THE OTHER BRAIN November 7 Th – 8Th , 2019, Ljubljana

THE OTHER BRAIN November 7th – 8th, 2019, Ljubljana

Slovenian Academy of Sciences and Arts Novi trg 3, Ljubljana, Slovenia

The Other Brain

PROGRAMME

Thursday, November 7th, 2019 8.30 – 9.00 Registration 9.00 – 9.15 Opening of the Conference SASA President Tadej Bajd Her Excellency the British Ambassador to Slovenia Dr. Sophie Honey His Excellency the Ambassador of the Czech Republic to Slovenia – Mr. Juraj Chmiel Robert Zorec: The Other Brain: a target for developing new therapies 9.15 – 9.45 Alexei Verkhratsky (Manchester): Neuroglia in health and disease: Astroglial atrophy and functional asthenia in the pathogenesis of neurological diseases 9.45 – 10.15 Dmitri Rusakov (UCL): Activity-dependent plasticity of perisynaptic astroglia 10.15 – 10.45 COFFEE BREAK 10.45 – 11.15 Kristian Franze (Cambridge): Mechanical signals regulate glia function in health and disease 11.15 – 11.45 Arthur Butt (Portsmouth): Astrocytes are direct cellular targets of lithium treatment 11.45 – 12.00 Matjaž Stenovec (Ljubljana) Vesicular mechanisms of interferon γ-induced MHCII-dependent antigen presentation in astroglia 12.00 – 12.15 Maja Potokar (Ljubljana): ZIKV Strains Differentially Affect Survival of Human Fetal Astrocytes versus Neurons and Traffic of ZIKV-Laden Endocytotic Compartments 12.15 – 12.45 Frances Platt (Oxford): Understanding and treating neurodegenerative diseases: insights from Niemann-Pick type C 12.45 – 14.15 LUNCH 14.15 – 14.45 Charles ffrench Constant (Edinburgh): Oligodendrocytes and multiple sclerosis 14.45 – 15.30 COFFEE BREAK 15.30 – 16.00 David Pitt (Yale, Brno): Dysfunctional astrocytes are drivers of multiple sclerosis (MS) susceptibility and progression 16.00 – 16.30 Marko Kreft (Ljubljana): Insulin and Adrenaline Modulate Cytoplasmic Glucose, Lactate and Glycogen Levels in Astrocytes 16:30-17:00 Nina Vardjan (Ljubljana): TDP-43 inclusions in astrocytes dysregulate glucose and lipid metabolisms

Friday, November 8th, 2019 8.30 – 9.00 Registration 9.00 – 9.30 Gorazd Bernard Stokin (ICRC, Brno): Axonal interactions with astrocytes 9.30 – 10.00 Wendy Noble (Kings’ College): Astrocytes mediate the beta-amyloid-driven toxic effects of synaptic tau in Alzheimer's disease 10.00 – 10.30 Boris Rogelj (Ljubljana): Protein binding RNAs in neurodegeneration 10.30 – 11.00 COFFEE BREAK 11.00 – 11.30 Eva Sykova (Bratislava): Astrocytes and stem cells in pathophysiology of neurodegenerative diseases 11.30 – 12.00 Jernej Ule (UCL, Ljubljana): RNA biology to understand neurodegeneration 12.00 – 12.30 Conference End & Communiqué Reception hosted by His Excellency the Ambassador of the Czech Republic to 12:30 - 14:00 Slovenia – Mr. Juraj Chmiel

The Other Brain

CONTENT

The Other Brain

Neuroglia in health and disease: Astroglial atrophy and functional asthenia in the pathogenesis of neurological diseases

Alexei Verkhratsky1,2

1Faculty of Biology, Medicine and Health,The University of Manchester, Manchester, UK

