Characterization of canine dorsal root ganglion neurons and growth promoting effects of GM1-gangliosides

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ISBN 978-3-86345-225-4

Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH 35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375

E-Mail: info@dvg.de · Internet: www.dvg.de Y imin W ang Hannover 2014

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Bibliografische Informationen der Deutschen Bibliothek Die Deutsche Bibliothek verzeichnet diese Publikation in der

Deutschen Nationalbibliografie;

Detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar.

1. Auflage 2014

Printed in Germany

ISBN 978-3-86345-225-4

Verlag: DVG Service GmbH Friedrichstraße 17

35392 Gießen 0641/24466 info@dvg.de www.dvg.de

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University of Veterinary Medicine Hannover Department of Pathology

Center for Systems Neuroscience Hannover

Characterization of canine dorsal root ganglion neurons and growth promoting effects of GM

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-gangliosides

THESIS

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD)

awarded by the University of Veterinary Medicine Hannover by

Yimin Wang Born in Jilin province/China

Hannover, Germany 2014

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Supervisor: Prof. Dr. Wolfgang Baumgärtner

Supervision Group: Prof. Dr. Wolfgang Baumgärtner Prof. Dr. Peter Claus

Prof. Dr. Herbert Hildebrandt

1^st Evaluation: Prof. Dr. Wolfgang Baumgärtner

Department of Pathology, University of Veterinary Medicine Hannover, Germany

Prof. Dr. Peter Claus

Department of Neuroanatomy, Medical School Hannover, Germany

Prof. Dr. Herbert Hildebrandt

Institute for Cellular Chemistry, Medical School Hannover, Germany

2^ndEvaluation Prof. Dr. Christiane Herden

Institute of Veterinary Pathology, Justus-Liebig-University, Giessen, Germany

Date of final exam 10^th October 2014

Yimin Wang has received financial support from the China Scholarship Council (CSC) under the file No. 2010617011

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To my family

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1. Aims of study

Dorsal root ganglia (DRGs) transmit sensory information from limbs and trunk to the central nervous system (CNS). They have been extensively used in fundamental neuroscience, pain, virus, and prion protein research, because cell cultures of DRG neurons and satellite cells represent a relevant model in neuroscience. Recent studies demonstrated that species-specific properties of human glia are closer related to dogs than rodents emphasizing the use of dogs as a translational animal model in research (OMAR et al., 2011; TECHANGAMSUWAN et al., 2008). However, detailed background knowledge about the structure of canine DRGs and the in vitro compared to the in vivo expression of different structural and functional proteins by resident cells is lacking. Therefore, the first aim of the present study was to describe light microscopical, ultrastructural, and immunohistochemical features of canine DRG neurons and satellite glial cells (SGCs) both in vivo and in vitro.

GM1-ganglioside is the prototype of different series of gangliosides that contains one sialic acid residue. Gangliosides are predominantly localized within the plasma membranes of neuronal processes and account for up to 10% of the total lipid content of neurons, which support their crucial role in the nervous system (LEDEEN, 1978).

Interestingly, GM1 has also been used in clinical trials to treat neurodegenerative diseases like Alzheimer’s (SVENNERHOLM et al., 2002) and Parkinson’s disease (SCHNEIDER et al., 2010). These studies demonstrated that GM1 treatment strongly inhibited the progression of these two important human CNS diseases and did not produce any significant changes in blood chemistry, hematologic indices, or urine chemistry. However, the molecular mechanisms of these positive GM1 effects on CNS function have still to be elucidated. Exogenously added gangliosides may mimic endogenous gangliosides by binding to the cells and insertion into membranes or by adhering as micelles (MOCCHETTI, 2005; OHMI et al., 2012; WU 2011). Their multimodal neurotrophic effects might be a potential therapeutic tool for the treatment of several neurodegenerative diseases including spinal cord injury. Consequently, the second aim of the study was to characterize the effects of GM1 on DRG neurons and SGCs focusing at different cytoskeletal and functional proteins such as synaptophysin.

Moreover, the interaction of GM1 with different growth factors and their effect on

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neuronal survival as well as process outgrowth and branching was investigated in detail.

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2. Introduction

2.1 Morphology and function of dorsal root ganglia of dogs and other species 2.1.1 Localization and function

The dorsal root ganglion (DRG) or spinal ganglion is a nodule that is located along the vertebral column in the dorsal root outside of the spinal cord and consists of neuronal cell bodies of afferent nerve fibers (ANDRES, 1961). In addition to neurons a DRG contains different glial cells. DRGs are necessary for the transmission of sensory information from the limbs and trunk to the CNS. Unlike most neurons in the CNS, DRG neurons have a pseudo-unipolar structure, which is characterized by one axon with a central and a peripheral branch and the absence of dendrites.

