Energy 2013 – Current Advances in Brain Maintenance

The Norwegian Academy of Science and Letters 29 August 2013

Cover: The lactate receptor GPR81 (green) is located in neurons, shown here in hippocampal cortex CA1. The receptor is concentrated in the pyramidal cell somatodendritic compartment including spines (white arrows in closeup), and to a lesser degree in vascular endothelium. Neurons were labelled for MAP2 (red), nuclei with DAPI (blue).

Electronmicroscopy shows lactate receptor labelling (10 nm immunogold, red arrowheads) at the postsynaptic membrane (between black arrowheads), Illustrated by a synapse between a nerve terminal (t) and a dendritic spine (s) in stratum radiatum of CA1.

See: Lauritzen KH, Morland C, Puchades M, Holm-Hansen S, Hagelin EM, Lauritzen F, Attramadal H, Storm-Mathisen J, Gjedde A, Bergersen LH (2013) Lactate Receptor Sites Link Neurotransmission, Neurovascular Coupling, and Brain Energy Metabolism. Cereb Cortex Epub 2013 May 21.

Brain Energy 2013 – Current Advances in Brain Maintenance

When: Thursday 29th August 2013 – Where: The Norwegian Academy of Science and Letters, Drammensveien 78, Oslo Why: Understanding mechanisms that keep the brain in good repair and able to adapt to new challenges, at all ages, will help address a major challenge facing mankind: how to retain an individual’s autonomy towards the end of life. Organized by: Linda H Bergersen, Vidar Gundersen, Jon Storm-Mathisen; Dep Oral Biology, and Inst Basic Medical Sciences, UiO Supported by: The Medical Faculty (equal opportunity grant to LHB), and Molecular Life Science - MLSUiO, University of Oslo Registration: Email to [email protected], [email protected] or [email protected] before 20th August

08:30 Registration, Coffee

08:50 The organizers Welcome - introduction

09:00 Linda H Bergersen Lactate is a volume transmitter – 'New Deal' for understanding brain function and brain disease

09:30 Pierre Magistretti Role of lactate as a signalling molecule for neuronal plasticity and memory

10:00 Albert Gjedde On the roles of energy metabolites as volume transmitters in brain: in vivo molecular imaging of neuroenergetics in aging and age-related neurodegenerative diseases

10:30 Ole Petter Ottersen Astrocytic endfeet: new insight in their function and organization

11:00 Coffee – Refreshments

11:30 Vidar Gundersen Glial signalling

12:00 Peter Somogyi Network state-dependent firing of distinct cell types in the awake and sleeping hippocampus

12:30 David Attwell Regulation of brain energy supply: where does the action start?

13:00 Martin Lauritzen Neurophysiology of local control of brain blood flow and energy metabolism

13:30 Lunch

14:15 Lene Juel Rasmussen Mitochondrial function regulates nucleotide metabolism and affects genomic stability: mechanisms and biomarkers for cognitive function

14:45 Vilhelm Bohr Base excision DNA repair and neurodegeneration

15:15 Tone Tønjum Glial responses to meningitis pathogens

15:45 Bjørnar Hassel When bacteria meet the brain: energy-metabolic interactions between abscess-forming bacteria and brain cells

16:15 Coffee – Refreshments

16:45 Magnar Bjørås Maintenance of brain function by Neil dependent repair of oxidative DNA damage

17:15 Arne Klungland Dynamics of methyl modifications in DNA and RNA: roles in brain and endurance running

17:45 Fred Gage (Rusty Gage) Functional consequences of exercise and enrichment on adult neurogenesis and cognition

18:15 Discussion Brain Maintenance (Moderator: Ole Petter Ottersen, Rector of the University of Oslo )

19:00 End of Symposium – Sparkling wine

Lactate is a volume transmitter – 'New Deal' for understanding brain function and brain disease

Linda Hildegard Bergersen Department of Oral Biology and Department of Anatomy, University of Oslo, Oslo, Norway, and Department of and Pharmacology, University of , Copenhagen, Denmark [email protected]

Lactate is a fuel for the brain, but of disputed importance. Some effects of lactate in the CNS, such as neuroprotection, are not easily explained in terms of energy metabolism, but suggest receptor mechanisms. In a recent study (Lauritzen KH et al 2013 Cereb Cortex), we show that the lactate receptor GPR81, also known as HCA1, downregulates cAMP production in the brain in response to physiologically increased lactate levels, such as occur in physical exercise, and by the specific GPR81 agonist 3,5-dihydroxybenzoate, which occurs in fruits and berries. The receptor is concentrated at the postsynaptic membranes of glutamatergic synapses on cortical pyramidal cells, including in the hippocampus. GPR81 is also enriched at the blood-brain barrier: the GPR81 density at endothelial cell membranes are about twice the GPR81 density at membranes of perivascular astrocytic processes, but about one seventh of that on synaptic membranes. There is only a slight signal in perisynaptic processes of astrocytes. In synaptic spines, as well as in adipocytes where the receptor was first identified, GPR81 immunoreactivity is located on subplasmalemmal vesicular organelles suggesting trafficking of the protein to and from the plasma membrane. In adipocytes, GPR81 is part of an autocrine / paracrine loop whereby increased production of lactate mediates the antilipolytic effect of insulin through downregulating cAMP. We suggest that, in the brain, the lactate receptor allows lactate released by activated neurons to act as a “volume transmitter” that tells brain cells to adjust their condition by curbing cAMP production. This would provide a means for linking neuronal activity, cerebral energy metabolism and energy substrate availability, including a neuronal glucose saving response to the supply of lactate. It is known that whereas brain cAMP levels increase acutely in arousal, augmenting cognition and memory, chronically increased cerebral cAMP such as in chronic stress and old age is associated with cognitive impairment. The new findings therefore point to the lactate receptor as a potential therapeutic target.

