| 1. |
- 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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| 2. |
- Cheng, Xinlai, et al.
(författare)
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Essential role of mitochondrial Stat3 in p38MAPK mediated apoptosis under oxidative stress
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Stat3 is an oncogene, frequently associated with malignant transformation. A body of evidence implicates that phospho-Stat3(Y705) contributes to its nucleic translocation, while phospho-Stat3(S727) leads to the accumulation in mitochondria. Both are of importance for tumor cell proliferation. In comparison to well-characterized signaling pathways interplaying with Stat3(Y705), little is known about Stat3(S727). In this work, we studied the influence of Stat3 deficiency on the viability of cells exposed to H2O2 or hypoxia using siRNA and CRISPR/Cas9 genome-editing. We found dysregulation of mitochondrial activity, which was associated with excessive ROS formation and reduced mitochondrial membrane potential, and observed a synergistic effect for oxidative stress-mediated apoptosis in Stat3-KD cells or cells carrying Stat3(Y705F), but not Stat3(S727D), suggesting the importance of functional mitochondrial Stat3 in this context. We also found that ROS-mediated activation of ASK1/p38(MAPK) was involved and adding antioxidants, p38(MAPK) inhibitor, or genetic repression of ASK1 could easily rescue the cellular damage. Our finding reveals a new role of mitochondrial Stat3 in preventing ASK1/p38(MAPK)-mediated apoptosis, wich further support the notion that selective inhibition mitochondrial Stat3 could provide a primsing target for chemotherapy.
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| 3. |
- Defourny, Jean, et al.
(författare)
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EphA4-ADAM10 Interplay Patterns the Cochlear Sensory Epithelium through Local Disruption of Adherens Junctions
- 2019
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Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 11, s. 246-257
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Tidskriftsartikel (refereegranskat)abstract
- The cochlear sensory epithelium contains a functionally important triangular fluid-filled space between adjacent pillar cells referred to as the tunnel of Corti. However, the molecular mechanisms leading to local cell-cell separation during development remain elusive. Here we show that EphA4 associates with ADAM10 to promote the destruction of E-cadherin-based adhesions between adjacent pillar cells. These cells fail to separate from each other, and E-cadherin abnormally persists at the pillar cell junction in EphA4 forward-signaling-deficient mice, as well as in the presence of ADAM10 inhibitor. Using immunolabeling and an in situ proximity ligation assay, we found that EphA4 forms a complex with E-cadherin and its sheddase ADAM10, which could be activated by ephrin-B2 across the pillar cell junction to trigger the cleavage of E-cadherin. Altogether, our findings provide a new molecular insight into the regulation of adherens junctions, which might be extended to a variety of physiological or pathological processes.
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| 4. |
- Defourny, Jean, et al.
(författare)
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Ephrin-A5/EphA4 signalling controls specific afferent targeting to cochlear hair cells
- 2013
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4, s. 1438-
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Tidskriftsartikel (refereegranskat)abstract
- Hearing requires an optimal afferent innervation of sensory hair cells by spiral ganglion neurons in the cochlea. Here we report that complementary expression of ephrin-A5 in hair cells and EphA4 receptor among spiral ganglion neuron populations controls the targeting of type I and type II afferent fibres to inner and outer hair cells, respectively. In the absence of ephrin-A5 or EphA4 forward signalling, a subset of type I projections aberrantly overshoot the inner hair cell layer and invade the outer hair cell area. Lack of type I afferent synapses impairs neurotransmission from inner hair cells to the auditory nerve. By contrast, radial shift of type I projections coincides with a gain of presynaptic ribbons that could enhance the afferent signalling from outer hair cells. Ephexin-1, cofilin and myosin light chain kinase act downstream of EphA4 to induce type I spiral ganglion neuron growth cone collapse. Our findings constitute the first identification of an Eph/ephrin-mediated mutual repulsion mechanism responsible for specific sorting of auditory projections in the cochlea.
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| 5. |
- Farnsworth, Bryn, et al.