The common and prevailing set of neurological thoughts considers neurones as the primary substrate of pathological progression. This "neurone-centric" concept, however, undergoes a dramatic change. It has become universally acknowledged that the homeostasis of the nervous tissue is regulated by a complex fabric of neuroglial cells. Astroglia in particular represent a main element in the maintenance of homeostasis and providing defense to the brain. Consequently, dysfunction of astrocytes underlies many, if not all, neurological, neuropsychiatric and neurodegenerative disorders. General astrogliopathy is evident in diametrically opposing morpho-functional changes in astrocytes, i.e. their hypertrophy along with reactivity or atrophy with asthenia. These complex plastic changes underlie pathophysiology of all neurological disorders including genetic (e.g. Alexander disease, which is a primary sporadic astrogliopathy), environmentally caused (e.g. heavy metal encephalopathies, hepatic encephalopathies or neuroinfections), neurodevelopmental (e.g. different forms of autistic spectrum disorder), neuropsychiatric (various forms of depression, bipolar syndromes and schizophrenia) and neurodegenerative (e.g. amyotrophic lateral sclerosis, Alzheimer’s and Huntington’s diseases). Astroglial atrophy with loss of function as a fundamental pathophysiological entity has been considered only very recently (Verkhratsky et al., 2010; 2017); nonetheless is has been since detected in several major neuropatholopgies including neurodegenerative diseases, in epilepsy, in psychiatric diseases and in cognitive disorders. Astroglial atrophy can be reversed by environmental stimulation including exposure to the enriched environment and caloric restriction of food intake; astrocytes can also be specifically targeted by various pharmacological agents such as ketamine or serotonin uptake inhibitors. Astroglial atrophy, asthenia and functional paralysis represent arguably a new focus for developing novel therapeutic strategies.

References

Verkhratsky, A. et al. (2010) Neurotherapeutics 7, 399-412.

Verkhratsky, A., Zorec, R. & Parpura, V. (2017). Brain Pathol 27, 629-644.

The Other Brain

Activity-dependent plasticity of perisynaptic astroglia

Dmitri A. Rusakov

UCL Queen Square Institute of Neurology, University College London, London, United Kingdom

Memory trace formation in the brain is thought to involve structural remodelling of synaptic connections. Whether and how this engages the astroglial microenvironment of synapses remains poorly understood. We combine patch-clamp electrophysiology with two-photon excitation microscopy, photolytic uncaging, super-resolution techniques, and correlational 3D electron microscopy, to monitor fine astroglial morphology during the induction of synaptic long-term potentiation (LTP). It appears that LTP induction protocols, either in multiple or in individual identified synapses, in acute slices or in vivo, trigger nanoscopic withdrawal of perisynaptic astroglial processes near potentiated synapses. The underlying cellular mechanisms do not depend on major cascades of astroglial Ca2+-dependent signalling but require the astroglial ion exchanger NCCK1, which in turn involves the actin-controlling protein cofilin. The LTP-associated reduction in synaptic astroglial coverage boosts extra- synaptic glutamate escape thus facilitating NMDA receptor-mediated cross-talk among neighbouring excitatory connections. Thus, LTP induction can engage astroglial remodelling altering the profile of neurotransmitter actions and thus signal integration rules in the proximity of potentiated synapses.

The Other Brain

Mechanical signals regulate glia function in health and disease

Kristian Franze

University of Cambridge

During development and pathological processes, central nervous system (CNS) glia are highly motile. Despite the fact that cell motion is driven by forces, our current understanding of the physical interactions between glial cells and their environment is very limited. We here show how nanometer deformations of CNS tissue caused by piconewton forces exerted by glial cells contribute to regulating CNS physiology and pathology. In vitro, glial cell morphology, migration, traction forces as well as gene and protein expression patterns all significantly depended on substrate stiffness. Moreover, when grown on substrates incorporating linear stiffness gradients, glial cells migrated towards stiffer, while axon bundles turned towards softer substrates. In vivo time-lapse atomic force microscopy revealed stiffness gradients in developing brain tissue. Interfering with brain stiffness and mechanosensitive ion channels in vivo both led to aberrant neuronal growth patterns as well as changes in glial cell reactivity. Importantly, CNS tissue significantly softened after traumatic injuries. Ultimately, mechanical signals not only directly impacted CNS cell behavior but also indirectly by regulating cellular responses to and the availability of chemical signals in the brain, strongly suggesting that chemical and mechanical signaling pathways are intimately linked, and that their interaction is crucial for CNS functioning.