2.1.2 Dorsal root ganglion neurons

DRG neurons from rats have been classified for a long time on the basis of morphology and size (ANDRES, 1961). DUCE and KEEN (1977) examined normal DRG neurons under the light microscope and separated them into two main subgroups: large light neurons (L or type A) and small dark neurons (SD or type B) (DUCE and KEEN, 1977). Neurofilament immunoreactivity was used to distinguish between the two cell types in rat DRGs, large light cells contain numerous neurofilament proteins in their cell bodies, small dark cells contain very few (SHARP et al., 1982).

In addition, rat DRG neurons were also divided based on the size and the distribution of their organelles into three groups named A, B, and C (RAMBOURG et al., 1983).

Type A cells were large neurons (40-75 µm in diameter) containing numerous small, block-like Nissl bodies uniformly distributed throughout the perikaryon except for the area of the axon hillock. Numerous small Golgi bodies and rod-like mitochondria were interspersed in the cytoplasm separating the Nissl bodies. The type B cells were smaller neurons (20-50 µm in diameter), which showed a characteristic zonation of their organelles. The Nissl substances formed a continuous web which completely filled the outer third of the perikaryon. The Golgi apparatus appeared as long and thick crescentic body arranged in a ring-like fashion in the mid-zone of the perikaryon. In contrast, mitochondria tended to accumulate next to and around the nucleus. The

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type C cells were the smallest of the ganglion cells (less than 20 µm diameter). Their perikaryon contained small, often poorly individualized Nissl bodies and dispersed mitochondria. The Golgi apparatus formed a perinuclear network in close contact with the nucleus (RAMBOURG et al., 1983).

Based on the overall distribution and three dimensional configuration of their ER, Golgi apparatus, mitochondria, and Nissl bodies, rat DRG neurons were further subdivided into 6 groups: A1, A2, A3, B1, B2, B3 (or C) (DUCE and KEEN, 1977;

RAMBOURG et al., 1983). The function of these different neurons was partly elucidated during the research of nociception, which prompted a subclassification according to their histochemical reaction pattern and capsaicin-, proton-, and ATP-activated currents (PETRUSKA et al., 2000). Small- and medium-diameter DRG neurons can be subclassified into at least nine distinct cell types based upon their patterns of voltage activated currents (PETRUSKA et al., 2000).

2.1.3 Dorsal root ganglion satellite glial cells

Satellite glial cells (SGCs) surround neuronal cell bodies within dorsal root ganglia of the peripheral nervous system (PNS). Similar to Schwann cells, they are derived from the neural crest of the embryo during development (HALL and LANDIS, 1992;

NELISSEN et al., 2009). They share many characteristics with CNS glia and may participate in the neuronal signaling process and transmission (HANANI, 2005).

SGCs have a variety of functions: they seem to have a similar role as astrocytes in the CNS including the protection and supply of nutrients to the surrounding neurons (HANANI, 2005) and the maintenance of homeostasis (NEWMAN, 2003). SGCs are important for the development of the nervous system and for the formation of synaptic contacts (GOLDMAN, 2003). Interestingly, the presence of SGCs in cultures of neonatal rat sensory neurons from nodose ganglia (of the vagus nerve) inhibit the extension of dendrites (DE KONINCK et al., 1993).

Modern research has clearly demonstrated that the nervous system constantly adapts to internal and external stimuli including growth, aging, environmental influences, or injury and disease. SGCs similar to CNS glia cells likely play a critical role in those processes. It has been demonstrated that SGCs proliferate after axotomy or inflammation and exert monocytic functions such as the phagocytosis of cell debris

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(ALDSKOGIUS and ARVIDSSON, 1978; LU and RICHARDSON, 1991). SGCs might also form a barrier to the spread of viruses like herpes simplex between infected and non-infected neurons and thereby inhibit a widespread infection of the ganglia (STEINER, 1996; LAVAIL et al., 1997).

2.1.4 Nociception

The DRG is pivotal in relaying somatosensory information from afferent sensory nerve fibers to the spinal cord and ultimately sensory cortex of the brain in the parietal cortex.

Nociception is based on the expression of pain receptors such as opioid receptors, which are synthesized in DRG neurons and transported toward the central terminal in the dorsal horn and the periphery (HUDSPITH et al., 2006). Abrupt opioid withdrawal may induce long-term potentiation in nociceptive spinal pathways leading to pain amplification in acute and chronic pain states (RUSCHEWEYH et al., 2011). Reduced inhibitory transmission in the spinal cord may contribute to hyperalgesia and allodynia in chronic pain (HUDSPITH et al., 2006). In addition, several studies demonstrated that DRGs are a source of ectopic afferent pain producing impulses in the aftermath of peripheral nerve lesions (DESANTIS and DUCKWORTH, 1982; WALL and DEVOR, 1983).

