Microscopy-based siRNA screen of microglia to identify neuroprotective drug targets

The uploaded accepted version corresponds to pages E419-E429 of the publication "GLIA Edinburgh 2017: Abstracts Oral Presentations, Posters, Indexes" available at https://onlinelibrary.wiley.com/doi/10.1002/glia.23157.Neuroinflammation is a fundamental process contributing to the death of neurons in neurodegenerative diseases, such as Parkinson ́s (PD) or Alzheimer ́s disease (AD). During this process, activated microglia secrete cytotoxic substances which lead to neuronal death (1). Therefore, we are looking for the molecular mechanism that reverses the inflammatory activation of microglia, since this knowledge would be essential to protect from neurodegeneration. Our previous data (2) indicate that adipose derived mesenchymal stem cells (ASCs) exert important anti-inflammatory actions on microglia. We observed that microglia exposed to ASCs or their secreted factors (conditioned medium, CM) underwent a dramatic cell shape change into a highly elongated morphology (Fig. 1A), similar to the phenotype of microglia observed in a healthy brain (3). The elongation induced by ASCs was associated with a decrease of the pro-inflammatory cytokine TNFalpha (Fig. 1B) as well as with an upregulation of neurotrophic factors (2). Thus, ASC stimulated microglia represent an ideal tool to study the intracellular events necessary for the transition from inflammatory activated to non-inflammatory neuroprotective microglia. Exploiting these anti-inflammatory properties of ASCs we set up a microscopy-based siRNA screen (Fig. 1C), identifying its hits by cell morphology (see Fig. 1A). In this light, we searched for molecules that inhibited the anti-inflammatory ASC-induced phenotype and thus are involved in the transition from neurotoxic microglia to neuroprotective ones. As changes in the cell shape are intrinsically related to changes of the cytoskeleton, we carried out the screen with the major cytoskeletal regulators. In addition, we included regulators of microglia-specific activation/inflammatory pathways as siRNA targets. Our project is the first siRNA screen performed in primary microglia and we have identified a list of molecules that are specifically implicated in the reversion from activated to neuroprotective microglia. Since our positive hits represent potential neuroprotective drug targets, the outcome of this screen opens up a variety of novel investigation lines and therapies in PD, AD or other neurodegenerative diseases.Michael J Fox Foundation research gran

siRNA screen of microglia to identify neuroprotective drug targets in Parkinson´s disease

The uploaded accepted version corresponds to pages E183-E184 of the publication "GLIA Bilbao 2015: Abstracts Oral Presentations, Posters, Indexes" available at https://doi.org/10.1002/glia.22870.Question: Neuroinflammation is a fundamental process contributing to the death of dopaminergic neurons in Parkinson´s Disease (PD). During this process, activated microglia secrete cytotoxic substances which lead to neuronal death. Therefore, we are looking for the molecular mechanism that reverses the inflammatory activation of microglia, since this knowledge would be essential to protect from neurodegeneration. Methods and Results: Very interestingly our previous data (Neubrand et al., 2014) indicate that adipose derived mesenchymal stem cells (ASCs) exert important anti-inflammatory actions on microglia. We observed that microglia exposed to ASCs or their secreted factors (conditioned medium, CM) underwent a dramatic cell shape change into a highly elongated morphology (Fig 1A), similar to the phenotype of microglia observed in a healthy brain. The elongation induced by ASCs was associated with a decrease of the pro-inflammatory cytokine TNFalpha (Fig 1B) as well as with an upregulation of neurotrophic factors. Thus, ASC stimulated microglia represent an ideal tool to study the intracellular events necessary for the transition from inflammatory activated to non-inflammatory neuroprotective microglia. In this way we have already identified the small RhoGTPases Rac1 and Cdc42, which are important regulators of the actin cytoskeleton, as essential molecules in this transition (Fig 1C). Since these molecules represent possible drug targets to induce the reversion of neurotoxic microglia to neuroprotective ones, we are currently performing an siRNA screen to identify the molecular players of this ASC-induced reversion. Because this transition is easily detectable by light microscopy (see Figs 1A and C) and changes in the cell shape are intrinsically related to changes of the cytoskeleton, we are carrying out a microscopy-based screen of the major cytoskeletal regulators. In addition, we are including in the screen the regulators of microglia-specific activation/inflammatory pathways as siRNA targets. Conclusion: Our project is the first siRNA screen performed in primary microglia and we aim to identify a list of molecules that are specifically implicated in the reversion from activated to neuroprotective microglia. Since positive hits would represent potential neuroprotective drug targets, the outcome of this screen opens up a variety of novel investigation lines and therapies in PD or other neurodegenerative diseases