The Other Brain

Astrocytes are direct cellular targets of lithium treatment

Andrea Riviera & Arthur Butt

Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and Biomedical Science, University of Portsmouth, UK

Astrocytes are multifunctional glial cells that play essential roles in supporting synaptic signalling and white matter-associated connectivity. There is increasing evidence that astrocyte dysfunction is involved in several brain disorders, including bipolar disorder (BD), depression and schizophrenia. The mood stabiliser lithium is a frontline treatment for BD, but the mechanisms of action remain unclear. Here, we demonstrate that astrocytes are direct targets of lithium and identify unique astroglial transcriptional networks that regulate specific molecular changes in astrocytes associated with BD and schizophrenia, together with Alzheimer's disease (AD). Using pharmacogenomic analyses, we identified novel roles for the extracellular matrix (ECM) regulatory enzyme lysyl oxidase (LOX) and peroxisome proliferator-activated receptor gamma (PPAR-γ) as profound regulators of astrocyte morphogenesis. This study unravels new pathophysiological mechanisms in astrocytes that have potential as novel biomarkers and potential therapeutic targets for regulating astroglial responses in diverse neurological disorders

The Other Brain

Vesicular mechanisms of interferon γ-induced MHCII-dependent antigen presentation in astroglia

Mićo Božić2, Alexei Verkhratsky1,3,4, Robert Zorec1,2, Matjaž Stenovec1,2

1Celica BIOMEDICAL, Tehnološki park 24, 1000 Ljubljana, Slovenia 2Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia 3Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK 4Achucarro Center for Neuroscience, IKERBASQUE, 48011 Bilbao, Spain

Astrocytes, the key homeostatic cells in the central nervous system, contribute to defensive responses via a process of reactive astrogliosis activated by inflammatory cytokines. Pro- inflammatory cytokine interferon γ (IFNγ) thus induces the expression of the major histocompatibility complex class II (MHCII) molecules, involved in astroglial antigen presentation. The exact pathway for MHCII delivery to the plasma membrane in reactive astrocytes is unknown. Cultured rat astrocytes and astrocytes in organotypic slices were therefore treated with IFNγ to induce reactive astrogliosis. Astrocytes were probed with optophysiological tools to investigate subcellular localization of immunolabeled MHCII, and with electrophysiology to characterize interactions of single vesicles with the plasmalemma. In culture and in organotypic slices, IFNγ augmented the astrocytic expression of MHCII, which prominently co-localized with lysosomal marker LAMP1-EGFP, modestly with Rab7, and negligibly with endosomal markers Rab4A, EEA1, and TPC1. Immunolabeling of live non-permeabilized cells revealed the surface presence of MHCII. In IFNγ-treated astrocytes an increased fraction of large-diameter exocytotic vesicles (lysosome-like vesicles) with prolonged fusion pore dwell time and larger pore conductance was recorded, whereas the rate of endocytosis was decreased. Stimulation with ATP, which elevates cytosolic calcium activity, increased the frequency of exocytotic events, whereas the frequency of full endocytosis was further reduced. In IFNγ-treated astrocytes, MHCII-linked antigen surface presentation is mediated by increased lysosomal exocytosis, whereas surface retention of antigens is prolonged by concomitant inhibition of endocytosis.

