Brain and Muscle Energy Group &Synaptic Neurochemistry Laboratory – Energy Supply and Synthesis, Recycling, Packaging and Action of Neuro- and Gliotransmitters

Linda Hildegard Bergersen, PhD

Associate Professor (Linjeleder), University of Oslo, NorwayProfessor Neurobiology of Aging, University of Copenhagen, Denmark

Affiliation details:SN Lab & Brain and Muscle Energy GroupCentre for Molecular Biology and Neuroscience (CMBN) &Department of Anatomy, Institute of Basic Medical Sciences (IMB)University of Oslo, NorwayProfessor Neurobiology of AgingCenter for Healthy Aging (CEHA) andDepartment of Neuroscience and Pharmacology (INF)University of Copenhagen, DenmarkNansen Neuroscience Network

Post: PO Box 1105 Blindern, 0317 Oslo, NorwayVisit: Domus Medica, Room 1293, Sognsvannsveien 9, Oslo, NorwayTelephone: +47 22851496 / Mobile telephone: +47 97032049Email: l.h.bergersen@medisin.uio.no

Current SN-Lab Members: Brain and Muscle EnergyLinda Hildegard Bergersen (Head)Johanne Egge Rinholm (Postdoc)Knut Husø Lauritzen (Postdoc)Fredrik Lauritzen (PhD student)Tirill Medin (PhD student)Christine E Regnell (PhD student)Marita Brandsar Hefte (Master student)

Glio- and NeurotransmittersVidar Gundersen (Head) Maja Amedjkouh Puchades (Postdoc)Lasse Ormel (PhD student)Cecilie Morland (PhD student)Kaja Nordengen (Medical research student)Mats Julius Stensrud (Medical research student)Carl Johan Sogn (Medical research student)

Funding: Work in the lab is currently funded by the Research Council of Norway (RCN)’s Centre of Excellence grant to the Centre of Molecular Biology and Neuroscience (CMBN), an RCN researcher grant to LHB, an Oslo University Hospital researcher grant to VG, an RCN postdoctoral grant to JHR, two UiO PhD student grants to LHB, a UiO PhD student grant to VG, a Nasjonalforeningen for folkehelsen (Norwegian National Association for Public Health) first PhD grant in dementia research to CR/LHB, three medical research student grants to VG, a joint RCN/UiO researcher grant to LHB/JSM.

Jon Storm-Mathisen (Emeritus Professor)

Photo: Elmer Laahne, for Nasjonalt medisinsk museum/Norsk teknisk museum

Overview

The function of both brain and muscle depends crucially on their energy supply. The brain uses most of its energy on electrical signalling, along and between neurons. Brain neurons contact each other at synapses. A synapse consists of a presynaptic terminal and a postsynaptic dendrite, separated by a 20 nm wide synaptic cleft, into which the neurotransmitter (eg, glutamate and GABA) is released from presynaptic vesicle stores to activate receptor proteins at outward facing binding sites. Glial cells, the astrocytes, tightly surround the synapse. Information transmission at synapses is based on ion fluxes through the postsynaptic cell membrane. The ions that enter or leave the cell must later be pumped out or in again, consuming energy.

Linda Hildegard Bergersen obtained her PhD at the University of Oslo in 2001 under the supervision of Ole Petter Ottersen. After a postdoc period in Lausanne with Pierre Magistretti and Luc Pellerin in 2003, she worked with Jon Storm-Mathisen and has now established her own group, in Oslo and Copenhagen.

Her joint positions at the Center for Healthy Aging (CEHA) University of Copenhagen and the CMBN, University of Oslo, provide optimized gain from her combined expertise in molecular biology and neuroscience.

Electron microscopic immunogold quantification

Our main research tool is quantitative immunogold electron microscopy (Bergersen et al. 2008 Nature Protoc) to decipher the microstructural localization of molecules, such as transporters and receptors that mediate ion movements and synaptic and extrasynaptic signalling, terminate signals, and provide energy to the cells.

Fig 1. Electron micrographs showing the principles of the immunogold quantification. For details of procedure and figure, see Protocol (Bergersen LH, Storm-Mathisen J, Gundersen V (2007) Nature Protocols). Link to Short legend

Our current research focuses on the following projects(Only select references to own work are cited, see below for extensive publication list linked from PubMed)

Lactate transporters and roles of lactate

Energy. Lactate is an important metabolic intermediate in all cells, including neurons in the brain. Although glucose is the major energy substrate for the central nervous system, lactate is produced within the brain itself and this lactate may be used by brain cells to produce energy. Unresolved questions are to what extent lactate is a required energy substrate for brain cells and which role lactate plays in disease states (Review: Bergersen 2007 Neuroscience).

