| 1. |
- Aresh, Bejan, 1984-, et al.
(författare)
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Characterization of preprodynorphin-expressing cells in the mouse nervous system
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Dynorphin is an endogenous opioid that has been implicated in maintaining chronic pain and gating of both itch and acute mechanical pain through acting on both opioid and non-opioid receptors. To improve our understanding of the complex and multifunctional population that expresses dynorphin, we have constructed a preprodynorphin Cre line (Pdyn-Cre) using BAC cloning. Single cell analysis of tdTomato;Pdyn-Cre cells revealed that 43% of the population expressed Pdyn mRNA, and no analyzed tdTomato;Pdyn-Cre negative cell expressed Pdyn mRNA, thus confirming that Cre had been inserted under the control of the Pdyn promoter. The Pdyn-Cre expressing population was found in the dorsal spinal cord, mainly in lamina II-IV and overlapped to 47% with Vglut2 mRNA, while co-expression with the inhibitory markers Viaat-egfp and Pax2 was 13% and 28%, respectively. The expression of Pdyn-Cre in the brain was extensive, marking virtually all cortical structures, including somatosensory and motor cortex. Furthermore, Pdyn-Cre was densely expressed in the striatum, amygdala and parts of the hippocampus, and expression was also observed in several pain and itch processing areas, including amygdala, lateral parabrachial nucleus, claustrum, insular cortex and raphe magnus nucleus. Our analysis indicates that the transgenic Pdyn-Cre line includes PDYN cells in the nervous system and will thus be useful as a transgenic tool for studies of the role and connectivity of the PDYN population.
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| 2. |
- Aresh, Bejan, 1984-, et al.
(författare)
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Characterization of preproenkephalin expressing neurons in the nervous system using a transgenic line
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Here we have constructed a Cre line to target preproenkephalin expressing cells (Penk-Cre) using a BAC cloning strategy. By crossing our Penk-Cre line with the tdTomato reporter, tdTomato;Penk-Cre expressing cells could be visualized. Penk-Cre was expressed throughout the dorsal spinal cord, where approximately 50% of the neurons were residing within lamina III. Furthermore, single-cell analysis of spinal Penk-Cre expressing cells showed that 41% was positive for Penk mRNA and that a majority (94%) of the population expressed Vglut2 mRNA. Immunohistochemical analysis showed that only 7% and 13% expressed the inhibitory markers Viaat-egfp and Pax2, respectively, hence identifying spinal Penk-Cre expressing neurons as mainly excitatory. The expression of Penk-Cre in the brain was extensive, including dense expression in the striatum, nucleus accumbens and several amygdaloid nuclei. Furthermore, Penk-Cre expression was also shown in insular cortex, cingulated cortex, primary and secondary somatosensory cortices and the trigeminal nuclei. The Penk-Cre line represents a useful genetic tool for future analysis of PENK expressing neurons in the nervous system.
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| 3. |
- Aresh, Bejan, 1984-, et al.
(författare)
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Dissection and Culture of Mouse Embryonic Kidney
- 2017
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; :123
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Tidskriftsartikel (refereegranskat)abstract
- The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant. One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.
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| 4. |
- Aresh, Bejan
(författare)
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Functional Aspects of Peripheral and Spinal Cord Neurons Involved in Itch and Pain
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- We have investigated the role of the metabotropic glutamate receptor 7 (mGluR7) and the gastrin releasing peptide receptor (Grpr) population that are involved at different levels of itch transmission. We found that mGuR7 deficient mice displayed an anaphylaxis-like behavior when provoked with histamine. Analysis of blood revealed elevated plasma levels of histamine and mouse mast cell protease-1 (mMCP1), two indicators of anaphylaxis, in mGluR7 deficient mice compared with control mice. Inhibition of the neurokinin 1 receptor, by preventing binding of the corresponding ligand substance P (SP), prior to provocation with histamine prevented the development of anaphylaxis in mGluR7 deficient animals. However, blocking GRPR (gastrin releasing peptide receptor) only resulted in decreased itch levels in mGluR7 deficient mice but did not prevent the systemic anaphylaxis-like behavior. Our findings indicate that mGluR7 normally functions as a brake on histaminergic itch that is mediated through GRPR as well as anaphylaxis through Substance P.Grpr has previously been shown to mediate both histaminergic and non-histaminergic itch but little is known about the GRPR neuronal population. We used a BAC cloning strategy to construct a Grpr-Cre line, which we crossed with the reporter lines tdTomato and Viaat-egfp as well as with Vglut2-lox. We could conclude that Grpr-Cre neurons are mainly excitatory interneurons located in lamina II-IV, that convey itch using VGLUT2-mediated glutamatergic transmission to the next, currently unknown, step in the labeled line of chemical itch.To eventually deduce the function of the endogenous opioids dynorphin and enkephalin, which are hypothesized to be involved in gating pain and itch in the spinal cord, we constructed two Cre lines using BAC cloning that targeted the precursor proteins preprodynorphin and preproenkephalin, respectively. Preprodynorphin-Cre neurons were mainly located in lamina II-IV and overlapped to 47% with Vglut2 mRNA, while the co-expression with the inhibitory markers Viaat-egfp and PAX2 was 13% and 28% respectively in the spinal cord. Preproenkephalin neurons were more localized to lamina III in the dorsal horn, furthermore single cell analysis showed that they overlapped to 94% with Vglut2 mRNA while 7% and 13% expressed Viaat-egfp and PAX2 respectively.