The Other Brain

ZIKV Strains Differentially Affect Survival of Human Fetal Astrocytes versus Neurons and Traffic of ZIKV-Laden Endocytotic Compartments

Maja Potokar1,3, Jernej Jorgačevski1,3, Miša Korva2, Marjeta Lisjak1, Tatjana Avšič-Županc2, Robert Zorec1,3,5

1Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

2Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

3Celica BIOMEDICAL, Ljubljana, Slovenia

An outbreak of Zika virus (ZIKV), which started roughly ten years ago, changed the epidemiology of this flavivirus, since ZIKV infection has been linked to malformations of the fetal CNS, known as microcephaly. However, the cellular responses of individual cell types to the infection with ZIKV remain poorly understood. Therefore, we infected primary human fetal astrocytes and neurons, as well as mammalian (SK-N-SH, Vero E6) and mosquito (C6/36) cell lines, with ZIKV strains Brazil 2016 (ZIKV-BR), French Polynesia 2013 (ZIKV-FP), and Uganda #976 1947 (ZIKV-UG) and studied viral production, cell viability, infectivity rate, and mobility of endocytotic ZIKV-laden vesicles. While all cell types released productive virus, astrocytes were more susceptible to ZIKV infection than neurons, released more progeny virus and tolerated higher virus load than neurons. Among the three ZIKV strains, ZIKV-UG proved to have the highest infection rate. Nonetheless, all ZIKV strains elicited differences in trafficking of ZIKV-laden endocytotic vesicles in all cell types, including astrocytes and neurons, except in mosquito cells. In the latter, ZIKV infection also failed to induce cell death. In summary, we have performed a thorough screening of cell viability, infection and production of three ZIKV strains in five different cell types and demonstrated that ZIKV affects vesicle mobility in all but mosquito cells.

The Other Brain

Understanding and treating neurodegenerative diseases: insights from Niemann-Pick type C

Frances Platt

Oxford University, UK

Lysosomal storage diseases (LSDs) are a group of over 70 inherited metabolic diseases that are individually rare but collectively affect 1 in 5,000 live births. Most LSDs have a progressive neurodegenerative clinical course, although symptoms in other organ systems are a common feature of these diseases. We have a major interest in the LSD Niemann-Pick disease type C. Two independent genes NPC1 and NPC2 cause this disease and how the NPC1 and NPC2 proteins they encode cooperate together within cells is not fully understood. Both proteins reside in the lysosome, NPC1 is a large multi-pass limiting membrane protein where as NPC2 is a soluble cholesterol binding protein. The storage material in NPC is a complex mixture of different lipids including cholesterol and sphingolipids. Within the CNS Purkinje cell loss leads to cerebellar ataxia and microglial activation is a prominent feature of the disease. In this talk I will review what is known about the complex cell biology of this disease and how this knowledge is translating into innovative new therapies.

The Other Brain

Oligodendrocytes and multiple sclerosis

Charles ffrench Constant

Center for Regenerative Medicine, University of Edinburgh

The Other Brain

Dysfunctional astrocytes are drivers of multiple sclerosis (MS) susceptibility and progression

David Pitt1,2

1Yale School of Medicine,

2ICRC Brno, Czech Republic

Genetic risk variants are predicted to mediate MS susceptibility predominantly by perturbing gene regulation in lymphocytes. We have demonstrated that genetic MS risk variants enhance astrocyte and microglia functions that lead to increased lymphocyte recruitment and activation in the CNS. This establishes that genetic susceptibility to MS results from variant- driven dysregulation of peripheral lymphocytes and CNS cells, where altered CNS cell function aids establishing local autoimmune inflammation. In addition, we have shown that chronically activated astrocytes in patients with progressive MS express high levels of adenosine 2A receptors. A2aR signaling in activated astrocytes is associated with oxidative damage in cell culture and lesion tissue and therefore represent a pathway by which astrocytes drive neurotoxic damage in progressive MS.

