylation, histone modifications, and more are ongoing. A number of microRNAs and other noncoding elements are known to be associated with hearing impairment and there is promise that regulatory elements will serve as future tools and targets of therapeutics and diagnostics. This review covers the current state of the field and considers future directions for the noncoding genome and implications for hearing loss.

Spatial mapping of the tumor immune microenvironment

Engineering Technologies and Clinical Translation

2021 Sep 03

Wu, Y;Pagacz, J;Emery, S;Kron, S;Lee, S;
| DOI: 10.1016/B978-0-323-90949-5.00009-7

To replace one-size-fits-all cancer immunotherapy with personalized treatment, biomarkers of response and resistance as well as assays to evaluate them in each patient are essential. Among likely determinants of response, the spatial locations and activation states of the immune infiltrate appear critical. Current clinical methods for tissue analysis such as immunohistochemistry are poorly matched to the heterogeneity of the tumor immune microenvironment (TIME). However, multiple tools for analysis of the TIME can now image panels of biomarkers in a single experiment, permit deep profiling to measure dozens of immune features in each sample, and/or facilitate unbiased multiomic analysis at high spatial resolution. Several assays are commercialized with some nearing clinical adoption. In this chapter, we present a broad overview of established and emerging technologies that enable multiplexed detection and spatial mapping of cellular and molecular features of the TIME, highlighting advantages and disadvantages as well as opportunities for future development.

Using single-nucleus RNA-sequencing to interrogate transcriptomic profiles of archived human pancreatic islets

Genome medicine

2021 Aug 10

Basile, G;Kahraman, S;Dirice, E;Pan, H;Dreyfuss, JM;Kulkarni, RN;
PMID: 34376240 | DOI: 10.1186/s13073-021-00941-8

Human pancreatic islets are a central focus of research in metabolic studies. Transcriptomics is frequently used to interrogate alterations in cultured human islet cells using single-cell RNA-sequencing (scRNA-seq). We introduce single-nucleus RNA-sequencing (snRNA-seq) as an alternative approach for investigating transplanted human islets.The Nuclei EZ protocol was used to obtain nuclear preparations from fresh and frozen human islet cells. Such preparations were first used to generate snRNA-seq datasets and compared to scRNA-seq output obtained from cells from the same donor. Finally, we employed snRNA-seq to obtain the transcriptomic profile of archived human islets engrafted in immunodeficient animals.We observed virtually complete concordance in identifying cell types and gene proportions as well as a strong association of global and islet cell type gene signatures between scRNA-seq and snRNA-seq applied to fresh and frozen cultured or transplanted human islet samples.We propose snRNA-seq as a reliable strategy to probe transcriptomic profiles of freshly harvested or frozen sources of transplanted human islet cells especially when scRNA-seq is not ideal.

Nociceptor subtypes are born continuously over DRG development

Developmental Biology

2021 Aug 01

Landy, M;Goyal, M;Lai, H;
| DOI: 10.1016/j.ydbio.2021.07.018

Sensory neurogenesis in the dorsal root ganglion (DRG) occurs in two waves of differentiation with larger, myelinated proprioceptive and low-threshold mechanoreceptor (LTMR) neurons differentiating before smaller, unmyelinated (C) nociceptive neurons. This temporal difference was established from early birthdating studies based on DRG soma cell size. However, distinctions in birthdates between molecular subtypes of sensory neurons, particularly nociceptors, is unknown. Here, we assess the birthdate of lumbar DRG neurons in mice using a thymidine analog, EdU, to label developing neurons exiting mitosis combined with co-labeling of known sensory neuron markers. We find that different nociceptor subtypes are born on similar timescales, with continuous births between E9.5 to E13.5, and peak births from E10.5 to E11.5. Notably, we find that thinly myelinated Aδ-fiber nociceptors and peptidergic C-fibers are born more broadly between E10.5 and E11.5 than previously thought and that non-peptidergic C-fibers and C-LTMRs are born with a peak birth date of E11.5. Moreover, we find that the percentages of nociceptor subtypes born at a particular timepoint are the same for any given nociceptor cell type marker, indicating that intrinsic or extrinsic influences on cell type diversity are occurring similarly across developmental time. Overall, the patterns of birth still fit within the classical “two wave” description, as touch and proprioceptive fibers are born primarily at E10.5, but suggest that nociceptors have a slightly broader wave of birthdates with different nociceptor subtypes continually differentiating throughout sensory neurogenesis irrespective of myelination.