2.1.5 Dorsal root ganglion differences between species

DRGs have been investigated in various species including fish, amphibians, reptiles, birds, and mammals (PANNESE et al., 1972; PANNESE et al., 1975; ALVAREZ et al., 1991; LEDDA et al., 2004; MATSUDA et al., 2005). However, approximately 50% and 20% of all studies have been performed in rats and mice, respectively, mainly due to technical and economic reasons. Consequently, background knowledge about DRG development, morphology, and function is predominantly based on findings in rodents and generalized to other species. A comparative light microscopic study of trigeminal ganglion neurons demonstrated similar cellular constituents (neurons and glia) and features of most of the neurons in investigated rats, rabbits, and goats (DILKASH et al., 2010). Similarly, the clustering of nerve cell bodies within a common connective tissue envelope was found in spinal ganglia of lizards and rats (PANNESE and LEDDA, 1991). The coupling of SGCs by gap junctions was observed in DRGs of

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mice, rats, and guinea pigs (HUANG et al., 2005). In addition, only minimal species-specific differences in neuropeptide expression patterns have been found in DRGs of different mammals including horse and pigs (MERIGHI et al., 1990).

Nevertheless, mice demonstrate species-specific morphological features in a high percentage of DRG neurons including the occurrence of binucleolate neurons (KHAN and DILKASH, 2011). The neuronal somatic size appears to have a direct relationship with the body size of the animal (DILKASH et al., 2010). A larger mean soma size of DRG neurons was found in cats compared to rats maybe reflecting different metabolic rates (ISHIHARA et al., 1996). Size-dependent differences of neurons in metabolic needs are also reflected in the mean volume of a nerve cell body corresponding to surrounding SGCs (higher in large compared to small neurons), since SGCs metabolically support spinal ganglion neurons (LEDDA et al., 2004). Significant smaller SGC numbers in lizards and geckos compared to mice, rats, and rabbits can also be explained by the lower metabolic rate in the nervous system of poikilotherms compared to mammals and could also have a phylogenetic significance (LEDDA et al., 2004). Interestingly, the phylogenetic development of DRGs is reflected by the morphology of sensory neurons, since DRG neurons of fish are bipolar in contrast to the pseudounipolar type of neurons found in amphibians, birds, and mammals (MATSUDA et al., 2005). The speed of unipolarization during DRG development also varies between different homeotherm species such as chicken and rats, which might affect the design and analysis of experiments on dorsal root ganglion neurons grown in tissue culture (MATSUDA et al., 1996). Moreover, there seems to be a progressive specialization of receptors such as delta opioid receptors on nociceptive neurons in DRGs during phylogeny from rodents to primates (MENNICKEN et al., 2003). In addition, peripheral axotomy induces a more pronounced decrease of μ-opioid receptor expression in DRG neurons of monkeys compared to rats (ZHANG et al., 1998). One study demonstrated that mammalian DRGs including horse, buffalo, cow, sheep, pig, dog, and rat vary in the percentage and size of neurons expressing neurocalcin, which is a calcium-binding protein present in a distinct subpopulation of sensory neurons and their peripheral mechanoreceptors (GALEANO et al., 2000).

There is also an inter-species variation in the distribution of binding sites for the plant lectin Ulex europaeus agglutinin I on primary sensory neurons between seven

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mammalian species such as rats, mice, guinea pigs, rabbits, flying foxes, cats, and marmoset monkeys, whereas these species show similarities in their binding patterns for Bandeiraea simplicifolia I-isolectin B4 (GERKE and PLENDERLEITH, 2002a, b).

In conclusion, various studies underline the necessity to analyze species-specific properties of DRG morphology and function, which provides the basis for a correct interpretation of data obtained from various animal disease conditions. Furthermore, the described differences in receptor expression of DRG neurons suggest that pharmacological effects of drugs in rodents are not entirely predictive of their action in humans (ROSSBACH and BÄUMER, 2014; ZHANG et al., 1998; MENNICKEN et al., 2003). Similar problems in the extrapolation of experimental rodent data have been described for several important human CNS diseases including multiple sclerosis (MS), epilepsy, and spinal cord injury (BOCK et al., 2013). Consequently, the use of dogs as large translational animal models might at least partly bridge the gap between highly experimental studies and heterogeneous clinical human CNS diseases (BOCK et al., 2013). The development, morphology, and function of canine DRGs have been investigated under various physiological and pathological conditions. For instance, the distribution of different neuropeptides, neuronal markers, and synaptic-vesicle-associated proteins was analyzed in DRGs of adult and newborn dogs (BONFANTI et al., 1991). The morphological and immunohistochemical changes in DRGs of mongrel dogs were described after mechanical compression of lumbar nerve roots (KOBAYASHI et al., 2004; KOBAYASHI et al., 2005). Aortic occlusion was shown to induce the vacuolization and hypertrophy of satellite cells within 1-2 days (FERCAKOVA and MARSALA, 1983). A cauda equine syndrome dog model indicated that brain-derived neurotrophic factor might play a role in the inflammatory and neuropathic pain (TAN et al., 2013). Canine bone marrow stromal cells were demonstrated to promote the neurite outgrowth from DRG neurons, which might be the basis to establish novel therapeutic strategies for several neurological disorders (KAMISHINA et al., 2009).