Biography Linda Hildegard Bergersen obtained her PhD at the University of Oslo in 2001 under the supervision of Ole Petter Ottersen. After postdoc periods, in Oslo with Jon Storm-Mathisen and in Lausanne with Pierre Magistretti and Luc Pellerin in 2003, she established the “Brain and Muscle Energy Group” in Oslo in 2004, and in Copenhagen in 2011. Bergersen’s main research interest is brain energy supply, under physiological as well as pathological conditions. She has done pioneering work on the localization of lactate transporters and in a recent breakthrough discovered the lactate receptor GPR81 to be present and active in the brain, opening new opportunities for basic and translational research. Bergersen is currently a Professor of Physiology at the University of Oslo and part time Professor of Neurobiology of Aging at the , and elected member of the Norwegian Academy of Science and Letters.

1 Role of lactate as a signaling molecule for neuronal plasticity and memory

Pierre J. Magistretti Brain Mind Intsitute, EPFL; Center for Psychiatric Neuroscience, CHUV/UNIL, Lausanne, Switzerland, and Biological and Environmental Sciences and Engineering Division, KAUST, Thuwal, Kingdom of Saudi Arabia [email protected]

A tight metabolic coupling between astrocytes and neurons is emerging as a key feature of brain energy metabolism (Allaman et al 2011; Bélanger et al 2011). Over the years we have described two basic mechanisms of neurometabolic coupling. First the glycogenolytic effect of VIP - restricted to cortical columns - and of noradrenaline - spanning across functionally distinct cortical areas - indicating a regulation of brain homeostasis by acting on astrocytes, as glycogen is exclusively localized in these cells (Magistretti, 2008). Second, the glutamate-stimulated aerobic glycolysis in astrocytes, mediated by the sodium-coupled reuptake of glutamate by astrocytes and the ensuing activation of the Na-K-ATPase, resulting in the release of lactate from astrocytes, which can then fuel the neuronal energy demands a mechanisms known as the ANLS (Pellerin and Magistretti 2011).

We have recently shown that lactate derived from astrocytic glycogen is necessary for long-term memory consolidation and for induction of plasticity genes in neurons such as Arc and for maintenance of LTP (Suzuki et al, 2011). This key role of L-lactate in neuronal plasticity mechanisms was demonstrated in experiments in which specific pharmacological and gene expression downregulation interventions were implemented to prevent the production of L-lactate from glycogen - which is exclusively localized in astrocytes - and its release from these cells in the hippocampus during behavioral training (Suzuki et al, 2011). Such interventions completely prevented the establishment of in vivo LTP and long term memory and their effect was fully reversed by the intrahippocampal administration of L-lactate during the training session. The fact that glucose at equicaloric concentrations only marginally mimicked the rescuing effect of L-lactate, was taken as an unexpected indication that the primary mechanism of action of L-lactate on plasticity mechanisms was independent of its ability to act as an energy substrate. We therefore set out to investigate the molecular mechanisms at the basis of the function of L-lactate on neuronal plasticity. We have found that L-lactate stimulates the expression of synaptic plasticity-related genes such as Arc and Zif268 through a mechanism involving NMDA receptor activity and its downstream signaling cascade Erk1/2. This effect is observed both in primary neurons in culture and in vivo in the sensory-motor cortex. L-lactate transport into neurons, coupled to changes in the cellular red-ox state, is necessary for the observed effects. These results provide novel insights for the understanding of the molecular mechanisms underlying the critical role of astrocyte-derived L-lactate in long term memory and reveal a novel action of L-lactate as a signaling molecule for neuronal plasticity.

Biography Pierre J. Magistretti received his MD from the University of Geneva and his, PhD in Biology from UCSD. He is Professor of Neuroscience at the Brain Mind Institute at EPFL and Professor of Psychiatry at the Center for Psychiatric Neuroscience at the University of Lausanne Medical School and Hospital (CHUV). He is currently on leave to be Dean of the Division of Biological and Environmental Sciences and Engineering at KAUST. Pierre J. Magistretti’s laboratory has discovered some of the cellular and molecular mechanisms that underlie the coupling between neuronal activity and energy consumption by the brain. These findings are particularly relevant for understanding the origin of the signals detected by functional brain imaging, and are revealing a role of astrocytes in neuronal plasticity. He is the author of over 180 publications in interntional peer-reviewed journals. He has been elected at the International Chair 2007-2008 of the Collège de France, . He is member of Academia Europeae and of the Swiss Academy of Medical Sciences. He is the Past- President of the Federation of European Neuroscience Societies (FENS) and past-Secretary General of The International Brain Research Organization (IBRO). Since 2010 he is Director of the National Center for Competence in Research (NCCR) of the Swiss National Science Foundation “The synaptic bases of mental diseases”. He is strongly engaged in the public understanding of neuroscience.