(författare)
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Gene Expression of Quaking in Sporadic Alzheimer’s Disease Patients is Both Upregulated and Related to Expression Levels of Genes Involved in Amyloid Plaque and Neurofibrillary Tangle Formation
- 2016
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Ingår i: Journal of Alzheimer's Disease. - : IOS Press. - 1387-2877 .- 1875-8908. ; 53:1, s. 209-219
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Tidskriftsartikel (refereegranskat)abstract
- Quaking (QKI) is a gene exclusively expressed within glial cells. QKI has previously been implicated in various neurological disorders and diseases, including Alzheimer’s disease (AD), a condition for which increasing evidence suggests a central role of glia cells. The objective of the present study was to investigate the expression levels of QKI and three QKI isoforms (QKI5, QKI6, and QKI7) in AD. Genes that have previously been related to the ontogeny and progression of AD, specifically APP, PSEN1, PSEN2, and MAPT, were also investigated. A real-time PCR assay of 123 samples from human postmortem sporadic AD patients and control brains was performed. The expression values were analyzed with an analysis of covariance model and subsequent multiple regressions to explore the possibility of related expression values between QKI, QKI isoforms, and AD-related genes. Further, the sequences of AD-related genes were analyzed for the presence of QKI binding domains. QKI and all measured QKI isoforms were found to be significantly upregulated in AD samples, relative to control samples. However, APP, PSEN1, PSEN2, and MAPT were not found to be significantly different. QKI and QKI isoforms were found to be predictive for the variance of APP, PSEN1, PSEN2, and MAPT, and putative QKI binding sites suggests an interaction with QKI. Overall, these results implicate a possible role of QKI in AD, although the exact mechanism by which this occurs remains to be uncovered.
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| 6. |
- Gezelius, Henrik, 1977-, et al.
(författare)
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Conditional genetic labeling of the Renshaw cell population for functional studies of motor control
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The Renshaw cells were among the first interneurons to be characterized in the mammalian spinal cord. Although the basic function of recurrent inhibition to motor neurons, as well as the Renshaw cell connectivity to other neurons have been thoroughly studied, the exact functional role of the Renshaw cells in motor control is still unknown. To further characterize the role of Renshaw cells in spinal cord circuitry, we searched for candidate genes useful in the Cre-loxP system. It has been reported that the mRNA expression of nicotinic cholinergic receptor alpha 2 (Chrna2) is found in a restricted number of cells at the ventral rim in adult rat and mouse spinal cord. In our own search for genes with distinct ventral expression, we noted a similar restricted Chrna2 mRNA expression pattern in the mouse spinal cord at postnatal day (P) 11 and during development at embryonic day 14.5. Based on the fact that the gene product is a cholinergic receptor and the pattern of expression, the neurons are predicted to be Renshaw cells. The possibility that these cells were motor neurons was excluded, since Chrna2 and Vesicular acetylcholine were not co-expressed at P11. To further study this cell population, we have generated a transgenic mouse expressing Cre recombinase (Cre) under the control of the Chrna2 promoter region. To visualize the Cre-expressing cells, the Chrna2-Cre transgenic mouse were bred with a reporter mouse expressing β-galactosidase (β-gal) in the nucleus after loxP excision. As expected, spinal cord β-gal immunoreactivity was observed in a limited number of ventrally located cells in the Cre-bearing offspring. Co-labeling of β-gal with calbindin-28K, a known marker for Renshaw cells, indicated that a majority of the calbindin positive cells were also β-gal positive at the ventral rim where calbindin is specific. In addition, β-gal positive cells without observable calbindin were also detected. It is conceivable that Chrna2 is expressed in additional cells apart from Renshaw cells or that a previously unidentified Renshaw cell subpopulation does not express calbindin. Nonetheless, a mouse with Cre-activity restricted to Chrna2-expressing cells opens the possibility to functionally study a limited population of spinal cord interneurons through genetic techniques, with the ambition to explore the specific role of Renshaw cells in spinal cord circuitry and motor control.
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| 7. |
- Johansson, Martin M., 1976-, et al.
