Glucagon-like peptide-2 stimulates S-phase entry of intestinal Lgr5+ stem cells

Cellular and molecular gastroenterology and hepatology

2022 Feb 23

Chen, ME;Naeini, SM;Srikrishnaraj, A;Drucker, DJ;Fesler, Z;Brubaker, PL;
PMID: 35218981 | DOI: 10.1016/j.jcmgh.2022.02.011

Leucine-rich repeat-containing G-protein coupled receptor-5 (Lgr5)+/olfactomedin-4 (Olfm4)+ intestinal stem cells (ISCs) in the crypt-base are crucial for homeostatic maintenance of the epithelium. The gut hormone, glucagon-like peptide-21-33 (GLP-2), stimulates intestinal proliferation and growth; however, the actions of GLP-2 on the Lgr5+ ISCs remain unclear. The aim of this study was to determine whether and how GLP-2 regulates Lgr5+ ISC cell cycle dynamics and number.Lgr5-eGFP-IRES-creERT2 mice were acutely administered human Gly2-GLP-2, or the GLP-2 receptor antagonist, GLP-23-33. Intestinal epithelial-insulin-like growth factor-1 receptor knockout and control mice were treated chronically with hGly2-GLP-2. Cell cycle parameters were determined by EdU, BrdU, Ki67 and phosphohistone-3 labeling and cell cycle gene expression.Acute hGly2-GLP-2 treatment increased the proportion of eGFP+EdU+/OLFM4+EdU+ cells by 11-22% (p<0.05), without affecting other cell cycle markers. hGly2-GLP-2 treatment also increased the ratio of eGFP+ cells in early-to-late S-phase by 97% (p<0.001), and increased the proportion of eGFP+ cells entering S-phase by 218% (p<0.001). hGly2-GLP-2 treatment induced jejunal expression of genes involved in cell cycle regulation (p<0.05), and increased expression of Mcm3 in the Lgr5-expressing cells by 122% (p<0.05). Conversely. GLP-23-33 reduced the proportion of eGFP+EdU+ cells by 27% (p<0.05), as well as the expression of jejunal cell cycle genes (p<0.05). Finally, chronic hGly2-GLP-2 treatment increased the number of OLFM4+ cells/crypt (p<0.05), in an intestinal epithelial insulin-like growth factor-1 receptor-dependent manner.These findings expand the actions of GLP-2 to encompass acute stimulation of Lgr5+ ISC S-phase entry through the GLP-2R, and chronic induction of Lgr5+ ISC expansion through downstream intestinal insulin-like growth factor-1 signaling.

Recombinant Lloviu virus as a tool to study viral replication and host responses

PLoS pathogens

2022 Feb 01

Hume, AJ;Heiden, B;Olejnik, J;Suder, EL;Ross, S;Scoon, WA;Bullitt, E;Ericsson, M;White, MR;Turcinovic, J;Thao, TTN;Hekman, RM;Kaserman, JE;Huang, J;Alysandratos, KD;Toth, GE;Jakab, F;Kotton, DN;Wilson, AA;Emili, A;Thiel, V;Connor, JH;Kemenesi, G;Cifuentes, D;Mühlberger, E;
PMID: 35120176 | DOI: 10.1371/journal.ppat.1010268

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.

Wnt signaling pathway in the human limbus: a comprehensive mapping by single mRNA detection

Investigative Ophthalmology & Visual Science

2022 Jan 01

Bonnet, C;Ruiz, M;Gonzalez, S;

RESULTS : All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were preferentially expressed in the basal layer of the cornea and limbus compared to the suprabasal layer (_P_

Identification of novel cross-talk between the neuroendocrine and autonomic stress axes controlling blood pressure

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

2021 Apr 14

Elsaafien, K;Kirchner, MK;Mohammed, M;Eikenberry, SA;West, C;Scott, KA;de Kloet, AD;Stern, JE;Krause, EG;
PMID: 33858944 | DOI: 10.1523/JNEUROSCI.0251-21.2021