2 On the role of energy metabolites as volume transmitters in brain: in vivo molecular imaging of neuroenergetics in aging and age-related neurodegenerative diseases

Albert Gjedde Dept of Neuroscience and Pharmacology, University of Copenhagen, Denmark, and Magnetic Resonance Research Center, Yale University, New Haven, Connecticut, USA, and Dept of Nuclear Medicine and PET Center, Hospital, Aarhus, Denmark [email protected]

The quantitative contributions of glucose metabolism to the generations of lactate, ATP, heat, and reactive oxygen species largely are unknown in vivo. The fraction of glucose that supplies lactate is disputed, and the fractions of glucose that fuel oxidative phosphorylation and the generation of heat, respectively, are in doubt as well. The term aerobic glycolysis applies to the part of glucose metabolism that ends in pyruvate, but recently was used to signify the production of lactate in the presence of oxygen. There is little support for recent claims that aerobic glycolysis serves brain plasticity, such that glucose moieties that are not oxidized to CO2 supply the tissue with structural components. The oxygen-glucose index (OGI) is less than 6 when some glucose supplied to brain is not fully oxidized, or more than 6 when substrates other than glucose enter brain and are fully oxidized there. There is little support for the claim that this index varies regionally in brain cortex, where the fraction of glucose oxidized to CO2 uniformly is close to 0.9. Recent meta-analysis of human cerebral metabolism and EEG data from several behavioral states suggests that glutamatergic neurotransmission in resting awake human brain depends on uniformly high oxidative energy metabolism [5,7]. Thus, we now claim that lactate serves neither mainly as alternative fuel nor mainly as a building block but rather as an active coordinator of brain metabolism and brain functions by means of volume transmission [2,8]. The ultimate effect of this coordination is the maintenance of constant metabolite concentrations when metabolism adapts to prevailing demands for energy turnover. A measure of the success of this adaptation is the coupling of cerebral blood flow to oxidative metabolism, expressed as the oxygen extraction fraction (OEF) signal. We now show that OGI and OEF measures in human brain are closely correlated across the regions of the cerebral cortex, with average values of CMRglc, CMRO2, and CBF in resting gray matter of 29±8 μmol/hg/min, 150±57 μmol/hg/min, and 39±5 ml/hg/min, respectively, yielding OGI and OEF values of 5.2 and 0.36. Nowhere in cortex did the excursions exceed 10% of the global mean. We believe that the considerable variability instead reflects individual differences related to different efficiencies of oxidative phosphorylation [4]. The general non-linear relation between CBF and CMRO2 appears to be regulated jointly by the glycolytic and oxidative components of CMRglc [8]. Both OEF and OGI decline in specific regions during activation, and both rise during aging in relation to the declining energy turnover [1,3,8]. In aging, we found significant increases of OEF in frontal and parietal cortices, excluding primary motor and somatosensory regions, and in the temporal cortex. Because of the inverse relation between OEF and capillary oxygen tension, increased OEF above the threshold mandated by metabolism in principle can compromise oxygen delivery to neurons, with possible perturbation of energy turnover in some parts of the tissue [4]. However, in a smaller group of patients, correction for the CO2 response suggested that oxygen delivery is uncompromised and the rise of OEF and OGI in aging hence a consequence of reduced mitochondrial function [6]. The results establish a possible mechanism of progression from healthy to unhealthy brain aging, with the most affected regions being vulnerable to neurodegeneration.

[1] Aanerud JA et al (2012) J Cereb Blood Flow Metab 32: 1177-1187, [2] Bergersen & Gjedde (2012) Front Neuroenerg 4:5 doi: 10.3389 / fnene.2012.00005, [3] Dastur DK (1985). J Cereb Blood Flow Metab 5: 1-9, [4] Gjedde A et al. (2011) Adv Exp Med Biol 701: 243-248, [5] Hyder F et al (2013) J Cereb Blood Flow Metab. 33:339-347, [6] Rodell AB et al. (2012) Front Neuroenerg 4:8 doi: 10.3389/fnene.2012.00008, [7] Shulman RG et al (2009) PNAS USA. 106:11096-11101, [8] Vafaee MS et al. (2012) J Cereb Blood Flow Metab 32: 1859-1868

Biography Albert Gjedde is Professor and Chairman at the Department of Neuroscience and Pharmacology at the University of Copenhagen. He previously served as director of laboratories in Aarhus, Denmark, and Montreal, Canada, with earlier sojourns to Berkeley and New York. Albert Gjedde is Fellow of the Royal Society of Canada, the American Association of the Advancement of Science, and the American College of Neuropsychopharmacology, and a member of the Academy of Europe. He holds adjunct professorships at McGill, Aarhus, and Johns Hopkins Universities. Albert Gjedde’s research focuses on the relations between brain energy metabolism and monoaminergic neurotransmission. In this work, Albert Gjedde uses PET to understand the binding of radioligand and tracer molecules that match the neurotransmitters and their actions under different functional conditions of the brain. With this data, he establishes the spatial and temporal relations among changes of cerebral blood flow and cerebral oxygen consumption as a more precise measure of brain work. Albert Gjedde’s collaborations focus on experiments with volunteer subjects and patients that reveal the lesions and degeneration of brain in disorders such as persistent vegetative state, , ludomania, Parkinson’s disease, , depression, and somaticizing disorders, as well as disorders related to addiction.