The Other Brain

Insulin and Adrenaline Modulate Cytoplasmic Glucose, Lactate and Glycogen Levels in Astrocytes

Marko Kreft1,2,3, Katja Fink1, Marko Muhič1, Helena H. Chowdhury1,2, Nina Vardjan1,2, Robert Zorec1,2

1Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, Ljubljana, Slovenia

2Celica Biomedical Center, Tehnoloski park 24, Ljubljana, Slovenia

3Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, Ljubljana, Slovenia

Astrocytes play a significant role in a number of processes, including the brain energy metabolism. Their anatomical position between blood vessels and neurons makes them an interface for effective glucose uptake from blood. Astrocytes contain glycogen, an energy buffer, which can bridge local short term energy requirements in the brain. Glycogen levels reflect a dynamic equilibrium between glycogen synthesis and glycogenolysis. Many factors that include hormones and neuropeptides, such as insulin and adrenaline likely modulate glycogen stores in astrocytes, but detailed mechanisms at the cellular level are sparse. We used a glucose nanosensor based on Förster resonance energy transfer to monitor cytosolic glucose and lactate concentration with high temporal resolution and a cytochemical approach to determine glycogen stores in single cells. We show that following the adrenaline or noradrenaline stimulation the availability of cytosolic glucose and lactate is increased promptly after stimulation. Insulin boosts the process of glycogen formation. Although astrocytes appear to express glucose transporter GLUT4, glucose entry across the astrocyte plasma membrane is not affected by insulin. Stimulation of cells with insulin decreased cytosolic glucose concentration, likely because of elevated glucose utilization for glycogen synthesis.

KEY WORDS: insulin, adrenaline, glycogen, astrocytes

The Other Brain

TDP-43 inclusions in astrocytes dysregulate glucose and lipid metabolism

Jelena Velebit1, Anemari Horvat1,2, Sonja Prpar Mihevc3, Boris Rogelj3,4,5, Robert Zorec1,2, Nina Vardjan1,2

1Laboratory of Cell Engineering, Celica Biomedical, 1000 Ljubljana, Slovenia 2Laboratory of Neuroendocrinology – Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia 3Department of Biotechnology, Jožef Stefan Institute, 1000 Ljubljana, Slovenia 4Biomedical Research Institute BRIS, 1000 Ljubljana, Slovenia 5Faculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, Slovenia

In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) pathological cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) are present in neurons and non-neuronal cells, including astrocytes, which metabolically support neurons with nutrients. During intense activity neuronal metabolism largely depends on the activation of the noradrenergic system releasing noradrenaline that activates astroglial β-adrenergic receptors (β-ARs) and triggers cAMP signaling. This augments aerobic glycolysis with production of lactate, an important neuronal energy fuel. Cytoplasmic TDP-43 inclusions in astrocytes alone can cause motor neuron death. Whether they alter astroglial metabolism and metabolic support of neurons is unclear. The effect of cytoplasmic TDP-43 inclusions on lipid droplet and glucose metabolisms was measured in astrocytes expressing the inclusion-forming C- terminal fragment of TDP-43 or the wild-type TDP-43 using fluorescent dyes or genetically encoded nanosensors. Astrocytes with TDP-43 inclusions exhibited increase in the accumulation of lipid droplets, indicating altered lipid droplet metabolism. In these cells the noradrenaline-triggered increase in intracellular cAMP levels was reduced due to the downregulation of β2-ARs, but the probability of activating aerobic glycolysis was facilitated. Thus, while in astrocytes with TDP-43 inclusions β-adrenergic cAMP signaling is reduced, glucose and lipid droplet metabolisms are facilitated, suggesting dysregulated astroglial metabolic support of neurons in TDP-43-associated ALS and FTD.