Cellular differences. According to a current idea lactate is produced primarily in astrocytes, from which it is exported for uptake and oxidation in neurons. This lactate flux is thought to be coupled to glutamatergic transmission, with release of lactate from astrocytes being stimulated by uptake of glutamate. We provided early evidence for the localization of MCTs in the brain in line with this notion (Bergersen 1999 Neuroscience; 2001 Exp Brain Res; 2002 Neurochem Res; 2003 Neuroscience). We found that MCT2 is located at fast acting glutamatergic synapses and that MCT4 is in the surrounding astroglia, compatible with a flux of lactate from astrocytes to metabolically active neurons. On the other hand, MCT4 localization in the perivascular glial endfeet and MCT1 in endothelial cells provide means for exchange of lactate between blood and brain and rationalize the finding that lactate exits from the brain only at rest and is taken up from the bloodstream when blood lactate rises, such as during exercise.

Like lactate produced in fast, glycolytic muscle cell is oxidized in enduring, oxidative muscle cells, lactate is produced and oxidized in different components of brain tissue, and indeed of tumours, and excess lactate produced in muscle can be oxidized in heart and brain.

Activity dependence. We have taken advantage of the fact that brain and the experimentally more easily accessible skeletal muscle share features of lactate production and consumption: we compare the localization of MCTs in brain and muscle to study how lactate and other monocarboxylates can influence energy supply under physiological and pathological conditions. Thus we have shown, by cross-reinnervation experiments of slow oxidative and fast glycolytic muscle, that the type and quantity of MCT expressed, is governed by the nerve, like is the contraction speed (Bergersen et al. 2006 Neuroscience). The idea that the activity pattern is a general regulator of MCT expression gains support from our observation that MCT2 is concentrated at fast acting synapses in the brain (Bergersen et al. 2005 Cereb Cortex).

Disease states. In experimental heart failure in rat we observed a dramatic upregulation of MCT1 in the cardiomyocytes, concomitant with increased lactate uptake for oxidation and presumably alleviation of energy deficit (Jóhannsson E et al. 2001 Circulation). Energy deficit has been invoked as contributing to the pathogenetic mechanisms of a variety of brain diseases, including epilepsy. We have recently shown that in human mesial temporal lobe epilepsy as well as in several rat models of the disease, MCT1 is downregulated in the vascular endothelium and upregulated in the astrocytic network in the epileptogenic hippocampus (Lauritzen F et al. 2011 Neurobiol Dis; 2012 Neurobiol Dis).

Fig 2. Electron microscopic immunogold localization and quantification of MCT1 in hippocampal blood vessel endothelium in human surgical specimens from patients with mesial temporal lobe epilepsy (MTLE) and non-MTLE. Less gold particle labeling for MCT1 is found on CA1 microvessel plasma membranes in MTLE, compared to non-MTLE. The reduced particle density in MTLE is evident both at abluminal (arrows in A, B) and luminal (arrows in C, D) plasma membranes. Abbreviations: bl, basal lamina; ec, endothelial cell; lu, blood vessel lumen; nu, nucleus; p, pericyte. The bar diagrams depict mean±SE, *Pb0.05. Scale bar: 500 nm. From Lauritzen F, de Lanerolle NC, Lee TS, Spencer DD, Kim JH, Bergersen LH, Eid T (2011) Monocarboxylate transporter 1 is deficient on microvessels in the human epileptogenic hippocampus. Neurobiol Dis 41(2):577-84

Myelination. In addition to regulatory roles and being an energy fuel, lactate may serve as a precursor of materials needed to build components of the brain. Thus lactate can be converted to lipid building blocks of myelin, the insulating sheaths that protect axons and boost their conduction velocity, essential for efficient communication between widely separated brain regions. We recently showed that lactate, taken up into oligodendrocytes through MCT1, is required to sustain myelin formation. In CNS diseases involving energy deprivation at times of myelination or remyelination, such as periventricular leukomalacia leading to cerebral palsy, stroke, and secondary ischemia after spinal cord injury, lactate transporters in oligodendrocytes may play an important role in minimizing the inhibition of myelination that occurs (Rinholm et al. 2011 J Neurosci).