2.1.6 Gangliosides

GM1 (monosialotetra-hexosylaganglioside) is the prototype of different series of gangliosides and contains one sialic acid (N-acetylneuraminic acid) residue.

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Gangliosides were first identified in the 1930s. Their name is derived from the association of these molecules with the ganglion cells in the brain gray matter (SVENNERHOLM, 1956). Gangliosides are predominantly localized with the plasma membranes of neuronal processes and account for up to 10% of the total lipid content of neurons, which support their crucial role in nervous system (LEDEEN, 1978).

Several in vitro studies demonstrated the dose-dependent effects of gangliosides on neuronal sprouting and neurite extension and arborization (LESKAWA and HOGAN, 1985). The growth-promoting and other neurotrophic effects of gangliosides are mediated by the activation of Trk receptors and the downstream Raf/MEK/ERK cascade and PI3-kinase/Akt pathway thereby demonstrating functional similarities to neurotrophins (FERRARI et al., 1995; RABIN and MOCCHETTI, 1995; DUCHEMIN et al., 2002; DUCHEMIN et al., 2008). Indeed, recent studies substantiated that exogenous gangliosides activate Trk receptors by inducing the release of neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) (RABIN et al., 2002; MOCCHETTI, 2005; LIM et al., 2011). The activation of the Raf/MEK/ERK cascade, which is implicated in complex cellular responses such as differentiation, survival, synaptic plasticity, long-term potentiation, and learning and memory, might explain the diverse actions of the ganglioside on immature and mature neurons (GREWAL et al., 1999; SWEATT, 2001).

The optimal responses to GM1 in tyrosine phosphorylation of known TrkA target proteins were found at concentration ranging from 80 to 100µM (RABIN and MOCCHETTI, 1995). Higher doses of GM1 caused negative effects by inducing a depletion of ER calcium stores and an activation of an unfolded protein response. This lead to an upregulation of the chaperone “Binding immunoglobulin protein” (BiP) and the transcription factor “C/EBP-homologous transcription factor” (CHOP), the activation of “c-Jun N-terminal kinases” (JNK) 2 and caspase-12, and finally neuronal apoptosis (TESSITORE et al., 2004). In addition, electron microscopic studies of cortical neurons in GM2-gangliosidosis demonstrated the formation of large neural processes that develop at storage sites of the accumulated undigestible substrates (PURPURA and SUZUKI, 1976). These meganeurites are found at the base of the perikaryon and the initial portion of the axon, frequently give rise to secondary neurites, and may possess spines, which are occasionally contacted by presynaptic

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processes. These meganeurites including their synapses might contribute to neuronal dysfunction in storage diseases by changing the electrical properties of neurons.

Gangliosides can rescue isolated neuronal cells from death in several conditions including neurotrophic factor deprivation and ethanol-induced, glutamate, and kainate neurotoxicity (FAVARON et al., 1988; FERRARI et al., 1993; SAITO et al., 1999). The pleiotropic neurotrophic activity of GM1 on cholinergic, dopaminergic, serotoninergic and noradrenergic neurons were confirmed in lesioned animal brains, which raised hope to use them as therapeutic agents for neurological diseases (CECCARELLI et al., 1976; ODERFELD-NOWAK et al., 1984; HADJICONSTANTINOU and NEFF, 1998). In addition, GM1-ganglioside increased the choline acetyltransferase activity in the brain of aged rats and improved their spatial learning and memory indicating that memory deficits associated with aging might be treatable with this sialoglycolipid (FONG et al., 1995a; FONG et al., 1995b; FONG et al., 1997). The trophic effects of exogenous gangliosides for the in vivo activity are dependent on the negative charge on the ganglioside molecule (CANNELLA et al., 1990). Clinical trials even demonstrated that GM1 might reduce neuronal damage after spinal cord injury (GEISLER, 1998). However, there are strong concerns about negative side effects of gangliosides especially the possible induction of the Guillain-Barré syndrome in patients (LANDI and CICCONE, 1992; SCHONHOFER, 1992). Consequently, further study is warranted and needed for a final evaluation of the positive and negative effects of ganglioside treatment.