3 Astrocytic endfeet: new insight in their function and organization

Ole Petter Ottersen Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway [email protected]

Astrocytic endfeet are highly specialized processes that abut on brain microvessels and pia. The endfeet provide a complete covering of cortical capillaries and contain a unique arrangement of proteins that sets them apart from other astroglial compartments. The specialization of the endfoot membrane depends on a dystrophin associated protein complex that is tethered to the pericapillary basal lamina through a dystroglycan bridge (Amiry-Moghaddam & Ottersen, Nat Rev Neurosci 2003). High resolution immunogold analyses (Amiry-Moghaddam & Ottersen, Nat Neurosci 2013) indicate that endfeet membranes are enriched with a number of transporters and channels, including aquaporin-4 (AQP4), the inwardly rectifying K+ channel Kir4.1, and the transient receptor receptor potential vanilloid TRPV4. Glial-conditional deletion of aquaporin-4 (Aqp4) reduces blood-brain water uptake and confers barrier function on perivascular astrocyte endfeet, and in vivo NADH fluorescence imaging indicates effect of aquaporin-4 deletion on oxygen microdistribution in cortical spreading depression. The molecular organization in endfoot membranes is disrupted in a number of pathological conditions, including mesial temporal lobe epilepsy and Alzheimer’s disease. Thus, endfeet are likely to play distinct physiological and pathophysiological roles in brain (Nagelhus & Ottersen, Physiol Rev, in press). Two of the endfoot proteins (AQP4 and TRPV4) appear to form a molecular complex involved in osmosensing and regulatory volume control (Benfenati et al., PNAS 2011). In vivo optical imaging is now being applied to further our insight in signalling and transport processes in astrocytic endfeet.

Biography Ole Petter Ottersen obtained his MD in 1980 and his PhD in 1982. In 1992 he was appointed Professor at the Institute of Basic Medical Sciences, University of Oslo. He served as Director of the Centre for Molecular Biology and Neuroscience (a Centre of Excellence sponsored by the Research Council of Norway) from 2002 until he took office as Rector (President) of the University of Oslo in 2009. Ottersen has coordinated a Nordic Center of Excellence in Molecular Medicine (Centre for Research on Water Imbalance Related Disorders - WIRED). He has worked on several subjects including synaptic signalling and water transport in brain.

4 Glial signalling

Vidar Gundersen Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway, and Department of , Oslo University Hospital, Oslo, Norway [email protected]

Abstract With their delicate processes astrocytes wrap around synapses. This morphological arrangement, dubbed the tripartite synapse, sets the stage for bi-directional neuron-astrocyte signalling. We have reported that perisynaptic astrocytes contain the machinery for vesicular release of glutamate. In these delicate processes glutamate is located in synaptic like microvesicles, which possess the transporters that pump glutamate into vesicles; the vesicular glutamate transporters (VGLUTs). Upon activation of e.g. P2Y1 ATP receptors glutamate is released from astrocytes to act on for instance the NMDA type of glutamate receptor in presynaptic terminals. Like perisynaptic astrocytes, quiescent microglia also contact synapses. Such microglia neuron contacts have been shown to regulate morphological spine plasticity and pruning during development. The ability of microglia to move in the neuropil is crucial for such a function. In the adult healthy brain we have characterised the contacts between delicate microglial processes and synapses. Such contacts are rather rare; only about 3-4% of the synapses receive direct contact from microglia. Compared to the cell bodies and larger processes, the delicate microglial processes contain high levels of Iba-1 and actin, explaining why they are able to move in the neuropil. Perisynaptic astrocytes wrap around most synapses. They are able to regulate synaptic transmission through release of glutamate at the tripartite synapse, while the proposed quadripartite synapse, due to its infrequent occurrence, probably does not play a major role in regulating synaptic function in the adult healthy brain.

Biography Vidar Gundersen is Leader of the Synaptic Neurochemistry Laboratory, Dept Anatomy, University of Oslo, a laboratory founded by Jon Storm-Mathisen, who was Gundersen’s PhD supervisor. Gundersen is also neurologist at the Dept of Neurology, Oslo University Hospital with a special interest in Parkinson’s disease. His basic neurobiological work has focused on release of transmitters from neurons and lately also from astrocytes. The latter topic was introduced during Gundersen’s post doc stay in the laboratory of Professor Andrea Volterra in Lausanne. Recently, Gundersen has also worked with uncovering the pathogenesis of Parkinson’s disease, with focus on the role of microglia.

5 Network state-dependent firing of distinct cell types in the awake and sleeping hippocampus

Péter Somogyi Medical Research Council Anatomical Neuropharmacology Unit, Dept. Pharmacology, University of Oxford, Oxford, UK [email protected]