Neurotransmitter receptor actions on neurons and glia

Glutamate-mediated damage to oligodendrocytes contributes to mental or physical impairment in periventricular leukomalacia (pre- or perinatal white matter injury leading to cerebral palsy), spinal cord injury, multiple sclerosis and stroke. Unlike neurons, white matter oligodendrocytes reportedly lack NMDA (N-methyl-D-aspartate) type glutamate receptors. In an interdisciplinary study with David Attwell, University College London, we showed that precursor, immature and mature oligodendrocytes in the white matter of the cerebellum and corpus callosum exhibit NMDA-evoked currents, mediated by receptors that are blocked only weakly by Mg2+ and that may contain NR1, NR2C and NR3 NMDA receptor subunits. NMDA receptors are present in the myelinating processes of oligodendrocytes, where the small intracellular space could lead to a large rise in intracellular ion concentration in response to NMDA receptor activation. Simulating ischaemia led to development of an inward current in oligodendrocytes, which was partly mediated by NMDA receptors. These results point to NMDA receptors of unusual subunit composition as a potential therapeutic target for preventing white matter damage in a variety of diseases (Káradóttir et al. 2005 Nature).

GABA actions in myelin. GABAA receptor subunits are expressed throughout compact myelin in vivo, at a density comparable to that at inhibitory synapses. GABA release might contribute to oligodendrocyte depolarization during energy deprivation (Attwell et al. In preparation).

Attention-deficit/hyperactivity disorder (ADHD) is the most common neurobehavioural disorder among children. ADHD children are hyperactive, impulsive and have problems with sustained attention. These cardinal features are also present in the best validated animal model of ADHD, the spontaneously hypertensive rat (SHR), which is derived from the Wistar Kyoto rat (WKY) (Sagvolden et al. 2009 Neuropharmacology). Current theories of ADHD relate symptom development to factors that alter learning. N-methyl-D-aspartate receptor (NMDAR) dependent long term changes in synaptic efficacy in the mammalian CNS are thought to represent underlying cellular mechanisms for some forms of learning. We therefore hypothesized that synaptic abnormality in excitatory, glutamatergic synaptic transmission might contribute to the altered behavior in SHRs. In interdisciplinary studies with the groups of Terje Sagvolden (deceased 2011) and Øyvind Hvalby, Dept of Physiology, we examined physiological and anatomical aspects of hippocampal CA3-to-CA1 synapses in age-matched SHR and WKY (controls). The results indicate that functionally altered NMDA-type glutamate receptors in with a subunit composition characteristic of early developmental stages causes functional impairments in glutamatergic synaptic transmission in SHR. This may be one of the underlying mechanisms leading to the abnormal behavior in SHR, and possibly in human ADHD (Jensen V, Rinholm et al. 2009 Neuroscience).

Transmitter release from astrocytes

There is growing evidence that astrocytes, like neurons, can release transmitters.

Glutamate has been shown to be released from astrocytes in a vast number of studies. Although astrocytic glutamate may be released by several mechanisms, the evidence in favour of exocytosis is most compelling. Astrocytes may respond to neuronal activity by such exocytotic release of glutamate. The astrocyte derived glutamate can in turn activate neuronal glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors. Morphological and functional data show that astrocytes possess the machinery for exocytosis of glutamate and contribute to the control of synaptic strength (Jourdain, Bergersen et al. 2007 Nature Neurosci). We describe the presence of

small synaptic-like microvesicles, SNARE proteins and vesicular glutamate transporters (VGLUTs) in astrocytes, as well as NMDA receptors (Fig 3) situated in the vicinity of the astrocytic vesicles (Bergersen & Gundersen 2009 Neuroscience). The content of VGLUT harbouring synaptic-like microvesicles appears to be a property of astrocytes in multiple brain regions (Ormel et al. 2012 Glia).

D-serine is present in brain in substantial quantities and this unusual amino acid is a ligand of the glycine binding site of the NMDA receptor, which must be occupied for the NMDA receptor to be activated by glutamate. We have shown that D-serine, like glutamate, is concentrated in clusters of small synaptic-like microvesicles forming microdomains with endoplasmic reticulum cisterns in the perisynaptic processes of astrocytes (Fig 4). The results indicate that D-serine may be co-released with glutamate to cause the astrocyte dependent activation of NMDA receptors at synapses (Bergersen et al. 2011 Cereb Cortex).