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2.2 Structural components and functional proteins of dorsal root ganglion neurons and satellite glial cells

2.2.1 Structural components 2.2.1.1 Microtubules

The shape of neurons is determined by their cytoskeleton, which consists of a three-dimensional network of cross-linked microtubules and neurofilaments (HIROKAWA, 1982). Microtubules are composed of alpha- and beta-tubulin heterodimers representing globular proteins expressed in all eukaryotic cells (MOHRI, 1968). There are six major classes of beta-tubulin (TUBB) in mammals. Three classes of beta-tubulin, namely class I (TUBB1), class II (TUBB2) and class III (TUBB3) are strongly and specifically expressed in neurons (SULLIVAN et al., 1986;

SULLIVAN, 1988; JOSHI and CLEVELAND, 1989). In addition, TUBB3 is widely regarded as a neuronal marker in developmental neurobiology and stem cell research (DE DONATO et al., 2012).

Microtubules are involved in mitosis, cell motility, intracellular transport, secretion, cell shape, and polarization. They are modified through posttranslational detyrosination/tyrosination, generation of ∆2-tubulin (non-tyrosinatable variant of tubulin that lacks a carboxy-terminal glutamyl-tyrosine group on its α-subunit), glutamylation, glycylation, and acetylation influencing their stability and function (JANKE and KNEUSSEL, 2010; SONG et al., 2013). As microtubules are fundamental to the morphology of neurons, defects in tubulin genes are likely to cause neuronal diseases (JAGLIN and CHELLY, 2009; TISCHFIELD and ENGLE, 2010).

Because microtubules modulate the functions of a variety of proteins, such as molecular motors and microtubule-associated proteins (MAPs), it is speculated that beta-tubulin mutations induce a variety of signs. The malfunctions of tubulins in the nervous system may be involved in neurodegenerative diseases, such as Huntington’s disease and Parkinson’s disease (DOMPIERRE et al., 2007; OUTEIRO et al., 2007; SUZUKI and KOIKE, 2007) . Mutations in beta-tubulin genes have been found to cause three different classes of neuronal disease: polymicrogyria, congenital fibrosis of extraocular muscle type 3 (CFEOM3), and malformation of cortical development (MCD; JAGLIN et al., 2009; POIRIER et al., 2010). In addition to the

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Introduction specific signs of these conditions, the TUBB3 mutations, E410K, D417H and D417N (single amino acid changes shown in single letter code), induce very severe neurological signs, such as peripheral neuropathy and loss of axons in many kinds of brain neurons (JAGLIN et al., 2009; TISCHFIELD and ENGLE, 2010). The stability and organization of microtubules also influences regenerative and degenerative processes of axonal stumps (ERTURK et al., 2007).

Kinesin superfamily proteins (KIFs) are molecular motors that transport vesicular organelles along microtubule filaments in axons (VERHEY and HAMMOND, 2009;

TERADA et al., 2010). In 2004, a standard kinesin nomenclature was established for KIFs (LAWRENCE et al., 2004). Until now 15 kinesin families have been found, namely kinesin 1 to 14B, according to the results of phylogenetic analyses (MIKI et al., 2001). KIFs are broadly classified into three types depending on the position of the motor domain in the molecule: N-kinesins have a motor domain in the N-terminal region, M-kinesins have one in the middle, and C-kinesins have a motor domain in the C-terminal region (HIROKAWA et al., 2009). Kinesin 5 (KIF5) seems to represent a good axonal marker for developing neurons and binds preferentially to microtubules in the initial segment of the axon (NAKATA and HIROKAWA, 2003; JACOBSON et al., 2006). KIFs have been shown to transport organelles, protein complexes and mRNA to specific destinations along the microtubules while hydrolyzing ATP for energy (HIROKAWA, 1996). Intracellular transport is essential for physiological cellular morphology and function. In neurons, there are two types of transport in the axon: a fast transport of cargo vesicles (50-400mm/day) and a slow transport of soluble proteins (less than 8mm/day; HIROKAWA et al., 2010). KIFs also participate in chromosomal and spindle movements during mitosis and meiosis (VALE and FLETTERICK, 1997).

There are approximately 15 forms of dynein, which have been divided into two groups: cytoplasmic dyneins and axonemal dyneins (HOLZBAUR and VALLEE, 1994).

Only two forms are cytoplasmic dyneins namely microtubule-associated protein 1C (MAP1C; PASCHAL and VALLEE, 1987) and dynein heavy chain 1b (DHC1b;

PAZOUR et al., 1999). All forms of dyneins contain one or more heavy chains (HC) and a variety of accessory subunits: light chains (LC), light-intermediate (LIC) and intermediate chains (IC; HOLZBAUR and VALLEE, 1994). The HC motor unit is

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constructed around a series of “ATPase associated with cellular activities” (AAA) domains and is fundamentally distinct from kinesin at both the structural and mechanistic levels (KING, 2000). LC, LIC, IC are responsible for binding cargo.