The hypothalamic paraventricular nucleus (PVN) controls neuroendocrine axes and the autonomic nervous system to mount responses that cope with the energetic burdens of psychological or physiological stress. Neurons in the PVN that express the angiotensin type 1a receptor (PVNAgtr1a) are implicated in neuroendocrine and autonomic stress responses; however, the mechanism by which these neurons coordinate activation of neuroendocrine axes with sympathetic outflow remain unknown. Here, we use a multidisciplinary approach to investigate intra-PVN signaling mechanisms that couple the activity of neurons synthesizing corticotropin-releasing-hormone (CRH) to blood pressure. We used the Cre-Lox system in male mice with in vivo optogenetics and cardiovascular recordings to demonstrate that excitation of PVNAgtr1a promotes elevated blood pressure that is dependent on the sympathetic nervous system. Next, neuroanatomical experiments found that PVNAgtr1a synthesize CRH, and intriguingly, fibers originating from PVNAgtr1a make appositions onto neighboring neurons that send projections to the rostral ventrolateral medulla (RVLM) and express CRH type 1 receptor (CRHR1) mRNA. We then used an ex vivo preparation that combined optogenetics, patch clamp electrophysiology and Ca2+ imaging to discover that excitation of PVNAgtr1a drives the local, intra-PVN release of CRH, which activates RVLM-projecting neurons via stimulation of CRHR1(s). Finally, we returned to our in vivo preparation and found that CRH receptor antagonism specifically within the PVN lowered blood pressure basally and during optogenetic activation of PVNAgtr1a Collectively, these results demonstrate that angiotensin II acts on PVNAgtr1a to conjoin hypothalamic-pituitary-adrenal axis activity with sympathetically mediated vasoconstriction in male mice.SIGNIFICANCE STATEMENTThe survival of an organism is dependent on meeting the energetic demands imposed by stressors. This critical function is accomplished by the central nervous system's ability to orchestrate simultaneous activities of neurosecretory and autonomic axes. Here, we unveil a novel signaling mechanism within the paraventricular nucleus of the hypothalamus that links excitation of neurons producing corticotropin-releasing-hormone with excitation of neurons controlling sympathetic nervous system activity and blood pressure. The implication is that chronic stress exposure may promote cardiometabolic disease by dysregulating the inter-neuronal cross-talk revealed by our experiments.

Decreased expression of synaptic genes in the vestibular ganglion of rodents following subchronic ototoxic stress

Neurobiology of disease

2023 Apr 24

Greguske, EA;Maroto, AF;Borrajo, M;Palou, A;Gut, M;Esteve-Codina, A;Barrallo-Gimeno, A;Llorens, J;
PMID: 37100209 | DOI: 10.1016/j.nbd.2023.106134

The vestibular ganglion contains primary sensory neurons that are postsynaptic to the transducing hair cells (HC) and project to the central nervous system. Understanding the response of these neurons to HC stress or loss is of great interest as their survival and functional competence will determine the functional outcome of any intervention aiming at repair or regeneration of the HCs. We have shown that subchronic exposure to the ototoxicant 3,3'-iminodipropionitrile (IDPN) in rats and mice causes a reversible detachment and synaptic uncoupling between the HCs and the ganglion neurons. Here, we used this paradigm to study the global changes in gene expression in vestibular ganglia using RNA-seq. Comparative gene ontology and pathway analyses of the data from both model species indicated a robust downregulation of terms related to synapses, including presynaptic and postsynaptic functions. Manual analyses of the most significantly downregulated transcripts identified genes with expressions related to neuronal activity, modulators of neuronal excitability, and transcription factors and receptors that promote neurite growth and differentiation. For choice selected genes, the mRNA expression results were replicated by qRT-PCR, validated spatially by RNA-scope, or were demonstrated to be associated with decreased expression of the corresponding protein. We conjectured that decreased synaptic input or trophic support on the ganglion neurons from the HC was triggering these expression changes. To support this hypothesis, we demonstrated decreased expression of BDNF mRNA in the vestibular epithelium after subchronic ototoxicity and also downregulated expression of similarly identified genes (e.g Etv5, Camk1g, Slc17a6, Nptx2, Spp1) after HC ablation with another ototoxic compound, allylnitrile. We conclude that vestibular ganglion neurons respond to decreased input from HCs by decreasing the strength of all their synaptic contacts, both as postsynaptic and presynaptic players.

