| 1. |
- Agholme, Lotta, et al.
(författare)
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Low-dose γ-secretase inhibition increases secretion of Aβ peptides and intracellular oligomeric Aβ.
- 2017
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Ingår i: Molecular and cellular neurosciences. - : Elsevier BV. - 1095-9327 .- 1044-7431. ; 85, s. 211-219
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Tidskriftsartikel (refereegranskat)abstract
- γ-Secretase inhibitors have been considered promising drug candidates against Alzheimer's disease (AD) due to their ability to reduce amyloid-β (Aβ) production. However, clinical trials have been halted due to lack of clinical efficacy and/or side effects. Recent in vitro studies suggest that low doses of γ-secretase inhibitors may instead increase Aβ production. Using a stem cell-derived human model of cortical neurons and low doses of the γ-secretase inhibitor DAPT, the effects on a variety of Aβ peptides were studied using mass spectrometry. One major focus was to develop a novel method for specific detection of oligomeric Aβ (oAβ), and this was used to study the effects of low-dose γ-secretase inhibitor treatment on intracellular oAβ accumulation. Low-dose treatment (2 and 20nM) with DAPT increased the secretion of several Aβ peptides, especially Aβx-42. Furthermore, using the novel method for oAβ detection, we found that 2nM DAPT treatment of cortical neurons resulted in increased oAβ accumulation. Thus, low dose-treatment with DAPT causes both increased production of long, aggregation-prone Aβ peptides and accumulation of intracellular Aβ oligomers, both believed to contribute to AD pathology.
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| 2. |
- Bergström, Petra, et al.
(författare)
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Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid precursor protein (APP) and its cleavage product amyloid beta (A beta) have been thoroughly studied in Alzheimer's disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. alpha-Cleaved soluble APP (sAPP alpha) was secreted early during differentiation, from neuronal progenitors, while beta-cleaved soluble APP (sAPP beta) was first secreted after deep-layer neurons had formed. Short A beta peptides, including A beta 1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as A beta 1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by A beta 1-40/42, is associated with mature neuronal phenotypes.
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| 3. |
- Bergström, Petra, et al.
(författare)
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Herpes Simplex Virus 1 and 2 Infections during Differentiation of Human Cortical Neurons
- 2021
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Ingår i: Viruses-Basel. - : MDPI AG. - 1999-4915. ; 13:10
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Tidskriftsartikel (refereegranskat)abstract
- Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) can infect the central nervous system (CNS) with dire consequences; in children and adults, HSV-1 may cause focal encephalitis, while HSV-2 causes meningitis. In neonates, both viruses can cause severe, disseminated CNS infections with high mortality rates. Here, we differentiated human induced pluripotent stem cells (iPSCs) towards cortical neurons for infection with clinical CNS strains of HSV-1 or HSV-2. Progenies from both viruses were produced at equal quantities in iPSCs, neuroprogenitors and cortical neurons. HSV-1 and HSV-2 decreased viability of neuroprogenitors by 36.0% and 57.6% (p < 0.0001), respectively, 48 h post-infection, while cortical neurons were resilient to infection by both viruses. However, in these functional neurons, both HSV-1 and HSV-2 decreased gene expression of two markers of synaptic activity, CAMK2B and ARC, and affected synaptic activity negatively in multielectrode array experiments. However, unaltered secretion levels of the neurodegeneration markers tau and NfL suggested intact axonal integrity. Viral replication of both viruses was found after six days, coinciding with 6-fold and 22-fold increase in gene expression of cellular RNA polymerase II by HSV-1 and HSV-2, respectively. Our results suggest a resilience of human cortical neurons relative to the replication of HSV-1 and HSV-2.
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| 4. |
- Bodda, C., et al.
(författare)
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HSV1 VP1-2 deubiquitinates STING to block type I interferon expression and promote brain infection
- 2020
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Ingår i: The Journal of experimental medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 217:7
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Tidskriftsartikel (refereegranskat)abstract
- Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS-STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain. © 2020 Bodda et al.
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| 5. |
- Cicognola, Claudia, et al.
(författare)
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Tauopathy-Associated Tau Fragment Ending at Amino Acid 224 Is Generated by Calpain-2 Cleavage.