Abstract The generation of spike trains and the recycling of neurotransmitters are energy demanding processes, which change depending on behavioural state. In particular, the firing rates of neurons in the cerebral cortex vary between total silence and over 300 Hz. The cortex is composed of a cooperative network of an unknown number of neuronal and glial cell types, each changing their activity patterns independently. We have explored how distinct cell types change their firing rates in the cortex by recording and labelling single neurons in the hippocampus of freely moving and naturally sleeping rats. During spatial navigation, or the offline replay of spatial representations, pyramidal cell firing is rhythmic and phase-related to the local field potential in the theta, gamma and sharp wave related ripple (SWR) frequency ranges. The rhythmic firing of GABAergic interneurons in the hippocampus contributes to the synchronization of neuronal activity. Interneurons, innervating specific postsynaptic domains, selectively discharge at different rates phase-locked to network oscillations in a cell type specific manner. All parts of pyramidal cells, except the axon initial segment, receive GABA from multiple interneuron types, each with distinct firing dynamics. Parvalbumin- expressing basket cells fire phase locked to field theta and gamma activity in both CA1 and CA3, and also strongly increase firing during SWRs in slow wave sleep, as do dendrite-innervating bistratified cells in CA1. Some other GABAergic cell types decrease their firing rate during sleep. The axon initial segment is exclusively innervated by axo-axonic cells, which preferentially fire after the peak of pyramidal layer theta when pyramidal cells are least active. Axo-axonic cells are inhibited during SWRs in both CA1 and CA3, when pyramidal cells fire most. This inverse correlation demonstrates the key inhibitory role of axo-axonic cells, and their cessation of firing is probably a requirement of memory consolidation. The evolution of domain-specific GABAergic innervation was probably driven by the need of coordinating multiple glutamatergic inputs to pyramidal cells through temporally-distinct GABAergic interneurons, which independently change firing during different network states. I will demonstrate the key mechanism of coordination: a network state-dependent temporal redistribution of inhibition over distinct subcellular domains of pyramidal cells.

Biography Péter Somogyi, FRS, is the director of the Medical Research Council Anatomical Neuropharmacology Unit and Professor of Neurobiology at the University of Oxford. His mentors were István Benedeczky in microscopy and cell biology, János Szentágothai in neuroanatomy and David Smith in biochemical pharmacology. The Unit investigates the synaptic organisation and temporal dynamics of neuronal systems in the mammalian brain. Educated as a biologist in Budapest, Hungary, he has explored identified neurons in the cerebellum, thalamus, brain stem, basal ganglia and the cerebral cortex. His vision is that explanations of normal and pathological events in the brain require the simultaneous definition of the neuronal organisation in space and time, the chronocircuit. In the last decade, he has focussed on exploring the brain- and network-state dependent activity patterns of identified hippocampal neurons. With his colleagues he discovered a highly specialised spatial and molecular organisation of interneurons, which has also led to the discovery of an unexpected temporal specialisation of GABA release to distinct domains of pyramidal cells.

6 Regulation of brain energy supply: where does the action start?

David Attwell Department of Neuroscience, Physiology & Pharmacology, University College London, London, UK [email protected]

Brain blood flow is regulated to ensure adequate power for neuronal computation. Blood flow is increased to areas where neurons are active, and this increase underlies non-invasive brain imaging using BOLD fMRI. Blood flow is controlled at the arteriole level by smooth muscle, but there is controversy over whether it is also regulated by pericytes at the capillary level. I will demonstrate that neuronal activity and the excitatory glutamate evoke an outward membrane current in pericytes and dilate capillaries, and that this dilation is caused by active relaxation of pericytes rather than a passive expansion of capillaries downstream of arteriole relaxation. Glutamate-evoked dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress synthesis of the vasoconstrictor 20-HETE. In vivo, when sensory input to the neocortex releases glutamate and increases blood flow, capillaries dilate first, followed by arterioles. Capillary dilation is estimated to generate ~89% of the increase of blood flow occurring. Capillaries also have a role in pathology. Ischaemia was previously shown to lead to a contraction of pericytes. I will report that this is followed by their death, which is expected to produce an irreversible constriction of capillaries. Pericyte death is reduced by block of glutamate receptors or calcium removal, and increases with reperfusion, but is not blocked by scavenging of reactive oxygen species. These data establish, for the first time, pericytes as major regulators of cerebral blood flow, and initiators of the BOLD fMRI response. They also focus attention on prevention of pericyte death as a therapeutic strategy to reduce the long-lasting blood flow decrease which contributes to neuronal death after stroke.

Biography David Attwell did a first degree in physics and a PhD on the electrophysiology of nerve and muscle cells (with Julian Jack) in Oxford, before spending 2 years in Berkeley studying the retina with Frank Werblin. On returning to the UK, he moved to the Department of Physiology at University College London, where he has remained ever since. He has worked on a wide range of subjects including the properties of glial cells, glutamate transporters, stroke, the formation of myelin by oligodendrocytes, and how neuronal computation is powered. He was made a Fellow of the Royal Society in 2001.

7 Neurophysiology of local control of brain blood flow and metabolism

Martin Lauritzen Department of Clinical Neurophysiology, Glostrup Hospital, and Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark [email protected]

Brain’s electrical activity correlates strongly to changes in cerebral blood flow (CBF) and the cerebral metabolic rate of oxygen (CMRO2). Subthreshold synaptic processes correlate better than the spike rates of principal neurons to CBF, CMRO2 and positive BOLD signals. . I will here present in vivo examples that support the view that brain oxygen consumption may be correlated to and controlled by Na,K-ATPase activity driven by transmembrane currents that are produced by neuronal signaling, and modulated by synaptic inhibition. I will furthermore provide evidence for Ca2+-dependent control of activity-dependent rises in cerebral blood flow in general, and at the level of single vessels in particular. The compiled data set from others and ourselves establish for the first time the dichotomy of metabolic (currents) and vascular 2+ (Ca ) control, which explains variations in the gap between the stimulation-induced rises in CMRO2 and CBF. The findings predict that activity-dependent rises in CBF and CMRO2 vary within and between brain regions due to differences in ATP turnover and Ca2+-dependent mechanisms, and that these variations determine the amplitude and polarity of the BOLD signal. Our results provide insights into the functional regulation of brain blood flow and metabolism, and the context-sensitivity of functional neuroimaging signals.