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| 5. |
- Aresh, Bejan, 1984-, et al.
(författare)
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Spinal Cord Interneurons Expressing the Gastrin-Releasing Peptide Receptor Convey Itch Through VGLUT2-Mediated Signaling
- 2017
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Ingår i: Pain. - : Ovid Technologies (Wolters Kluwer Health). - 0304-3959 .- 1872-6623. ; 158:5, s. 945-961
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Tidskriftsartikel (refereegranskat)abstract
- Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin-releasing peptide receptor (GRPR) have been identified as specific mediators of itch. However, our understanding of the GRPR population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in laminae II-IV. Application of the specific agonist gastrin-releasing peptide induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA, and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox; Grpr-Cre mice) displayed less spontaneous itch and attenuated responses to both histaminergic and nonhistaminergic agents. We could also show that application of the itch-inducing peptide, natriuretic polypeptide B, induces calcium influx in a subpopulation of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch.
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| 6. |
- Peuckert, Christiane, 1975-, et al.
(författare)
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Multimodal Eph/Ephrin signaling controls several phases of urogenital development
- 2016
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Ingår i: Kidney International. - : Elsevier BV. - 0085-2538 .- 1523-1755. ; 90:2, s. 373-388
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Tidskriftsartikel (refereegranskat)abstract
- A substantial portion of the human population is affected by urogenital birth defects resulting from a failure in ureter development. Although recent research suggests roles for several genes in facilitating the ureter/bladder connection, the underlying molecular mechanisms remain poorly understood. Signaling via Eph receptor tyrosine kinases is involved in several developmental processes. Here we report that impaired Eph/Ephrin signaling in genetically modified mice results in severe hydronephrosis caused by defective ureteric bud induction, ureter maturation, and translocation. Our data imply that ureter translocation requires apoptosis in the urogenital sinus and inhibition of proliferation in the common nephric duct. These processes were disturbed in EphA4/EphB2 compound knockout mice and were accompanied by decreased ERK-2 phosphorylation. Using a set of Eph, Ephrin, and signaling-deficient mutants, we found that during urogenital development, different modes of Eph/Ephrin signaling occur at several sites with EphrinB2 and EphrinA5 acting in concert. Thus, Eph/Ephrin signaling should be considered in the etiology of congenital kidney and urinary tract anomalies.
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| 7. |
- Rogoz, Katarzyna, et al.
(författare)
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Identification of a Neuronal Receptor Controlling Anaphylaxis
- 2016
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 14:2, s. 370-379
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Tidskriftsartikel (refereegranskat)abstract
- Allergic reactions can in severe cases induce a state of circulatory shock referred to as anaphylaxis. Histamine, the primary mediator of this condition, is released from immune cells, and, therefore, anaphylaxis has so far been considered an immune system disorder. However, we here show that the glutamatergic receptor mGluR7, expressed on a subpopulation of both peripheral and spinal cord neurons, controls histamine-induced communication through calcium-dependent autoinhibition with implications for anaphylaxis. Genetic ablation of mGluR7, and thus altered regulation of histamine-sensing neurons, caused an anaphylaxis-like state in mGluR7(-/-) mice, which could be reversed by antagonizing signaling between neurons and mast cells but not by antagonizing a central itch pathway. Our findings demonstrate the vital role of nervous system control by mGluR7 in anaphylaxis and open up possibilities for preventive strategies for this life-threatening condition.
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| 8. |
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| 9. |
- Steinz, Maarten M, et al.
(författare)
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Oxidative hotspots on actin promote skeletal muscle weakness in rheumatoid arthritis
- 2019
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Ingår i: JCI Insight. - : American Society for Clinical Investigation (ASCI). - 2379-3708 .- 2324-7703 .- 2325-4556. ; 4:9
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Tidskriftsartikel (refereegranskat)abstract
- Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here, we show that oxidative posttranslational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spectrometry, we identified the actin residues targeted by oxidative 3-nitrotyrosine (3-NT) or malondialdehyde (MDA) adduct modifications in weakened skeletal muscle from mice with arthritis and patients afflicted by RA. The residues were primarily located in 3 distinct regions positioned at matching surface areas of the skeletal muscle actin molecule from arthritic mice and patients with RA. Moreover, molecular dynamics simulations revealed that these areas, here coined "hotspots," are important for the stability of the actin molecule and its capacity to generate filaments and interact with myosin. Together, these data demonstrate how oxidative modifications on actin promote muscle weakness in RA patients and may provide novel leads for targeted therapeutic treatment to improve muscle function.
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