Airway basal stem cells reutilize the embryonic proliferation regulator, Tgfβ-Id2 axis, for tissue regeneration

Developmental cell

2021 Jun 08

Kiyokawa, H;Yamaoka, A;Matsuoka, C;Tokuhara, T;Abe, T;Morimoto, M;
PMID: 34129836 | DOI: 10.1016/j.devcel.2021.05.016

During development, quiescent airway basal stem cells are derived from proliferative primordial progenitors through the cell-cycle slowdown. In contrast, basal cells contribute to adult tissue regeneration by shifting from slow cycling to proliferating and subsequently back to slow cycling. Although sustained proliferation results in tumorigenesis, the molecular mechanisms regulating these transitions remain unknown. Using temporal single-cell transcriptomics of developing murine airway progenitors and genetic validation experiments, we found that TGF-β signaling decelerated cell cycle by inhibiting Id2 and contributed to slow-cycling basal cell specification during development. In adult tissue regeneration, reduced TGF-β signaling restored Id2 expression and initiated regeneration. Id2 overexpression and Tgfbr2 knockout enhanced epithelial proliferation; however, persistent Id2 expression drove basal cell hyperplasia that resembled a precancerous state. Together, the TGF-β-Id2 axis commonly regulates the proliferation transitions in basal cells during development and regeneration, and its fine-tuning is critical for normal regeneration while avoiding basal cell hyperplasia.

Single-cell RNA sequencing reveals Nestin+ active neural stem cells outside the central canal after spinal cord injury

Science China. Life sciences

2021 May 28

Shu, M;Xue, X;Nie, H;Wu, X;Sun, M;Qiao, L;Li, X;Xu, B;Xiao, Z;Zhao, Y;Fan, Y;Chen, B;Zhang, J;Shi, Y;Yang, Y;Lu, F;Dai, J;
PMID: 34061300 | DOI: 10.1007/s11427-020-1930-0

Neural stem cells (NSCs) in the spinal cord hold great potential for repair after spinal cord injury (SCI). The ependyma in the central canal (CC) region has been considered as the NSCs source in the spinal cord. However, the ependyma function as NSCs after SCI is still under debate. We used Nestin as a marker to isolate potential NSCs and their immediate progeny, and characterized the cells before and after SCI by single-cell RNA-sequencing (scRNA-seq). We identified two subgroups of NSCs: the subgroup located within the CC cannot prime to active NSCs after SCI, while the subgroup located outside the CC were activated and exhibited the active NSCs properties after SCI. We demonstrated the comprehensive dynamic transcriptome of NSCs from quiescent to active NSCs after SCI. This study reveals that Nestin+ cells outside CC were NSCs that activated upon SCI and may thus serve as endogenous NSCs for regenerative treatment of SCI in the future.

ATAK receptors, harnessing innate immunity to program myeloid cells to kill cancer

Cytotherapy

2021 May 01

Wang, Y;Diwanji, N;Nicholson, T;Mukherjee, S;Getts, D;
| DOI: 10.1016/S1465324921004217

Background & Aim: T cells therapies have revolutionized cancer treatment for many patients. However, for the majority of patients with advanced solid tumors, sustained clinical benefit has not been achieved. Unlike T cells, myeloid cells readily accumulate in tumors, in some cases contributing up to 50% of the tumor mass. More recently, the potential for engaging innate immune signaling sensors such as Toll-like receptors and STING-cGAS have been investigated as important pathways to drastically upregulate pro-inflammatory anti-tumor immune response and are associated with anti-tumor immunity. These approaches, using synthetic agonists to activate these pathways, can be potent but delivering a localized and tumor specific activation of innate immune signaling is difficult to achieve. Methods, Results & Conclusion: Here we designed and engineered a new class of chimeric antigen receptors that couple tumor recognition with innate immune signaling, referred to as Activate, Target, Attack & Kill (ATAK™) receptors. By combining cancer recognition domains with intracellular signaling domains from innate immune receptors such as Fcg, TLR and cytokine receptors, we show that myeloid cells can be programmed to recognize cancer and elicit a broad and tunable immune response. Our data show the versatility of building ATAK receptors by harnessing innate immune pathways and support their clinical development in cell and direct in vivo therapies.

Spatial Transcriptomics: Molecular Maps of the Mammalian Brain

Annual review of neuroscience

2021 Apr 29

Ortiz, C;Carlén, M;Meletis, K;
PMID: 33914592 | DOI: 10.1146/annurev-neuro-100520-082639

Maps of the nervous system inspire experiments and theories in neuroscience. Advances in molecular biology over the past decades have revolutionized the definition of cell and tissue identity. Spatial transcriptomics has opened up a new era in neuroanatomy, where the unsupervised and unbiased exploration of the molecular signatures of tissue organization will give rise to a new generation of brain maps. We propose that the molecular classification of brain regions on the basis of their gene expression profile can circumvent subjective neuroanatomical definitions and produce common reference frameworks that can incorporate cell types, connectivity, activity, and other modalities. Here we review the technological and conceptual advances made possible by spatial transcriptomics in the context of advancing neuroanatomy and discuss how molecular neuroanatomy can redefine mapping of the nervous system. Expected final online publication date for the Annual Review of Neuroscience, Volume 44 is July 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Pro-Epicardial Cells are Heterogeneous with Specified Smooth Muscle-Like and Pacemaker Progenitor Cells