Biography Martin Lauritzen obtained his MD from University of Copenhagen, and completed his doctoral thesis with Niels A.Lassen and Christian Crone in Copenhagen, before spending a year studying the turtle cerebellum with Charles Nicholson at New York University. He is a medical specialist of clinical neurophysiology, senior consultant at Glostrup Hospital, and professor at the University of Copenhagen where he has worked since 1974. ML has worked on a range of clinical, translational and basic scientific subjects including cortical spreading depression, the neurovascular unit, and control of brain energy metabolism.

8 Mitochondrial function regulates nucleotide metabolism and affects genomic stability: mechanisms and biomarkers for cognitive function

Lene Juel Rasmussen Center for Healthy Aging, and Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark [email protected]

Mitochondria are the powerhouse of the cell and where cellular energy supplies in the form of ATP are generated. Because of this pivotal role, mitochondrial dysfunction is very damaging for the cell and can lead to numerous pathological conditions in humans. Most notably, mitochondrial dysfunction is associated with cognitive decline, neurological abnormalities and aging.

Balanced levels of dNTP are important for genomic stability. Accordingly, imbalance of the cytosolic dNTP pool has been demonstrated to decrease the genetic stability. We have previously shown that depletion of mtDNA of human cell lines results in an imbalance of the cytosolic dNTP pools and a decrease of chromosomal stability. MtDNA primarily encodes peptides essential for the activity of the mitochondrial electron transport chain and, therefore, also ATP produced by oxidative phosphorylation. The ETC is also linked to the de novo synthesis of pyrimidines through the enzyme dihydroorotate dehydrogenase (DHODHase) located in the inner membrane of the mitochondria. We show that both an inhibition of ATP synthase and of DHODHase results in a decrease of cytosolic levels of dNTP. We find that inhibition of either ATP synthase or DHODHase leads to increased sensitivity DNA damage. Our findings support a model for the initiation of genome instability through a mitochondrial dysfunction and resulting imbalance of the cytosolic dNTP levels. This places fitness of mitochondria as an important determinant of genomic instability.

Cognitive impairment in adults may be an early indicator of later life dementia. Therefore it is important to search for early biomarkers of cognitive decline. We have tested peripheral blood mononuclear cells (PBMCs) isolated from low- and high-cognitive score subjects, for correlates to cognitive function that could potentially serve as biomarkers. Cognitive score is assessed by the Isaacs Set Test (IST) (1) and the Danish Military Draft Board Intelligence Test (Børge Priens Prøve, BPP). The population is drawn from the Metropolit Cohort of men born in 1953 as part of the Copenhagen Aging and Midlife Biobank (CAMB) data collection. The extremes of high and low cognitive measure are being used in this study. We have measured oxygen consumption rate (OCR), dNTP ratios, reactive oxygen species (ROS) levels, and DNA damage for the all cohort participants who participate. Our data promote investigation into mitochondrial activities and DNA damage as potential correlates or predictors of cognitive decline, which may lead to early treatment initiatives in order to delay or prevent later life dementia.

Biography Lene Juel Rasmussen did a MSc in chemistry and a PhD in molecular biology at the Technical University of Denmark, before spending 5 years in US studying DNA repair at University of Massachusetts with Martin Marinus and at Harvard University with Leona Samson. On returning to Denmark, she took up a position at Roskilde University and moved to the University of Copenhagen in 2009, where she has remained ever since. She is currently Managing Director of Center for Healthy Aging at University of Copenhagen. She has worked on a wide range of subjects including the properties of bacterial and mammalian DNA mismatch repair and how mitochondrial function affects genomic instability.

9 Base excision DNA repair and neurodegeneration

Vilhelm A. Bohr Laboratory of Molecular Gerontology, National Institutes on Aging, NIH, Bethesda, , USA, and Center for Healthy Aging, University of Copenhagen, Copenhagen, Denmark [email protected]

Proficient mitochondrial function is essential in the maintenance of the nervous system. Oxidation and stress constantly cause macromolecular damage which affect DNA, RNA and proteins. The damage to DNA is particularly threatening as the genetic material needs to remain intact. An intricate system of proteins maintain genome integrity and this system of DNA repair differs between the nucleus and the mitochondria. While much is known about how these pathways operate in the nucleus, much less is understood about these functions in mitochondria. Recent developments have disclosed that a number of proteins that are defective in diseases of neurodegeneration appear to operate in the mitochondria, and thus these neurodegenerations may in part be due to mitochondrial dysfunction.

We find that physiological levels of glutamate can stimulate a DNA repair pathway and that this is increased in mice after execise.

We are interested in whether deficiencies in nuclear or mitochondrial DNA base excision repair may be a cause of neurodegeneration. This is studied in mouse models deficient in DNA repair and in mice with Alzheimer’s.disease-like features where we have also depleted a critical DNA base excision repair protein, DNA polymerase beta. Preliminary findings suggest that these mice have significant memory defects.