The dyneins play a fundamental role in axonal transport and ciliary and flagellar movement (HOLZBAUR and VALLEE, 1994). They are responsible for the retrograde transport of a variety of cargos including golgi vesicles, peroxisomes, mitochondria, endosomes, lysosomes, transcription factors, aggregated protein, messenger ribonucleoproteins (mRNPs), and viruses (HARADA et al., 1998; JOHNSTON et al., 2002; HARRELL et al., 2004; VALLEE et al., 2004; KURAL et al., 2005; MACASKILL and KITTLER, 2010). They also participate in mitotic spindle orientation, centrosome centering, nuclear migration, and cell migration (SHEKHAR et al., 2013) and influence neuronal development through the retrograde transport of nerve growth factors (HIROKAWA and TAKEMURA, 2004). In addition, LC has been shown to bind to TrkA, TrkB, and TrkC neurotrophin receptors (YANO et al., 2001). Some studies indicated that dyneins are involved in the pathogenesis of many neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS; HIROKAWA and TAKEMURA, 2004;

BANKS and FISHER, 2008). Dyneins also play a role in the retrograde transport of viruses such as herpes simplex virus from nerve termini to the cell body (DIEFENBACH et al., 2008).

The microtubule-associated protein (MAP) family contains major cytoskeletal proteins in neurons, which are divided into two groups. Type I includes MAP1 and type II includes MAP2, MAP4, and Tau (MATUS, 1990). The three isoforms of MAP2 (MAP2a, MAP2b, MAP2c) are produced from a single gene by alternative splicing and post-translational modifications (GOEDERT et al., 1991). MAP2c is found during neuronal development and is later replaced by MAP2a. MAP2b is expressed both during development and adulthood (TUCKER et al., 1988; DEHMELT and HALPAIN, 2005). MAP2 is mainly expressed in neurons, but can also be detected in some non-neuronal cells such as oligodendrocytes, Schwann cells and satellite glial cells. It is involved in microtubule assembly and stability, regulates microtubule dynamics (GAMBLIN et al., 1996), takes part in cellular signaling (LIM and HALPAIN, 2000), and plays an important role in neuronal morphogenesis, neurite outgrowth, dendrite development (CACERES et al., 1992; MACCIONI and CAMBIAZO, 1995), cell

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Introduction division, and maintenance of cellular architecture (GUO et al., 2012). In the CNS, MAP-2 seems to be mainly expressed in neuronal cell bodies and dendrites and only rarely in axons (CACERES et al., 1984; BINDER et al., 1986). In cultured neurons MAP-2 is initially uniformly distributed within the cell but later accumulates in dendrites (CACERES et al., 1986; DOTTI et al., 1987). MAP2 can inhibit the kinesin and dynein dependent transport along microtubules (LOPEZ and SHEETZ, 1993; SEITZ et al., 2002). Interestingly, MAP2 is significantly reduced during several prion diseases including scrapie and Creutzfeldt-Jakob disease by calpain-mediated degradation leading to the disruption of the microtubule structure (GUO et al., 2012).

Tau belongs to the MAP family and is strongly expressed in the CNS and the PNS (TOLNAY and PROBST, 1999). It is a scaffolding protein involved in microtubule assembly and stabilization. Together with other MAPs Tau plays a fundamental role in the axoplasmic transport, particularly in axonal morphology, growth, and polarity (MANDELKOW and MANDELKOW, 1995). Tau proteins also play a role in the specialization of the adult peripheral nervous system (COUCHIE et al., 1992) and can be expressed in glial cells, but mainly in pathological conditions (CHIN and GOLDMAN, 1996). The tau gene is localized on chromosome 17 and contains 16 exons (ANDREADIS et al., 1992). Six tau isoforms differ in their number of binding domains resulting from an alternative splicing in exons 2, 3, and 10 of the tau gene.

Three isoforms have three or four tubulin binding domains, respectively. They are differentially expressed during the development executing different roles in physiological processes. The degree of tau phosphorylation is also developmentally regulated (SERGEANT et al., 2005; KOLAROVA et al., 2012). Aggregation of abnormally phosphorylated tau proteins in filamentous inclusions is a common feature of numerous neurodegenerative disorders, such as Alzheimer’s disease (AD), argyrophilic grain disease, Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration (BUEE et al., 2000). Consequently, Tau proteins have been defined as possible biomarkers of AD (KHACHATURIAN, 2002).