SSRN Electronic Journal

2021 Apr 29

Miao, L;Li, J;Lu, Y;Yin, C;Sun, D;Lo, E;Song, R;Cai, C;Huang, W;Long, X;McConnell, B;Fan, Z;Singer, H;Schwartz, R;Munshi, N;Wu, M;
| DOI: 10.2139/ssrn.3832987

The heterogeneity and specification of pro-epicardial cells (pro-EpiCs) to fibroblast and smooth muscle cell (SMC) are unknown. We applied single-cell RNA sequencing (scRNA-seq) paired with RNAScope, bioinformatics, and lineage tracing in an unbiased manner to identify the previously uncharacterized molecular heterogeneity of the pro-EpiCs isolated from pro-epicardium (PE). We found that pro-EpiCs labeled by _Tbx18 Cre/+ _ are heterogeneous, with three clusters displaying differential expression profiles and distinct spatial locations. Cluster 1 are mesothelial cells, and Cluster 2 express SMC markers. Surprisingly, Cluster 3 express _Isl1_ and markers of pacemaker progenitor cells (PMPC) but not marker of atrial cardiomyocytes. Our studies conclude that pro-EpiCs are heterogeneous and SMC-like cells are specified in the PE, while fibroblasts are not specified in PE but epicardium. We identified a region in PE that contains PMPCs, which translocate through the inflow tract to the sinoatrial node. The expression profile of Cluster 3 cells unifies previous studies regarding the origins and markers of PMPCs.

A Method to Pre-Screen Rat Mammary Gland Whole-Mounts Prior To RNAscope

Journal of mammary gland biology and neoplasia

2021 Apr 17

Duderstadt, EL;Sanders, MA;Samuelson, DJ;
PMID: 33866475 | DOI: 10.1007/s10911-021-09484-5

RNAscope is a quantitative in situ gene expression measurement technique that preserves the spatial aspect of intact tissue; thus, allowing for comparison of specific cell populations and morphologies. Reliable and accurate measurement of gene expression in tissue is dependent on preserving RNA integrity and the quantitative nature of RNAscope. The purpose of this study was to determine if the quantitative nature of RNAscope was retained following processing and carmine staining of mammary gland whole-mounts, which are commonly used to identify lesions, such as hyperplasia and ductal carcinoma in situ (DCIS). We were concerned that handling and procedures required to visualize microscopic disease lesions might compromise RNA integrity and the robustness of RNAscope. No effect on the quantitative abilities of RNAscope was detected when mammary gland whole-mounts were pre-screened for lesions of interest prior to RNAscope. This was determined in comparison to tissue that had been formalin-fixed and paraffin embedded (FFPE) immediately after collection. The ability to pre-screen whole-mounts allowed unpalpable diseased lesions to be identified without labor-intensive serial sectioning of tissue samples to find diseased tissue. This method is applicable to evaluate mammary gland whole-mounts during normal mammary gland development, function, and disease progression.

Coordination of escape and spatial navigation circuits orchestrates versatile flight from threats

Neuron

2021 Apr 13

Wang, W;Schuette, PJ;Nagai, J;Tobias, BC;Cuccovia V Reis, FM;Ji, S;de Lima, MAX;La-Vu, MQ;Maesta-Pereira, S;Chakerian, M;Leonard, SJ;Lin, L;Severino, AL;Cahill, CM;Canteras, NS;Khakh, BS;Kao, JC;Adhikari, A;
PMID: 33861942 | DOI: 10.1016/j.neuron.2021.03.033

Naturalistic escape requires versatile context-specific flight with rapid evaluation of local geometry to identify and use efficient escape routes. It is unknown how spatial navigation and escape circuits are recruited to produce context-specific flight. Using mice, we show that activity in cholecystokinin-expressing hypothalamic dorsal premammillary nucleus (PMd-cck) cells is sufficient and necessary for context-specific escape that adapts to each environment's layout. In contrast, numerous other nuclei implicated in flight only induced stereotyped panic-related escape. We reasoned the dorsal premammillary nucleus (PMd) can induce context-specific escape because it projects to escape and spatial navigation nuclei. Indeed, activity in PMd-cck projections to thalamic spatial navigation circuits is necessary for context-specific escape induced by moderate threats but not panic-related stereotyped escape caused by perceived asphyxiation. Conversely, the PMd projection to the escape-inducing dorsal periaqueductal gray projection is necessary for all tested escapes. Thus, PMd-cck cells control versatile flight, engaging spatial navigation and escape circuits.

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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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