1.
Izsak, Julia, et al.
(författare)
Differential acute impact of therapeutically effective and overdose concentrations of lithium on human neuronal single cell and network function
2021
Ingår i: Translational Psychiatry. - : Springer Science and Business Media LLC. - 2158-3188. ; 11
Tidskriftsartikel (refereegranskat) abstract
Lithium salts are used as mood-balancing medication prescribed to patients suffering from neuropsychiatric disorders, such as bipolar disorder and major depressive disorder. Lithium salts cross the blood-brain barrier and reach the brain parenchyma within few hours after oral application, however, how lithium influences directly human neuronal function is unknown. We applied patch–clamp and microelectrode array technology on human induced pluripotent stem cell (iPSC)-derived cortical neurons acutely exposed to therapeutic (<1 mM) and overdose concentrations (>1 mM) of lithium chloride (LiCl) to assess how therapeutically effective and overdose concentrations of LiCl directly influence human neuronal electrophysiological function at the synapse, single-cell, and neuronal network level. We describe that human iPSC-cortical neurons exposed to lithium showed an increased neuronal activity under all tested concentrations. Furthermore, we reveal a lithium-induced, concentration-dependent, transition of regular synchronous neuronal network activity using therapeutically effective concentration (<1 mM LiCl) to epileptiform-like neuronal discharges using overdose concentration (>1 mM LiCl). The overdose concentration lithium-induced epileptiform-like activity was similar to the epileptiform-like activity caused by the GABAA-receptor antagonist. Patch–clamp recordings reveal that lithium reduces action potential threshold at all concentrations, however, only overdose concentration causes increased frequency of spontaneous AMPA-receptor mediated transmission. By applying the AMPA-receptor antagonist and anti-epileptic drug Perampanel, we demonstrate that Perampanel suppresses lithium-induced epileptiform-like activity in human cortical neurons. We provide insights in how therapeutically effective and overdose concentration of lithium directly influences human neuronal function at synapse, a single neuron, and neuronal network levels. Furthermore, we provide evidence that Perampanel suppresses pathological neuronal discharges caused by overdose concentrations of lithium in human neurons.
2.
Delsing, Louise, et al.
(författare)
Models of the blood-brain barrier using iPSC-derived cells
2020
Ingår i: Molecular and Cellular Neuroscience. - : Elsevier BV. - 1044-7431 .- 1095-9327. ; 107
Tidskriftsartikel (refereegranskat) abstract
The blood-brain barrier (BBB) constitutes the interface between the blood and the brain tissue. Its primary function is to maintain the tightly controlled microenvironment of the brain. Models of the BBB are useful for studying the development and maintenance of the BBB as well as diseases affecting it. Furthermore, BBB models are important tools in drug development and support the evaluation of the brain-penetrating properties of novel drug molecules. Currently used in vitro models of the BBB include immortalized brain endothelial cell lines and primary brain endothelial cells of human and animal origin. Unfortunately, many cell lines and primary cells do not recreate physiological restriction of transport in vitro. Human-induced pluripotent stem cell (iPSC)-derived brain endothelial cells have proven a promising alternative source of brain endothelial-like cells that replicate tight cell layers with low paracellular permeability. Given the possibility to generate large amounts of human iPSC-derived brain endothelial cells they are a feasible alternative when modelling the BBB in vitro. iPSC-derived brain endothelial cells form tight cell layers in vitro and their barrier properties can be enhanced through co-culture with other cell types of the BBB. Currently, many different models of the BBB using iPSC-derived cells are under evaluation to study BBB formation, maintenance, disruption, drug transport and diseases affecting the BBB. This review summarizes important functions of the BBB and current efforts to create iPSC-derived BBB models in both static and dynamic conditions. In addition, it highlights key model requirements and remaining challenges for human iPSC-derived BBB models in vitro.
3.
Zhang, Yuehui, et al.