2.2.1.2 Intermediate filaments

Intermediate filaments (IFs) are a major component of the cellular cytoskeleton and provide a scaffold for animal cells. In contrast to others parts of the cellular

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cytoskeleton such as microfilaments (actin) and microtubules, whose components are highly evolutionarily conserved and very similar within cells of a particular species, IFs display much diversity in their numbers, sequences, and abundance (FUCHS and CLEVELAND, 1998). The family of IFs is divided into 5 major classes: the keratins form type I and II; Type III includes desmin, glial fibrillary acidic protein (GFAP), and vimentin; type IV includes neurofilaments (NFs), α-internexin and nestin; type V contains the nuclear lamins (ENG et al., 2000). Some studies indicate that the different IF proteins are specifically expressed in the various stages of neuronal maturation: whereas neuroepithelial stem cells express vimentin and nestin (LENDAHL et al., 1990), postmitotic neurons express α-intermexin and down-regulate vimentin and nestin (KAPLAN et al., 1990). The expression of neurofilament proteins begins during axon elongation and overlaps with vimentin expression for a short period of time (COCHARD and PAULIN, 1984; JULIEN et al., 1986).

GFAP was discovered in lipid studies of MS patients and subsequently purified from MS brain tissue (ENG et al., 1971; UYEDA et al., 1972; DAHL et al., 1989). GFAP is a specific marker for astrocytes (ENG, 1985; ENG et al., 2000). In the CNS, GFAP plays a very important role in cell communication including astrocyte-neuron interaction, participates in the formation and maintenance of the blood brain barrier, and has also been shown to be important in the repair after CNS injury (ENG et al., 2000). In the PNS, though some studies only detected GFAP in developing tissues, a GFAP expression has been described in Schwann cells, satellite cells, and other glial cells.

These inconsistent results might be caused by the use of monoclonal versus polyclonal anti-GFAP antibodies (ENG, 1985; ENG et al., 2000).

Vimentin is predominantly expressed in mesenchymal cells, maintains cell shape and the integrity of the cytoskeleton and cytoplasm (GOLDMAN et al., 1996), and plays an important role in the transport and metabolism of lipoprotein-derived cholesterol (SARRIA et al., 1992; SHEN et al., 2012a). In addition to mesenchymal cells, this cytoskeletal component can be found in less differentiated astrocytes (SEEHUSEN et al., 2007). Surprisingly, homozygous vimentin knock-out mice develop and reproduce without obvious phenotype (COLUCCI-GUYON et al., 1994). However, mice lacking vimentin display a decrease of adrenal and ovarian steroidogenesis (SHEN et al., 2012a).

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Introduction NFs are a major component of the neuronal cytoskeleton and mainly act as a molecular scaffold providing structural integrity in neuronal cells. It has been suggested that NFs determine the shape and architecture of axons in DRGs (HELFAND et al., 2004). Recently many diverse functions have been shown for NFs including relaying signals from the plasma membrane to the nucleus (CHANG and GOLDMAN, 2004), orchestrating the positioning and function of cellular organelles (TOIVOLA et al., 2005), and regulating protein synthesis (KIM et al., 2006). Based on the molecular weight of NFs, three major NF subunits have been discovered: the light or lowest (NF-L, about 68 KDa), the medium or middle (NF-M, about 150 KDa), and the heavy or highest (NF-H, about 200 KDa). Whereas all DRG neurons seem to express NF-M and NF-H, NF-L was only detected in large-light neurons (GOLDSTEIN et al., 1991; BRUINING et al., 2009b). Abnormal accumulation of neurofilaments is a central pathological feature leading to subsequent axonal swellings and degeneration.

It has been suggested that Charcot-Marie-Tooth disease (CMT), Parkinson’s disease (PD), and ALS are associated with mutations of NF proteins (ROOKE et al., 1996;

LAVEDAN et al., 2002; JORDANOVA et al., 2003).

2.2.1.3 Myelin proteins

Myelin basic protein (MBP) has been described as the second most abundant protein in the CNS after the myelin proteolipid protein (PLP), comprising approximately 30% of CNS myelin protein and approximately 18% of PNS myelin protein (GIVOGRI et al., 2000; GREER and LEES, 2001). However, a recent proteome analysis of peripheral nerve myelin demonstrated that P0, periaxin, and MBP constitute 21, 16, and 8% of the total myelin protein, respectively (PATZIG et al., 2011). The “Genes of Oligodendrocyte Lineage-Myelin basic protein” (Golli-MBP) gene complex is located on chromosome 18 in mice and humans (SPARKES et al., 1987), and alternative splicing gives rise to two protein families. Whereas the MBP proteins are exclusively expressed in myelin forming cells, the Golli proteins can be found in both oligodendrocytes and neurons in the CNS (LANDRY et al., 1996;

LANDRY et al., 1997). The molecular mass of MBP isoforms ranges from 14 to 21.5 kDa. The 18.5 and 21.5 kDa isoforms predominate in adult myelin and are responsible for compaction of the myelin sheath in the CNS (SMITH et al., 2012; HARAUZ and

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BOGGS, 2013).