Biography VB received his MD and D.Sci. from the University of Copenhagen. Was a postdoc and research scholar at Stanford University 1982-86. Then developed a research group at the National Cancer Institute, NIH, Washington 1986-92, and then became Department chair at the National Institute on Aging, where he has remained since 1992. Author of more than 500 peer reviewed papers in the area of DNA repair and aging.

10 Glial responses to meningitis pathogens

Tone Tønjum Department of Microbiology, Oslo University Hospital (Rikshospitalet), and University of Oslo, Oslo, Norway [email protected]

Neisseria meningitidis, the meningococcus, is a leading cause of meningitis worldwide. This pathogen faces up to the environment in its exclusive human host with a small but hyperdynamic genome. We constructed meningococcal mutants inactivating genes involved in virulence and DNA repair. Distinct differences between the meningococcus and the E. coli paradigm were identified. At the same time, the meningococcus is competent for uptake of exogenous DNA by transformation throughout its lifecycle. Our analyses of the DNA uptake sequence (DUS) and components required for transformation show that the outcome of transformation predominantly has a conservative nature, rather than mediating genomic variation, and is well adapted between bacterial species expressing dialects of DUS.

Although meningitis is a serious disease, little is known about the interaction between microbe and host in the meninges and brain. We are therefore monitoring the infection process to elucidate bacterial pathogenesis relevant for inflammation and brain edema. We have established a mouse model for meningitis by direct inoculation of bacteria into the subarachnoid space, in which we monitor host responses. Aquaporins are water-channels that are highly expressed in the brain and involved in the development of brain edema. The degree of inflammation and aquaporin expression in the brain was quantified morphologically and by assessing gene expression. Meningococcal meningitis was associated with pronounced brain edema with a unique AQP expression profile. In addition to defining roles for meningococcal virulence and genome dynamics, these findings are important in the context of models linking bacterial mutator phenotypes to cellular fitness, pathogenesis, drug resistance development and CNS disease.

Biography Tone Tønjum is professor and chief in clinical microbiology at the University of Oslo and Oslo University Hospital. She has an MD and a PhD from the University of Oslo and was a postdoc at the and Rocky Mountain Laboratory, NIH, USA. She is the leader of an active research group, focusing on the role of genome dynamics in microbes and humans. Tønjum plays a key role in several multidisciplinary research networks, is a member of the Norwegian Academy of Science and Letters, and and serves on a number of international scientific advisory boards.

11 When bacteria meet the brain: energy-metabolic interactions between abscess-forming bacteria and brain cells

Bjørnar Hassel Department of Neurology, Oslo University Hospital-Rikshospitalet, Oslo, Norway Norwegian Defence Research Establishment, Kjeller, Norway [email protected]

The extracellular fluid is, for most bacteria, the first tissue compartment they encounter when they infect a human being. Most bacteria that cause pneumonia will have to survive in the fluid that lines the alveoli. Most bacteria that enter the brain will find themselves in the cerebral extracellular fluid. In the lungs, the extracellular fluid is enriched in amino acids, providing an ideal environment for many bacterial species. In the brain, in contrast, the extracellular fluid is relatively poor in amino acids, partly because of restricted passage of amino acids across the blood-brain barrier, partly because of avid amino acid uptake into neurons and glia. Bacteria that enter the brain, may respond to this nutritional challenge by producing exotoxins that insert themselves into cell membranes and form pores. This response probably evolved because it kills other microbes that compete for the same limited nutrients. In the brain, pore-forming toxins may cause the death of brain cells and the formation of abscesses. Staphylococcus aureus is a pore-forming toxin-producing bacterium that may cause brain abscesses. I will report on the effect of staphylococcal infection on extracellular levels of amino acids in the rat brain and on the effect of a staphylococcal pore-forming toxin, α-hemolysin, on the nutritonal situation of staphylococci in the brain (which improves quite dramatically). I will further report on the levels of amino acids in longer standing brain abscesses in human patients. These abscesses contain amino acids, including glutamate, in the millimolar range. Such high levels of neuroactive amino acids probably contribute to the symptoms of brain abscesses in humans, e.g. seizures and altered sensorium.

Biography Bjørnar Hassel began studies on brain energy metabolism and amino acid neurotransmission with Frode Fonnum at The Norwegian Defence Research Establishment, Kjeller, i.a. focussing on how brain energy metabolism and glutamatergic neurotransmission can be visualized in experimental animals by the combined use of 13C-labeled energy substrates and NMR spectroscopy or GC/MS. Effects of drugs, toxins, and bacteria on brain energy metabolism and neurotransmission were studied with the same techniques. He works clinically as a neurologist at Oslo University Hospital-Rikshospitalet, Oslo, and experimentally at The Norwegian Defence Research Establishment, Kjeller.

12 Maintenance of brain function by Neil dependent repair of oxidative DNA damage

Magnar Bjørås Department of Microbiology, Oslo University Hospital, Oslo Norway [email protected]

Accumulation of oxidative DNA damage has been proposed as a potential cause of age-related cognitive decline. The major pathway for removal of oxidative DNA base lesions is Base Excision Repair (BER), which is initiated by DNA glycosylases. In mammalian cells, at least six different DNA glycosylases remove a broad spectrum of oxidative DNA lesions. Adult neurogenesis is crucial for maintenance of hippocampus- dependent functions involved in behaviour. Herein, behavioural studies of Neil DNA glycosylase deficient mice revealed significant differences in learning, memory and anxiety-like behavior. By example, neural stem/progenitor cells from Neil3 deficient mice showed impaired proliferative capacity. Further, hippocampal neurons in adult Neil3-/- mice displayed synaptic irregularities. It appears that BER of oxidative DNA damage in neural cells is required for maintenance of induced and adult neurogenesis to counteract the age associated deterioration of cognitive performance.