Fig 3. Glutamate from astrocytes stimulates nerve endings in hippocampus. Electron micrograph showing NMDA type glutamate receptor subunits NR2B (immunogold particles) at the synapse as well as in extrasynaptic membranes (arrows) of nerve terminals (ter) that synapse on dendritic spines (sp). NR2Bs face astrocytic processes (ast) containing glutamate laden synaptic-like microvesicles (arrowheads in enlarged inset). Scale bars, 100 nm. From: Jourdain P, Bergersen LH, Bhaukaurally K, Bezzi P, Santello M, Domercq M, Matute C, Tonello F, Gundersen V, Volterra A (2007) Glutamate exocytosis from astrocytes controls synaptic strength. Nature Neuroscience 10(3):331-9

Neurotransmitter transporters

The vertebrate neuromuscular junction (NMJ) is known to be a cholinergic synapse at which acetylcholine (ACh) is released from the presynaptic terminal to act on postsynaptic nicotinic ACh receptors. There is now growing evidence that glutamate, which is the main excitatory transmitter in the CNS and at invertebrate NMJs, may have a signalling function together with ACh also at the vertebrate NMJ. In the CNS, the extracellular concentration of glutamate is kept at a subtoxic level by Na+-driven high-affinity glutamate transporters such as GLT and GLAST located in plasma membranes of astrocytes and neurons. The glutamate transporters are also pivotal for shaping glutamate receptor responses at synapses. Analyses in rat showed that GLAST and GLT are enriched in the junctional folds of the postsynaptic membrane at the NMJ. GLT was relatively higher in the slow-twitch muscle soleus than in the fast-twitch muscle extensor digitorum longus, whereas GLAST was relatively higher in extensor digitorum longus than in soleus. The findings show - together with previous demonstration of vesicular glutamate, a vesicular glutamate transporter and glutamate receptors - that mammalian NMJs contain the machinery required for synaptic release and action of glutamate. This indicates a signalling role for glutamate at the normal NMJ and provides a basis for the ability of denervated muscle to be reinnervated by glutamatergic axons from the CNS (Rinholm JE et al. 2007 Neuroscience).

Mitochondria

Mitochondria trafficking. Energy use, mainly to reverse ion movements in neurons, is a fundamental constraint on brain information processing. Because nerve cells are so large, they need special

Fig 4. An astrocyte process (Ast) has synaptic-like microvesicles (red arrowheads) containing D-serine (small particles); the Ast is identified as having glutamate transporter EAAT2 in its plasma membrane (large particles) and is adjacent to a nerve ending (Term) synapsing on a spine (Sp). From: Bergersen LH, Morland C, Ormel L, Rinholm JE, Larsson M, Wold JF, Røe AT, Stranna A, Santello M, Bouvier D, Ottersen OP, Volterra A, Gundersen V. Immunogold detection of L-glutamate and D-serine in small synaptic-like microvesicles in adult hippocampal astrocytes. Cereb Cortex. 2011 Sep 12. [Epub ahead of print; ISI Impact Factor 6.8]

mechanisms to locate mitochondria at locations where the energy is needed. Synapses are sites of particularly large energy consumption. Mitochondria can be moved around cells by kinesin motors on intracellular microtubule tracks (for a movie visit http://www.ucl.ac.uk/npp/jk.html and look at the bottom of the page). The Group member, Johanne Egge Rinholm (then PhD student of LHB), collaborated with the groups of David Attwell and Josef Kittler at the University College London discovering that the linkage of mitochondria to these motors is mediated by a protein called Miro. Importantly, this linkage is uncoupled by calcium ions, which enter through NMDA receptors when synapses are activated. The resulting uncoupling of mitochondria from their motors leads to them being "parked" near the active synapses where they are needed to generate energy. This work advances our understanding of how neurons are powered. It also explains why in pathological conditions like stroke, which dramatically raise the internal calcium concentration in nerve cells, all movements of mitochondria within the cell halt (Macaskill AF, Rinholm JE et al. 2009 Neuron).