- 2020
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Ingår i: Journal of Alzheimer's disease : JAD. - 1875-8908. ; 74:4, s. 1143-1156
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Tidskriftsartikel (refereegranskat)abstract
- Tau aggregation in neurons and glial cells characterizes tauopathies as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Tau proteolysis has been proposed as a trigger for tau aggregation and tau fragments have been observed in brain and cerebrospinal fluid (CSF). Our group identified a major tau cleavage at amino acid (aa) 224 in CSF; N-terminal tau fragments ending at aa 224 (N-224) were significantly increased in AD and lacked correlation to total tau (t-tau) and phosphorylated tau (p-tau) in PSP and CBD.Previous studies have shown cleavage from calpain proteases at sites adjacent to aa 224. Our aim was to investigate if calpain-1 or -2 could be responsible for cleavage at aa 224.Proteolytic activity of calpain-1, calpain-2, and brain protein extract was assessed on a custom tau peptide (aa 220-228), engineered with fluorescence resonance energy transfer (FRET) technology. Findings were confirmed with in-gel trypsination and mass spectrometry (MS) analysis of brain-derived bands with proteolytic activity on the FRET substrate. Finally, knock-down of the calpain-2 catalytic subunit gene (CAPN2) was performed in a neuroblastoma cell line (SH-SY5Y).Calpain-2 and brain protein extract, but not calpain-1, showed proteolytic activity on the FRET substrate. MS analysis of active gel bands revealed presence of calpain-2 subunits, but not calpain-1. Calpain-2 depletion and chemical inhibition suppressed proteolysis of the FRET substrate. CAPN2 knock-down caused a 76.4% reduction of N-224 tau in the cell-conditioned media.Further investigation of the calpain-2 pathway in the pathogenesis of tauopathies is encouraged.
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| 6. |
- Domert, Jakob, et al.
(författare)
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Aggregated Alpha-Synuclein Transfer Efficiently between Cultured Human Neuron-Like Cells and Localize to Lysosomes
- 2016
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 11:12
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Tidskriftsartikel (refereegranskat)abstract
- Parkinson's disease and other alpha-synucleinopathies are progressive neurodegenerative diseases characterized by aggregates of misfolded alpha-synuclein spreading throughout the brain. Recent evidence suggests that the pathological progression is likely due to neuron-to-neuron transfer of these aggregates between neuroanatomically connected areas of the brain. As the impact of this pathological spreading mechanism is currently debated, we aimed to investigate the transfer and subcellular location of alpha-synuclein species in a novel 3D co-culture human cell model based on highly differentiated SH-SY5Y cells. Fluorescently-labeled monomeric, oligomeric and fibrillar species of alpha-synuclein were introduced into a donor cell population and co-cultured with an EGFP-expressing acceptor-cell population of differentiated neuron-like cells. Subsequent transfer and colocalization of the different species were determined with confocal microscopy. We could confirm cell-to-cell transfer of all three alpha-synuclein species investigated. Interestingly the level of transferred oligomers and fibrils and oligomers were significantly higher than monomers, which could affect the probability of seeding and pathology in the recipient cells. Most alpha-synuclein colocalized with the lysosomal/endosomal system, both pre- and postsynaptically, suggesting its importance in the processing and spreading of alpha-synuclein.
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| 7. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 8. |
- Mogren, Å, et al.
(författare)
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Orofacial function in children with Speech Sound Disorders (SSD) persisting after the age of six years
- 2020
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Ingår i: International Journal of Speech-Language Pathology. - : Informa UK Limited. - 1754-9515 .- 1754-9507. ; 22:5, s. 526-536
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: The aim was to investigate, describe and analyse speech characteristics, intelligibility, orofacial function and co-existing neurodevelopmental symptoms in children with SSD of unknown origin, persisting after six years of age. Method: The study included 61 children with SSD (6–17 years) of unknown origin, referred for a speech and oral motor examination. The severity of SSD was estimated using Percentage Consonants Correct (PCC) and Percentage Vowels Correct (PVC) and assessments of resonance based on Swedish Articulation and Nasality Test (SVANTE). Orofacial function was screened using the Nordic Orofacial Test-Screening (NOT-S). Parents completed the Intelligibility in Context Scale (ICS) and a questionnaire including questions about heredity, medical and neurodevelopmental conditions, and speech development. Result: SSD varied according to PCC (8–95%) and PVC (55–100%) measurements. Percentages of co-occurring disorders included: 51% resonance deviations, 90% intelligibility issues, and 87% orofacial difficulties. The most affected orofacial domains were “Chewing and swallowing” (41%), “Masticatory muscles and jaw function” (38%) and “Sensory function” (38%). The majority (64%) had co-existing dysfunctions relating to general motor and neurodevelopmental disorders. Conclusion: Children with persistent SSD are at risk for orofacial dysfunction, general motor difficulties and other neurodevelopmental disorders and therefore should be screened for co-occurring disorders. © 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
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| 9. |
- Nazir, Faisal Hayat, et al.