Single-cell transcriptomic atlas of Alzheimer's disease middle temporal gyrus reveals region, cell type and sex specificity of gene expression with novel genetic risk for MERTK in female

medRxiv : the preprint server for health sciences

2023 Feb 23

Zhang, L;He, CH;Coffey, S;Yin, D;Hsu, IU;Su, C;Ye, Y;Zhang, C;Spurrier, J;Nicholson, L;Rothlin, CV;Ghosh, S;Gopal, PP;Hafler, DA;Zhao, H;Strittmatter, SM;
PMID: 36865305 | DOI: 10.1101/2023.02.18.23286037

Alzheimer's disease, the most common age-related neurodegenerative disease, is closely associated with both amyloid-ß plaque and neuroinflammation. Two thirds of Alzheimer's disease patients are females and they have a higher disease risk. Moreover, women with Alzheimer's disease have more extensive brain histological changes than men along with more severe cognitive symptoms and neurodegeneration. To identify how sex difference induces structural brain changes, we performed unbiased massively parallel single nucleus RNA sequencing on Alzheimer's disease and control brains focusing on the middle temporal gyrus, a brain region strongly affected by the disease but not previously studied with these methods. We identified a subpopulation of selectively vulnerable layer 2/3 excitatory neurons that that were RORB-negative and CDH9-expressing. This vulnerability differs from that reported for other brain regions, but there was no detectable difference between male and female patterns in middle temporal gyrus samples. Disease-associated, but sex-independent, reactive astrocyte signatures were also present. In clear contrast, the microglia signatures of diseased brains differed between males and females. Combining single cell transcriptomic data with results from genome-wide association studies (GWAS), we identified MERTK genetic variation as a risk factor for Alzheimer's disease selectively in females. Taken together, our single cell dataset revealed a unique cellular-level view of sex-specific transcriptional changes in Alzheimer's disease, illuminating GWAS identification of sex-specific Alzheimer's risk genes. These data serve as a rich resource for interrogation of the molecular and cellular basis of Alzheimer's disease.

Boldine modulates glial transcription and functional recovery in a murine model of contusion spinal cord injury

bioRxiv : the preprint server for biology

2023 Feb 15

Toro, CA;Johnson, K;Hansen, J;Siddiq, MM;Vásquez, W;Zhao, W;Graham, ZA;Sáez, JC;Iyengar, R;Cardozo, CP;
PMID: 36824813 | DOI: 10.1101/2023.02.15.528337

Membrane channels such as connexins (Cx), pannexins (Panx) and P2X 7 receptors (P2X 7 R) are permeable to calcium ions and other small molecules such as ATP and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx hemichannels (HC) and Panx. To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X 7 R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing (of the spinal cord revealed that boldine modulated a large number of genes involved in neurotransmission in in spinal cord tissue just below the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.

SARS-CoV2 infects pancreatic beta cells in vivo and induces cellular and subcellular disruptions that reflect beta cell dysfunction

Research square

2021 Jul 20

Millette, K;Cuala, J;Wang, P;Marks, C;Woo, V;Hayun, M;Kang, H;Martin, M;Dhawan, S;Chao, L;Fraser, S;Junge, J;Lewis, M;Georgia, S;
PMID: 34312617 | DOI: 10.21203/rs.3.rs-592374/v1

Increasing evidence of new-onset diabetes during the COVID19 pandemic indicates that the SARS-CoV2 virus may drive beta-cell dysfunction leading to diabetes, but it is unclear if it is a primary or secondary effect. Here, we present evidence of SARS-CoV-2 infection of pancreatic beta cells in vivo using a robust and reproducible non-human primates model of mild to moderate COVID19 pathogenesis. Pancreas from SARS-CoV-2 infected subjects were positive for the SARS-CoV2 spike protein by immunohistochemistry and structures indicative of viral replication were evident by electron microscopy. Total beta cell area was decreased in SARS-CoV-2-infected pancreas, attributable to beta cell atrophy. Beta cell granularity was decreased. These histologic phenotypes persisted beyond the duration of the clinical disease course. Detailed electron microscopy of SARS-CoV-2 infected beta-cells revealed ultrastructural hallmarks of beta cell stress that are seen in islets of patients with Type 2 diabetes, including disrupted mitochondria and dilated endoplasmic reticulum. To assess the metabolic status of beta cells from SARS-CoV-2-infected subjects, we used fluorescence life-time imaging to measure the ratio of free and bound NADH as a surrogate of glycolytic and oxidative metabolism. We report an increase in free NADH levels, suggesting that beta cells from SARS-CoV-2-infected subjects adopt a more glycolytic metabolic profile. Taken together, we conclude that SARS-CoV-2 infection induces beta cell stress that may compromise beta-cell function beyond the duration of the disease course. This raises the possibility that the beta cell stress and injury may have clinical implications of the long-term future health of patients that have recovered from COVID19.