Mitochondria in myelin? JE Rinholm, now a Postdoc in the Group (RCN fellowship 2012-14 as one of four awarded among 64 applicants), pursues this line of research along with the complementary one on lactate and myelination (see above: Rinholm et al. 2011 J Neurosci) to solve the puzzle of energy supply in myelin and myelinated axons. For this we collaborate with the labs of David A Clayton and Eric Betzig (Howard Hughes Medical Institute, Janelia Farm, Ashburn, Virginia), where Rinholm will use newly developed super-resolution imaging technique called structured illumination microscopy (SIM) to test her hypothesis that mitochondria are located in cytoplasmic loops in myelin, as suggested by her preliminary observations with confocal imaging (Rinholm JE et al. in preparation).

Mitochondrial dysfunction. Mitochondrial dysfunction is considered to underly changes in neurodegenerative diseases and is often associated with apoptosis and a progressive loss of neurons. Such pathologies are proposed to be caused by damage to the mitochondrial genome. In a current collaborative study with Arne Klungland, Rikshospitalet, we have designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in forebrain structures. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis, neuronal death and progressive atrophy in the hippocampus leading to decreased synaptic efficiency associated with a reduction in the size of the postsynaptic membrane (ie overlying the postsynaptic density, PSD) on dendritic spines in hippocampal CA1 pyramidal cells. The mice subsequently show abnormal behaviour, including increased activity, reduced cognitive abilities and lack of anxiety responses (Lauritzen KH et al. 2010 Mol Cell Biol). Further studies suggested that the accumulating pathology was caused by reduced mtDNA copy number and transcription, increased oxidative stress, and disturbed mitochondrial trafficking, thus mimicking changes observed in neurodegenerative diseases (Lauritzen KH et al. 2011 DNA Repair (Amst)).

Impact of gene repair on brain structure and function

Accumulation of oxidative DNA damage, not only in mtDNA but also in nuclear DNA, has been proposed as a potential cause of age-related cognitive decline. The major pathway for removal of oxidative base lesions is base excision repair, which is initiated by DNA glycosylases. In mice, Neil3 is the main DNA glycosylase for repair of hydantoin lesions in single stranded DNA of neural stem/progenitor cells (NSPC), that sustain neurogenesis. Adult neurogenesis is considered crucial for maintenance of hippocampus dependent functions involved in normal behavior. In Niel3-deficient mice, we demonstrated learning and memory deficits and reduced anxiety-like behavior. NSPCs from aged Neil3-/- mice showed impaired proliferative capacity. Further, hippocampal neurons in aged Neil3-/- mice displayed synaptic irregularities, comprising reduced synaptic membrane size, and selective increase (NR1, NR2A/B, GABAAα1) or reduction (GluR2/3) of transmitter receptor subunits. The results indicate that repair by Neil3 of oxidative DNA damage in NSPCs is required for the maintenance of adult neurogenesis to counteract age associated synaptic changes and deterioration of cognitive performance (Regnell et al. 2012 Submitted).

CollaborationsWe currently have active collaborations including the labs of:

David Attwell http://www.ucl.ac.uk/npp/research/da Magnar Bjørås http://www.cmbn.no/group-bjoras.html & http://www.ous-research.no/bjoras/ David A. Clayton http://www.hhmi.org/research/groupleaders/clayton_bio.html Tore Eid http://labmed.yale.edu/people/tore_eid.profile Albert Gjedde http://forskning.ku.dk/search/profil/?id=52543 Øyvind Hvalby http://www.med.uio.no/imb/english/people/aca/oivind/index.html Arne Klungland http://www.cmbn.no/group-klungland.html & http://www.ous-research.no/klungland/ Erik Pettersen http://www.mn.uio.no/fysikk/english/people/aca/erikop/index.html Lene Juul Rasmussen http://icmm.ku.dk/english/icmm-staff/lene_rasmussen/ Andrea Volterra http://www.unil.ch/fbm/page28867_en.html

Publications listed in PubMedClick here Display Settings, Items per page: Select 50

http:// www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&term=bergersen-lh[auth]|bergersen-h[auth]%20AND %20Blindern[Affiliation]|bergersen-l[auth]%20NOT%20(bergersen-lc[auth]%20OR%20bergersen-lj[auth]%20OR%20Boston%20[Affiliation]%20OR%20Lebanon%20[Affiliation] %20OR%20Vermont%20[Affiliation]%20OR%20Columbus%20[Affiliation])

CitationsH-index 17 (Thomson Reuters ISI Web of Science (8th February 2012), Author=(Bergersen L*) minus 4 extraneous Bergersen L) Twelve of my publications have been cited 30 times or more.

Updated 8th February 2012 by lindabe