Biography Focus of Bjørås research has been on the repair mechanisms of endogenous DNA base lesions. Bjørås started his research career in the laboratory of Professor Erling Seeberg in 1989 and was trained in molecular biology and protein biochemistry. He has been involved in cloning, purification and characterization of several DNA repair enzymes from bacteria, yeast and human. He spent two years (2002-2004) in the lab of Professor John Tainer at Scripps Research Institute, California, to improve on his expertise in protein structure analysis. Since 2004, when the Bjørås group joined the CoE in Molecular Biology and Neuroscience, he has built up research competence and expertise on understanding the impact of oxidative DNA damage repair for brain function with emphasis on neuronal stem cells and neurogenesis.

13 Dynamics of methyl modifications in DNA and RNA: roles in brain and endurance running

Arne Klungland Department of Molecular Medicine, Oslo University Hospital and University of Oslo, Oslo, Norway [email protected]

A broad repertoire of modifications is known to underlie adaptable coding and structural function of proteins, DNA and various RNA species. These include epigenetic modifications that are heritable alterations, and deregulation of these modifications contributes to the development of several human pathologies, including cancer. Methylations of mammalian DNA and histone residues are known to regulate transcription and the discoveries of demethylases that remove methylation in DNA and histones provide a basis for the understanding of dynamic regulation of mammalian gene expression. The reversions of methyl marks on DNA and proteins have been extensively studied the last decade. On the contrary, reversal of N6- methyladenosine (6mA) to adenosine (A) in messenger RNA (mRNA) was only identified recently (for the obesity risk gene, FTO). 6-methyladenine (m6A) is the most abundant internal base modification of messenger RNA (mRNA) in higher eukaryotes and we recently identified a second 6mA demethylase for mRNA. This demethylase, ALKBH5, reverses 6mA in mRNA in vitro and in vivo. The discovery of this RNA demethylase strongly suggests that the reversible m6A modification has fundamental and broad functions in mammalian cells and in human disease. Notable, the role of other AlkB enzymes (ALKBH1- ALKBH9), as well as the TET homologs (TET1-TET3), seem to be particularly relevant for the brain. The deletion of these enzymes causes multiple phenotypes including infertility, obesity and a remarkable running endurance.

Biography Arne Klungland did his PhD on DNA repair at the Norwegian Defence Research Establishment with Erling Seeberg, before spending 5 years with Cancer Research UK with Tomas Lindal studying oxidative DNA damage. On returning to Oslo he has worked on the identification and analysis of known and novel dynamic methyl groups in DNA, RNA and proteins.

14 Functional consequences of exercise and enrichment on adult neurogenesis and cognition

Fred. H. Gage Laboratory of Genetics, The Salk Institute, La Jolla, California, USA [email protected]

Most neurons in the adult central nervous system (CNS) are terminally differentiated, but evidence now exists that small populations of neurons are generated in the adult olfactory bulb and hippocampus. In the adult hippocampus, newly born neurons originate from putative stem cells that exist in the subgranular zone of the dentate gyrus. Progeny of these putative stem cells differentiate into neurons in the granular layer within a month of the cells’ birth, and this late neurogenesis continues throughout the adult life of all mammals. The timing of the process of neurogenesis in the adult dentate gyrus is now well characterized. In addition, environmental stimulation can differentially affect the proliferation, migration, and differentiation of these cells in vivo. These environmentally induced changes in the structural organization of the hippocampus, result in changes in electrophysiological responses in the hippocampus, as well as in hippocampal related behaviors. We are studying the cellular, molecular, as well as environmental influences that regulate neurogenesis in the adult brain and spinal cord. Computational model of adult neurogenesis has generated novel hypotheses with regard to its function with the hippocampus. The functional and practical significance of these findings will be discussed in light of their implications for alternative or expanded views of structural plasticity in the adult brain.

Aimone JB, Deng W, Gage FH (2010) Adult neurogenesis: integrating theories and separating functions. Trends Cogn Sci 14:325-37 Deng W, Aimone JB, Gage FH (2010) New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 11:339-50

Biography Fred H. Gage, Ph.D., a Professor in the Laboratory of Genetics, joined The Salk Institute in 1995. He received his Ph.D. in 1976 from The . Dr. Gage's work concentrates on the adult central nervous system and unexpected plasticity and adaptability to environmental stimulation that remains throughout the life of all mammals. In addition, he models human neurological and psychiatric disease in vitro using human stem cells. Finally his lab studies the genomic mosaicism that exists in the brain as a result of mobile elements that are active during neurogenesis. Prior to joining Salk, Dr. Gage was a Professor of Neuroscience at the University of California, San Diego. Before that he worked in Lund, Sweden. He is a Fellow of the American Association for the Advancement of Science, a Member of the National Academy of Sciences and the Institute of Medicine, and American Philosophical Society, a foreign member of the European Molecular Biology Organization and a Member of the American Academy of Arts and Sciences. He has served as President of the Society for Neuroscience in 2002, and past President for the International Society for Stem Cell Research 2012.

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