(författare)
Increased uterine androgen receptor protein abundance results in implantation and mitochondrial defects in pregnant rats with hyperandrogenism and insulin resistance. : Deciphering the role of uterine AR in PCOS during early pregnancy
2021
Ingår i: Journal of molecular medicine (Berlin, Germany). - : Springer Science and Business Media LLC. - 1432-1440 .- 0946-2716. ; 99, s. 1427-1446
Tidskriftsartikel (refereegranskat) abstract
In this study, we show that during normal rat pregnancy, there is a gestational stage-dependent decrease in androgen receptor (AR) abundance in the gravid uterus and that this is correlated with the differential expression of endometrial receptivity and decidualization genes during early and mid-gestation. In contrast, exposure to 5α-dihydrotestosterone (DHT) and insulin (INS) or DHT alone significantly increased AR protein levels in the uterus in association with the aberrant expression of endometrial receptivity and decidualization genes, as well as disrupted implantation. Next, we assessed the functional relevance of the androgen-AR axis in the uterus for reproductive outcomes by treating normal pregnant rats and pregnant rats exposed to DHT and INS with the anti-androgen flutamide. We found that AR blockage using flutamide largely attenuated the DHT and INS-induced maternal endocrine, metabolic, and fertility impairments in pregnant rats in association with suppressed induction of uterine AR protein abundance and androgen-regulated response protein and normalized expression of several endometrial receptivity and decidualization genes. Further, blockade of AR normalized the expression of the mitochondrial biogenesis marker Nrf1 and the mitochondrial functional proteins Complexes I and II, VDAC, and PHB1. However, flutamide treatment did not rescue the compromised mitochondrial structure resulting from co-exposure to DHT and INS. These results demonstrate that functional AR protein is an important factor for gravid uterine function. Impairments in the uterine androgen-AR axis are accompanied by decreased endometrial receptivity, decidualization, and mitochondrial dysfunction, which might contribute to abnormal implantation in pregnant PCOS patients with compromised pregnancy outcomes and subfertility. KEY MESSAGES: The proper regulation of uterine androgen receptor (AR) contributes to a normal pregnancy process, whereas the aberrant regulation of uterine AR might be linked to polycystic ovary syndrome (PCOS)-induced pregnancy-related complications. In the current study, we found that during normal rat pregnancy there is a stage-dependent decrease in AR abundance in the gravid uterus and that this is correlated with the differential expression of the endometrial receptivity and decidualization genes Spp1, Prl, Igfbp1, and Hbegf. Pregnant rats exposed to 5α-dihydrotestosterone (DHT) and insulin (INS) or to DHT alone show elevated uterine AR protein abundance and implantation failure related to the aberrant expression of genes involved in endometrial receptivity and decidualization in early to mid-gestation. Treatment with the anti-androgen flutamide, starting from pre-implantation, effectively prevents DHT + INS-induced defects in endometrial receptivity and decidualization gene expression, restores uterine mitochondrial homeostasis, and increases the pregnancy rate and the numbers of viable fetuses. This study adds to our understanding of the mechanisms underlying poor pregnancy outcomes in PCOS patients and the possible therapeutic use of anti-androgens, including flutamide, after spontaneous conception.
4.
Deland, Lily, et al.
(författare)
Novel TPR::ROS1 Fusion Gene Activates MAPK, PI3K and JAK/STAT Signaling in an Infant-type Pediatric Glioma.
2022
Ingår i: Cancer genomics & proteomics. - : Anticancer Research USA Inc.. - 1109-6535 .- 1790-6245. ; 19:6, s. 711-726
Tidskriftsartikel (refereegranskat) abstract
Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors.Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed.Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3' end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient.We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse.
5.
Chebli, Jasmine
(författare)
Physiological roles of amyloid precursor protein in vivo - zebrafish as a model
2021
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Amyloid-beta precursor protein (APP) is an evolutionarily conserved transmembrane protein expressed in many different tissues. APP belongs to a gene family consisting of two other APP-like proteins (APLP1 and APLP2). APP has been shown to be involved in biological processes such as neurite outgrowth, neuronal migration, synapse formation and plasticity, and cell-cell interactions. APP also plays a central role in the development of Alzheimer's disease (AD). APP's physiological role has been difficult to understand and despite all research is not yet completely understood. The purpose of this thesis was to study the role of APP during early development with zebrafish as the main model system. We have focused on the zebrafish's Apps and have tried to understand their function with the help of genetic knockout models created using the CRISPR / Cas9 method. We report that appb mutants have weakened cell adhesions that give rise to changes in cell organization. We also report that the appb mutants are smaller but develop into fertile and healthy adult individuals. We also found defects in the formation of the trigeminal ganglia (TG) and that Appb seems to have a role in cell-cell interaction. The more widespread TG also consisted of fewer nerve cells, indicating that Appb promotes nerve cell formation. Furthermore, our studies demonstrate APP expression in cilia on sensory nerve cells and ependymal cells covering the brain chambers. The conserved expression of APP in ependymal cilia in mice and humans suggest an important and preserved function. Zebrafish with mutated App were found to have defects in the formation of both cilia and cerebral ventricles. To identify new signalling pathways through which Appb controls these functions, we studied protein changes in appb mutants using mass spectrometry. These studies highlight changes that both confirm known and suggest new regulations by appb, especially in neural development, cell adhesion and in gene regulation. Finally, we tried to answer the underlying mechanisms behind compensation within the App family. We found that mutations in the app genes activate expression of homologous genes via so-called transcriptional adaptation. In conclusion, the findings reported in this thesis showed that App is implicated already in early cellular adhesion and sensory neuronal differentiation processes and is located to several sensory cilia in vivo. The use of zebrafish as a model organism allowed us to gain valuable knowledge on the physiological roles of App.