Microglia and Microglia-Like Cells: Similar but Different

We want to thank all people for fighting in the front line of the COVID-19 pandemic, during which most parts of this article was written. We also acknowledge the task of the reviewers who contributed to improve the quality of this article.Microglia are the tissue-resident macrophages of the central nervous parenchyma. In mammals, microglia are thought to originate from yolk sac precursors and posteriorly maintained through the entire life of the organism. However, the contribution of microglial cells from other sources should also be considered. In addition to “true” or “bonafide” microglia, which are of embryonic origin, the so-called “microglia-like cells” are hematopoietic cells of bone marrow origin that can engraft the mature brain mainly under pathological conditions. These cells implement great parts of the microglial immune phenotype, but they do not completely adopt the “true microglia” features. Because of their pronounced similarity, true microglia and microglia-like cells are usually considered together as one population. In this review, we discuss the origin and development of these two distinct cell types and their differences. We will also review the factors determining the appearance and presence of microglia-like cells, which can vary among species. This knowledge might contribute to the development of therapeutic strategies aiming at microglial cells for the treatment of diseases in which they are involved, for example neurodegenerative disorders like Alzheimer’s and Parkinson’s diseases.University of Granada, Spain, and FEDER-Junta de Andalucía, Spain (grant number A1-CTS-324- UGR18

Switching Roles: Beneficial Effects of Adipose Tissue-Derived Mesenchymal Stem Cells on Microglia and Their Implication in Neurodegenerative Diseases

This research was funded by the Andalusian Government, Spain (grant no. P20-01255 to M.D. and FEDER program grant no. A1-CTS-324-UGR18 to M.R.S.) and by the Spanish Ministry for Economy and Competition, Spain (grant no. SAF2017-85602-R and PID2020-119638RB-I00, both to E.G.-R.). A.I.S.-C. was the awardee of a Research Starting Fellowship for master´s students at the University of Granada, Spain. The APC was funded by MDPI.Neurological disorders, including neurodegenerative diseases, are often characterized by neuroinflammation, which is largely driven by microglia, the resident immune cells of the central nervous system (CNS). Under these conditions, microglia are able to secrete neurotoxic substances, provoking neuronal cell death. However, microglia in the healthy brain carry out CNS-supporting functions. This is due to the ability of microglia to acquire different phenotypes that can play a neuroprotective role under physiological conditions or a pro-inflammatory, damaging one during disease. Therefore, therapeutic strategies focus on the downregulation of these neuroinflammatory processes and try to re-activate the neuroprotective features of microglia. Mesenchymal stem cells (MSC) of different origins have been shown to exert such effects, due to their immunomodulatory properties. In recent years, MSC derived from adipose tissue have been made the center of attention because of their easy availability and extraction methods. These cells induce a neuroprotective phenotype in microglia and downregulate neuroinflammation, resulting in an improvement of clinical symptoms in a variety of animal models for neurological pathologies, e.g., Alzheimer’s disease, traumatic brain injury and ischemic stroke. In this review, we will discuss the application of adipose tissue-derived MSC and their conditioned medium, including extracellular vesicles, in neurological disorders, their beneficial effect on microglia and the signaling pathways involved.Andalusian Government, Spain P20-01255FEDER program grant no. A1-CTS-324-UGR18Spanish Ministry for Economy and Competition, Spain (grant no. SAF2017-85602-R and PID2020-119638RB-I00)Research Starting Fellowship for master´s students at the University of Granada, SpainMDP

Illuminating the human genome

Supplementary material for this paper can be obtained electronically by using Springer LINK Server located at http://dx.doi.org/10.1007/s004180000236Acknowledgements The Advanced Light Microscopy Facility at EMBL is supported by BioRad (UK), Bitplane (Switzerland), Eppendorf (Germany), Evotec (Germany), Improvision (UK), Leica (Germany), Nikon (Europe), Olympus (Europe), Perkin Elmer (UK), T.I.L.L. Photonics (Germany) and Zeiss (Germany). The Molecular Genome Analysis Department is supported by the BMBF as part of the German Genome Project. J.C.S. is supported by an EMBO Long Term Fellowship.The identification and analysis of novel genes and their encoded protein products remains a vigorous area of research in biology today. Worldwide genomic and cDNA sequencing projects are now identifying new molecules every day and the need for methodologies to functionally characterise these proteins has never been greater. The distinct compartmental arrangement of eukaryotic cells helps define the processes which occur within or in proximity to these membranes, and as such provides one means of inferring protein function. We describe here some of the methods recently reported in the literature, which use the subcellular localisation of proteins as a first step towards their further characterisation.BioRad (UK)Bitplane (Switzerland)Eppendorf (Germany)Evotec (Germany)Improvision (UK)Leica (Germany)Nikon (Europe)Olympus (Europe)Perkin Elmer (UK)T.I.L.L. Photonics (Germany)Zeiss (Germany)The Molecular Genome Analysis Department is supported by the BMB