(författare)
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Expression and secretion of synaptic proteins during stem cell differentiation to cortical neurons.
- 2018
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Ingår i: Neurochemistry international. - : Elsevier BV. - 1872-9754 .- 0197-0186. ; 121, s. 38-49
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Tidskriftsartikel (refereegranskat)abstract
- Synaptic function and neurotransmitter release are regulated by specific proteins. Cortical neuronal differentiation of human induced pluripotent stem cells (hiPSC) provides an experimental model to obtain more information about synaptic development and physiology in vitro. In this study, expression and secretion of the synaptic proteins, neurogranin (NRGN), growth-associated protein-43 (GAP-43), synaptosomal-associated protein-25 (SNAP-25) and synaptotagmin-1 (SYT-1) were analyzed during cortical neuronal differentiation. Protein levels were measured in cells, modeling fetal cortical development and in cell-conditioned media which was used as a model of cerebrospinal fluid (CSF), respectively. Human iPSC-derived cortical neurons were maintained over a period of at least 150 days, which encompasses the different stages of neuronal development. The differentiation was divided into the following stages: hiPSC, neuro-progenitors, immature and mature cortical neurons. We show that NRGN was first expressed and secreted by neuro-progenitors while the maximum was reached in mature cortical neurons. GAP-43 was expressed and secreted first by neuro-progenitors and its expression increased markedly in immature cortical neurons. SYT-1 was expressed and secreted already by hiPSC but its expression and secretion peaked in mature neurons. SNAP-25 was first detected in neuro-progenitors and the expression and secretion increased gradually during neuronal stages reaching a maximum in mature neurons. The sensitive analytical techniques used to monitor the secretion of these synaptic proteins during cortical development make these data unique, since the secretion of these synaptic proteins has not been investigated before in such experimental models. The secretory profile of synaptic proteins, together with low release of intracellular content, implies that mature neurons actively secrete these synaptic proteins that previously have been associated with neurodegenerative disorders, including Alzheimer's disease. These data support further studies of human neuronal and synaptic development in vitro, and would potentially shed light on the mechanisms underlying altered concentrations of the proteins in bio-fluids in neurodegenerative diseases.
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| 10. |
- Reinert, Line S, et al.
(författare)
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Brain immune cells undergo cGAS-STING-dependent apoptosis during herpes simplex virus type 1 infection.
- 2020
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Ingår i: The Journal of clinical investigation. - 1558-8238. ; 131:1
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Tidskriftsartikel (refereegranskat)abstract
- Protection of the brain from viral infections involves the type I interferon (IFN-I) system, defects in which renders humans susceptible to herpes simplex encephalitis (HSE). However, excessive cerebral IFN-I levels leads to pathologies, suggesting the need for tight regulation of responses. Based on data from mouse models, human HSE cases, and primary cell culture systems, we here show that microglia and other immune cells undergo apoptosis in the HSV-1-infected brain through a mechanism dependent on the cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway, but independent of IFN-I. HSV-1 infection of microglia induced cGAS-dependent apoptosis at high viral doses, while lower viral doses led to IFN-I responses. Importantly, inhibition of caspase activity prevented microglial cell death and augmented IFN-I responses. Accordingly, HSV-1-infected organotypic brain slices, or mice treated with caspase inhibitor, exhibited lower viral load and improved outcome of infection. Collectively, we identify an activation-induced apoptosis program in brain immune cells which down-modulates local immune responses.
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