Drosophila Fezf functions as a transcriptional repressor to direct layer-specific synaptic connectivity in the fly visual system

Proceedings of the National Academy of Sciences of the United States of America

2021 Mar 30

Santiago, IJ;Zhang, D;Saras, A;Pontillo, N;Xu, C;Chen, X;Weirauch, MT;Mistry, M;Ginty, DD;Pecot, MY;Peng, J;
PMID: 33766917 | DOI: 10.1073/pnas.2025530118

The layered compartmentalization of synaptic connections, a common feature of nervous systems, underlies proper connectivity between neurons and enables parallel processing of neural information. However, the stepwise development of layered neuronal connections is not well understood. The medulla neuropil of the Drosophila visual system, which comprises 10 discrete layers (M1 to M10), where neural computations underlying distinct visual features are processed, serves as a model system for understanding layered synaptic connectivity. The first step in establishing layer-specific connectivity in the outer medulla (M1 to M6) is the innervation by lamina (L) neurons of one of two broad, primordial domains that will subsequently expand and transform into discrete layers. We previously found that the transcription factor dFezf cell-autonomously directs L3 lamina neurons to their proper primordial broad domain before they form synapses within the developing M3 layer. Here, we show that dFezf controls L3 broad domain selection through temporally precise transcriptional repression of the transcription factor slp1 (sloppy paired 1). In wild-type L3 neurons, slp1 is transiently expressed at a low level during broad domain selection. When dFezf is deleted, slp1 expression is up-regulated, and ablation of slp1 fully rescues the defect of broad domain selection in dFezf-null L3 neurons. Although the early, transient expression of slp1 is expendable for broad domain selection, it is surprisingly necessary for the subsequent L3 innervation of the M3 layer. DFezf thus functions as a transcriptional repressor to coordinate the temporal dynamics of a transcriptional cascade that orchestrates sequential steps of layer-specific synapse formation.

Single-cell transcriptomic profiling of the mouse cochlea: An atlas for targeted therapies

Proceedings of the National Academy of Sciences of the United States of America

2023 Jun 27

Jean, P;Wong Jun Tai, F;Singh-Estivalet, A;Lelli, A;Scandola, C;Megharba, S;Schmutz, S;Roux, S;Mechaussier, S;Sudres, M;Mouly, E;Heritier, AV;Bonnet, C;Mallet, A;Novault, S;Libri, V;Petit, C;Michalski, N;
PMID: 37339214 | DOI: 10.1073/pnas.2221744120

Functional molecular characterization of the cochlea has mainly been driven by the deciphering of the genetic architecture of sensorineural deafness. As a result, the search for curative treatments, which are sorely lacking in the hearing field, has become a potentially achievable objective, particularly via cochlear gene and cell therapies. To this end, a complete inventory of cochlear cell types, with an in-depth characterization of their gene expression profiles right up to their final differentiation, is indispensable. We therefore generated a single-cell transcriptomic atlas of the mouse cochlea based on an analysis of more than 120,000 cells on postnatal day 8 (P8), during the prehearing period, P12, corresponding to hearing onset, and P20, when cochlear maturation is almost complete. By combining whole-cell and nuclear transcript analyses with extensive in situ RNA hybridization assays, we characterized the transcriptomic signatures covering nearly all cochlear cell types and developed cell type-specific markers. Three cell types were discovered; two of them contribute to the modiolus which houses the primary auditory neurons and blood vessels, and the third one consists in cells lining the scala vestibuli. The results also shed light on the molecular basis of the tonotopic gradient of the biophysical characteristics of the basilar membrane that critically underlies cochlear passive sound frequency analysis. Finally, overlooked expression of deafness genes in several cochlear cell types was also unveiled. This atlas paves the way for the deciphering of the gene regulatory networks controlling cochlear cell differentiation and maturation, essential for the development of effective targeted treatments.