The Other Brain

Axonal interactions with astrocytes

Gorazd Bernard Stokin

ICRC Brno, Czech Republic

In contrast to dendrites, axons are significantly fewer, lengthier and poorly branched. Axonal prime role is to convey messages across long distances. To achieve this goal axons developed unique axonal transport system, which relies on strictly unipolar microtubule tracks. Molecular motors exploit chemical energy to fuel mechanical movement of axonal cargoes along microtubule tracks. Although regulation of axonal transport remains to be fully elucidated, accumulating evidence suggests that axonal transport plays a role in a plethora of neurological diseases ranging from traumatic brain injury to Alzheimer’s disease. Wealth of studies revealed close interaction between axons and astrocytes, however, functional implications of this interaction remain poorly understood. In order to critically test the axon astrocyte interaction, we developed a method to differentiate human stem cells into glutaminergic neurons and astrocytes. This method allowed us to uncover basic principles of the axon astrocyte interaction in biology and disease.

The Other Brain

Astrocytes mediate the beta-amyloid-driven toxic effects of synaptic tau in Alzheimer's disease

Wendy Noble

Institute of Psychiatry, King's College London, London, United Kingdom

Neuroinflammation contributes to the pathogenesis of most late-onset neurodegenerative diseases including Alzheimer’s disease (AD). Microglia and astrocytes are the predominant cells that mediate neuroinflammatory responses. Glial function is altered in disease in response to local environmental signals including danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). In AD, this includes binding of discrete Aβ species and other insults to toll-like receptors, purinoceptors including P2X7 and other cell surface receptors. The result is a change in glial phenotype, leading to induction of immune responses, activation of the inflammasome, and increased synthesis and release of inflammatory molecules such as cytokines and chemokines that interact in potentially damaging inter-cellular cascades. Astrocytes and microglia appear to work in conjunction to cause neurotoxicity; pro-inflammatory mediators released by microglia induce an active A1 phenotype in astrocytes that is neurotoxic. We have investigated the role of astrocytes in mediating the toxic effects of physiological concentrations of Aβ at synapses. We find that mouse and human astrocytes secrete soluble molecules, including various cytokines, upon exposure to human Aβ that engage neuronal receptors and act in a tau-dependent manner to induce synapse loss. I will discuss this work showing that astrocytes contribute to damaging neuroinflammatory responses in AD.

The Other Brain

Protein binding RNAs in neurodegeneration

Boris Rogelj1,2,3

1Department of Biotechnology, Jozef Stefan Institute, Ljubljana, Slovenia

2Chair of Biochemistry, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia

3Biomedical Research Institute, Ljubljana, Slovenia

Ribonucleoprotein (RNP) granules are membraneless organelles formed of RNA binding proteins (RBP) on a scaffold of RNA that have an increasing range of functions in cells from physiological such as stress response and synaptic plasticity to pathological in cancer and neurodegeneration. The expansion mutation of the GGGGCC (G4C2) repeat in the gene C9ORF72 is the most common genetic cause of FTD and ALS. It is transcribed both from the sense and the antisense strands forming scaffolds that lead to the formation of nuclear RNA foci. They may sequester specific RBPs, form pathological RNP granules and affect various steps of post-transcriptional gene regulation. Core paraspeckle proteins FUS, hnRNPH, SFPQ, NONO and RBM14 as well as PSPC1 bind to (G4C2)n repeat RNA in vitro, and colocalize with nuclear RNA foci in fibroblasts and brain tissue of C9ORF72 mutant carriers. (G4C2)n RNA foci lead to an increased number of SFPQ-stained RNP granules, which form independently of the known paraspeckle platform long non-coding RNA NEAT1. Furthermore, (G4C2)n RNA foci are also dependent on expression of SFPQ. Our results suggest that (G4C2)n RNA foci form paraspeckle-like structures, which function in similar fashion as paraspeckles and modulate nuclear compartmentalization of paraspeckle-bound RBPs and RNA.