6.
7.
Nguyen, Tho D. K., et al.
(författare)
Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells
2022
Ingår i: Acs Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 16:3, s. 4831-4842
Tidskriftsartikel (refereegranskat) abstract
For decades, "all-or-none"and "kiss-and-run"were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I-, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I- in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically. © 2022 The Authors. Published by American Chemical Society.
8.
Rodriguez, Juan, 1983
(författare)
Targeting apoptosis-inducing factor as a novel therapeutic strategy for preventing perinatal brain injury
2020
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Perinatal complications such as asphyxia can cause brain injuries that are often associated with subsequent neurological deficits. The mechanisms of perinatal brain injury are not fully understood, but mitochondria play a prominent role, not only due to their central function in metabolism, but also because many proteins with apoptosisrelated functions are located in the mitochondrion. Among these proteins, CHCHD4 and apoptosis-inducing factor (AIF) have already been shown to make important contributions to neuronal cell death upon hypoxia-ischemia (HI), but a better understanding of the mechanisms behind these processes is required for the development of improved treatments. By inducing HI in 9-day-old mice, leading to moderate brain injury, we studied these mechanisms from multiple perspectives. First, we determined the effect of chchd4 haploinsufficiency, and we showed that neonatal mice with this genotype experienced less brain damage due to reduced translocation of AIF and Cytochrome c from the mitochondrion. Second, we characterized the role of a newly discovered AIF isoform (AIF2), which is only expressed in the brain and the functions of which are unknown. By using Aif2 knockout mice, we showed that under physiological conditions there is an increase in Aif1 expression (the ubiquitously expressed isoform) due to a compensatory effect of loss of Aif2 expression. As a result, these mice showed a higher degree of brain damage after HI and were more vulnerable to oxidative stress. Third, we used another transgenic mouse in which Aif was overexpressed by knocking in a proviral insertion of Aif, leading to an increased expression of Aif1 without affecting the expression of Aif2. This mouse also showed a higher degree of brain damage and higher levels of oxidative stress. Finally, we used a peptide designed to block the apoptotic function of AIF. The results in young mice showed that the neuroprotective effect of the peptide was greater in male mice than in female mice. In summary, this PhD project has opened new perspectives in the comprehension of the mechanisms by which CHCHD4 and AIF are crucial proteins for brain damage after HI, and it has showed that AIF is a promising therapeutic target for improving outcome after perinatal brain injury.
9.
Bally, Marta, 1981, et al.
(författare)
Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context
2021
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 413, s. 7157-7178
Tidskriftsartikel (refereegranskat) abstract
The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.
10.
Omer, Abubakr A. M., 1982-, et al.
(författare)
Plantaricin NC8 αβ rapidly and efficiently inhibits flaviviruses and SARS-CoV-2 by disrupting their envelopes
2022
Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 17:11
Tidskriftsartikel (refereegranskat) abstract
Potent broad-spectrum antiviral agents are urgently needed to combat existing and emerging viral infections. This is particularly important considering that vaccine development is a costly and time consuming process and that viruses constantly mutate and render the vaccine ineffective. Antimicrobial peptides (AMP), such as bacteriocins, are attractive candidates as antiviral agents against enveloped viruses. One of these bacteriocins is PLNC8 αβ, which consists of amphipathic peptides with positive net charges that display high affinity for negatively charged pathogen membrane structures, including phosphatidylserine rich lipid membranes of viral envelopes. Due to the morphological and physiological differences between viral envelopes and host cell plasma membranes, PLNC8 αβ is thought to have high safety profile by specifically targeting viral envelopes without effecting host cell membranes. In this study, we have tested the antiviral effects of PLNC8 αβ against the flaviviruses Langat and Kunjin, coronavirus SARS-CoV-2, influenza A virus (IAV), and human immunodeficiency virus-1 (HIV-1). The concentration of PLNC8 αβ that is required to eliminate all the infective virus particles is in the range of nanomolar (nM) to micromolar (μM), which is surprisingly efficient considering the high content of cholesterol (8–35%) in their lipid envelopes. We found that viruses replicating in the endoplasmic reticulum (ER)/Golgi complex, e.g. SARS-CoV-2 and flaviviruses, are considerably more susceptible to PLNC8 αβ, compared to viruses that acquire their lipid envelope from the plasma membrane, such as IAV and HIV-1. Development of novel broad-spectrum antiviral agents can significantly benefit human health by rapidly and efficiently eliminating infectious virions and thereby limit virus dissemination and spreading between individuals. PLNC8 αβ can potentially be developed into an effective and safe antiviral agent that targets the lipid compartments of viral envelopes of extracellular virions, more or less independent of virus antigenic mutations, which faces many antiviral drugs and vaccines.