(författare)
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Novel Y-Chromosome Long Non-Coding RNAs Expressed in Human Male CNS During Early Development
- 2019
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Ingår i: Frontiers in Genetics. - : Frontiers Media S.A.. - 1664-8021. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Global microarray gene expression analyses previously demonstrated differences in female and male embryos during neurodevelopment. In particular, before sexual maturation of the gonads, the differences seem to concentrate on the expression of genes encoded on the X- and Y-chromosomes. To investigate genome-wide differences in expression during this early developmental window, we combined high-resolution RNA sequencing with qPCR to analyze brain samples from human embryos during the first trimester of development. Our analysis was tailored for maximum sensitivity to discover Y-chromosome gene expression, but at the same time, it was underpowered to detect X-inactivation escapees. Using this approach, we found that 5 out of 13 expressed gametolog pairs showed unbalanced gene dosage, and as a consequence, a male-biased expression. In addition, we found six novel non-annotated long non-coding RNAs on the Y-chromosome with conserved expression patterns in newborn chimpanzee. The tissue specific and time-restricted expression of these long non-coding RNAs strongly suggests important functions during central nervous system development in human males.
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| 8. |
- Larhammar, Martin, 1985-, et al.
(författare)
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SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis
- 2015
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Ingår i: Biological Psychiatry. - : Elsevier BV. - 0006-3223 .- 1873-2402. ; 77:6, s. 526-536
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundThe neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis.MethodsWe used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine-regulated behaviors.ResultsWe show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice overexpressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine.ConclusionsOur results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders.
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| 9. |
- Mota, Ana, et al.
(författare)
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FRET-FISH probes chromatin compaction at individual genomic loci in single cells
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Chromatin compaction is a key biophysical property that influences multiple DNA transactions. Lack of chromatin accessibility is frequently used as proxy for chromatin compaction. However, we currently lack tools for directly probing chromatin compaction at individual genomic loci. To fill this gap, here we present FRET-FISH, a method combining fluorescence resonance energy transfer (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at select loci in single cells. We first validate FRET-FISH by comparing it with ATAC-seq, demonstrating that local compaction and accessibility are strongly correlated. FRET-FISH also detects expected differences in compaction upon treatment with drugs perturbing global chromatin condensation. We then leverage FRET-FISH to study local chromatin compaction on the active and inactive X chromosome, along the nuclear radius, in different cell cycle phases, and during increasing passage number. FRET-FISH is a robust tool for probing local chromatin compaction in single cells.
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| 10. |
- Olivera, Gabriela Carina, et al.
(författare)
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Blood-brain barrier-restricted translocation of Toxoplasma gondii from cortical capillaries
- 2021
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Ingår i: eLIFE. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The cellular barriers of the central nervous system proficiently protect the brain parenchyma from infectious insults. Yet, the single-celled parasite Toxoplasma gondii commonly causes latent cerebral infection in humans and other vertebrates. Here, we addressed the role of the cerebral vasculature in the passage of T. gondii to the brain parenchyma. Shortly after inoculation in mice, parasites mainly localized to cortical capillaries, in preference over post-capillary venules, cortical arterioles or meningeal and choroidal vessels. Early invasion to the parenchyma (days 1-5) occurred in absence of a measurable increase in blood-brain barrier (BBB) permeability, perivascular leukocyte cuffs or hemorrhage. However, sparse focalized permeability elevations were detected adjacently to replicative parasite foci. Further, T. gondii triggered inflammatory responses in cortical microvessels and endothelium. Pro- and anti-inflammatory treatments of mice with LPS and hydrocortisone, respectively, impacted BBB permeability and parasite loads in the brain parenchyma. Finally, pharmacological inhibition or Cre/loxP conditional knockout of endothelial focal adhesion kinase (FAK), a BBB intercellular junction regulator, facilitated parasite translocation to the brain parenchyma. The data reveal that the initial passage of T. gondii to the central nervous system occurs principally across cortical capillaries. The integrity of the microvascular BBB restricts parasite transit, which conversely is exacerbated by the inflammatory response.
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