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Astrocytes and stem cells in pathophysiology of neurodegenerative diseases

Eva Sykova1,2

1Institute of Neuroimmunology, Slovak Academy of Sciences, Bratislava, Slovakia 2Department of Research and Development, Scimed Biotechnologies, Prague - Zlatníky, Czech Republic

Neurodegenerative diseases (ND) such as Alzheimer’s disease (AD), spinal cord injury (SCI), and amyotrophic lateral sclerosis (ALS) represent a group of diseases with human protein-misfolding and ECM disorders. Astrocytes play a key role in these pathologies as they secrete neurotrophic factors that stimulate neurogenesis, stimulate synaptogenesis and maintain optimal PNNs which are important for neuronal vulnerability and CNS plasticity. Reactive astrocytes are closely associated with β-amyloid plaques (AD), gliotic scars formation (SCI) and a disfunction of perineuronal nets (ALS).

Stem cells have been investigated for their therapeutic potential in ND. Implantations of human mesenchymal stem cells (MSCs), a conditionally immortalized stem cell line from fetal spinal cord (SPC-01) and induced pluripotent stem cell-derived neural precursors (iPS- NPs), labelled in culture with iron-oxide nanoparticles for MRI tracking, were studied for their capacity to migrate towards lesion sites, differentiate, induce better regeneration, preserve PNNs and stimulate neural plasticity [3]. Our human induced pluripotent stem cells (NP-iPS) after multiple intraspinal grafting into asymptomatic and early symptomatic SOD1 G93A transgenic rats preserved motoneurons number, slowed disease progression and extended survival of all cell-treated animals. We found that SOD1 G93A rats at the terminal stage have dysregulation of some components of ECM as is versican, has-1, tenascin-R and hapln-1 and spinal chondroitin sulphate proteoglycans (CSPGs). NP-iPS grafting led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2 and casp-3) and to a restoration of perineuronal nets around the preserved motoneurones. In the host spinal cord, transplanted cells adopted a glial phenotype or remained as progenitors, many in contact with motoneurons. Current study implicates dysfunction of ECM-related structures in SOD1 G93A rats and unveil novel mechanisms of NP-iPS action via regulation of host ECM-related genes and proteins. NP-iPS have potent neuroprotective properties and are able to preserve motoneurons and normal structure of the CNS extracellular matrix.

Supported by APVV-17-0642.

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RNA biology to understand neurodegeneration

Jernej Ule1,2,3

1The Francis Crick Institute, London, UK

2Department of Neuromuscular Diseases, UCL Institute of Neurology, London, UK

3National Institute of Chemistry, Ljubljana, Slovenia

My talk will introduce the methods that integrate transcriptomics, imaging and bioinformatics to study the roles of ribonucleoprotein complexes (RNPs) in neurodegenerative diseases. I will present our study of the interplay of molecular events and cellular changes that take place upon aging in different brain regions, which could be relevant for the various age- related neurodegenerative diseases. Here we found that astrocyte and oligodendrocyte- specific shift their regional expression patterns upon aging, particularly in the hippocampus and substantia nigra, whereas the expression of microglia and endothelial-specific genes increase in all brain regions. In line with these changes, high-resolution immunohistochemistry demonstrated decreased numbers of oligodendrocytes and of neuronal subpopulations in the aging brain cortex, and we showed that glial-specific genes predict age with greater precision than neuron-specific genes.

Next, I will present our studies of faulty RNPs in amyotrophic lateral sclerosis (ALS), where mutations in several RNA binding proteins (RBPs) cause ALS, including TDP43, hnRNPA1, hnRNPA2/B1, FUS and MATRIN3. All of these proteins are recruited to a nuclear compartment, called paraspeckles, which forms through a process called liquid-liquid phase separation (LLPS). I will also present our work on TDP-43, and its role in regulating paraspeckles through a non-coding RNA called NEAT1, thereby forming a dynamic cross- regulatory network that is relevant for ALS. Finally, I will also present our study of disease- causing mutations in RBPs, which are most often located within intrinsically disordered regions (IDRs). I will demonstrate how the IDRs serve as a docking platform for protein- protein interactions, which fine-tunes the RNA binding properties and functions of RBPs. Taken together, I will discuss how RNPs might affect the cellular fates in aging and in neurodegenerative diseases.