A leptin-responsive hypothalamic circuit inputs to the circadian feeding network

bioRxiv : the preprint server for biology

2023 Feb 26

Tang, Q;Godschall, E;Brennan, CD;Zhang, Q;Abraham-Fan, RJ;Williams, SP;Güngül, TB;Onoharigho, R;Buyukaksakal, A;Salinas, R;Olivieri, JJ;Deppmann, CD;Campbell, JN;Podyma, B;Güler, AD;
PMID: 36865258 | DOI: 10.1101/2023.02.24.529901

Salient cues, such as the rising sun or the availability of food, play a crucial role in entraining biological clocks, allowing for effective behavioral adaptation and ultimately, survival. While the light-dependent entrainment of the central circadian pacemaker (suprachiasmatic nucleus, SCN) is relatively well defined, the molecular and neural mechanisms underlying entrainment associated with food availability remains elusive. Using single nucleus RNA sequencing during scheduled feeding (SF), we identified a leptin receptor (LepR) expressing neuron population in the dorsomedial hypothalamus (DMH) that upregulates circadian entrainment genes and exhibits rhythmic calcium activity prior to an anticipated meal. We found that disrupting DMH LepR neuron activity had a profound impact on both molecular and behavioral food entrainment. Specifically, silencing DMH LepR neurons, mis-timed exogenous leptin administration, or mis-timed chemogenetic stimulation of these neurons all interfered with the development of food entrainment. In a state of energy abundance, repetitive activation of DMH LepR neurons led to the partitioning of a secondary bout of circadian locomotor activity that was in phase with the stimulation and dependent on an intact SCN. Lastly, we discovered that a subpopulation of DMH LepR neurons project to the SCN with the capacity to influence the phase of the circadian clock. This leptin regulated circuit serves as a point of integration between the metabolic and circadian systems, facilitating the anticipation of meal times.

Probing regenerative heterogeneity of corticospinal neurons with scRNA-Seq

Research square

2023 Feb 21

Kim, H;Saikia, J;Monte, K;Ha, E;Romaus-Sanjurjo, D;Sanchez, J;Moore, A;Hernaiz-Llorens, M;Chavez-Martinez, C;Agba, C;Li, H;Lusk, D;Cervantes, K;Zheng, B;
PMID: 36865182 | DOI: 10.21203/rs.3.rs-2588274/v1

The corticospinal tract (CST) is clinically important for the recovery of motor functions after spinal cord injury. Despite substantial progress in understanding the biology of axon regeneration in the central nervous system (CNS), our ability to promote CST regeneration remains limited. Even with molecular interventions, only a small proportion of CST axons regenerate1. Here we investigate this heterogeneity in the regenerative ability of corticospinal neurons following PTEN and SOCS3 deletion with patch-based single cell RNA sequencing (scRNA-Seq)2,3, which enables deep sequencing of rare regenerating neurons. Bioinformatic analyses highlighted the importance of antioxidant response and mitochondrial biogenesis along with protein translation. Conditional gene deletion validated a role for NFE2L2 (or NRF2), a master regulator of antioxidant response, in CST regeneration. Applying Garnett4, a supervised classification method, to our dataset gave rise to a Regenerating Classifier (RC), which, when applied to published scRNA-Seq data, generates cell type- and developmental stage-appropriate classifications. While embryonic brain, adult dorsal root ganglion and serotonergic neurons are classified as Regenerators, most neurons from adult brain and spinal cord are classified as Non-regenerators. Adult CNS neurons partially revert to a regenerative state soon after injury, which is accelerated by molecular interventions. Our data indicate the existence of universal transcriptomic signatures underlying the regenerative abilities of vastly different neuronal populations, and further illustrate that deep sequencing of only hundreds of phenotypically identified CST neurons has the power to reveal new insights into their regenerative biology.

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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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