MBP is a multifunctional protein, which is interacting with a number of polyanionic proteins including actin, Ca²⁺-calmodulin, tubulin, and clathrin, and negatively charged lipids. Due to the interaction with actin MBP might participate in transmission of extracellular signals to the cytoskeleton. In addition, extracellular signals cause changes in the phosphorylation status of MBP and some size isoforms of MBP are transported into the nucleus, where they bind polynucleotides (BOGGS, 2006).

Myelin protein zero (MPZ, P0) is a major structural protein of the myelin sheath in the PNS, which is encoded by the MPZ gene, not found in the CNS myelin, and exclusively expressed by Schwann cells (KIRSCHNER and GANSER, 1980; LEMKE et al., 1988). P0 accounts for approximately 50% of all PNS myelin proteins (GREENFIELD et al., 1973) and is essential for the normal wrapping, compaction, and maintenance of the myelin sheath and consequently the fast conduction of action potentials and the integrity of axons in the PNS (GARBAY et al., 2000, SHERMAN and BROPHY, 2005; SIMONS and TROTTER, 2007). P0 also seems to regulate a series of genes, which are important for myelin formation including peripheral myelin 22 (p22), MBP, and myelin associated glycoprotein (MAG; GIESE et al., 1992). P0 contains a large extracellular immunoglobulin-like domain, which can associate with P22 and act as a homophilic adhesion molecule necessary for the compaction and stabilization of adjacent membrane layers in the myelin sheath (FILBIN et al., 1990;

HASSE et al., 2004). The impact of P0 on myelin structure and function has been demonstrated by mutations in the P0 gene, which cause peripheral neuropathies such as Charcot-Marie-tooth disease type 1B (CMT1B) leading to an abnormal myelin sheath and axonal degeneration (SHY, 2006).

Periaxin (PRX) is after P0 the second most abundant protein within PNS myelin, which is in contrast to P0 not incorporated into compact myelin (PATZIG et al., 2011).

It is encoded by the PRX gene, which can produce two PTX isoforms namely long-periaxin (L-periaxin, 147 kDa) and short-periaxin (S-periaxin, 16 kDa; DYTRYCH et al., 1998). Whereas S-periaxin is distributed diffusely in the cytoplasm, L-periaxin is predominantly localized to the plasma membrane of myelinating Schwann cells linking the Schwann cell cytoskeleton to the extracellular matrix (DYTRYCH et al., 1998;

WILLIAMS and BROPHY, 2002). PRX contains “PSD-95, Discs large, ZO-1”

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Introduction (PDZ)-like domains at its N-terminus, which are among the most common protein–

protein-interacting modules (HAN and KURSULA, 2013). These domains allow PRX to participate in membrane-protein, protein-protein, and axon-glial interactions (SCHERER et al., 1995). PRX plays an important role in the myelination process and the stabilization of myelin (GILLESPIE et al., 1994). PTX-null mice initially myelinate normally, but develop a demyelinating peripheral neuropathy later in life and show an increased thickness of peripheral myelin during remyelination (GILLESPIE et al., 2000). In addition, PTX gene mutations cause a broad spectrum of demyelinating peripheral sensory neuropathies including the autosomal recessive demyelinating Charcot-Marie-Tooth (CMT4F) and Dejerine-Sottas disease. (TAKASHIMA et al., 2002;

WILLIAMS and BROPHY, 2002).

2.2.2 Functional proteins

GAP-43 was initially named F1 or B-50, then GAP-43 or pp46, and finally neuromodulin (BENOWITZ and ROUTTENBERG, 1987; BIFFO et al., 1990). It represents a membrane phosphoprotein, which is implicated in CNS development, axonal regeneration, and synaptic plasticity (BENOWITZ and ROUTTENBERG, 1987;

BIFFO et al., 1990). Though the level of GAP-43 expression declines in most neurons after the formation of mature synapses, the limbic system and associative regions of the neocortex continue to express it throughout whole life. GAP-43 is also present at low levels in primary sensory and motor areas and may contribute to their remodeling (DENNY, 2006).

Glutamate is the major excitatory neurotransmitter in the CNS and PNS and also serves as the precursor for the synthesis of γ-aminobutyric acid (GABA). Glutamate is produced by glutaminase in the cell body, translocated to mitochondria, and shipped to nerve terminals by excitatory amino acid transporters (EAAT) and sodium coupled neural amino acid transporters (SNAT; CAROZZI et al., 2008). Peripheral inflammation can induce the production of glutamate in DRG neurons, which might contribute to central and peripheral sensitization (MILLER et al., 2012). In addition, increased levels of glutamate might trigger or contribute to the maintenance of an inflammatory response (MILLER et al., 2011).

Glutamate receptors are located on presynaptic and postsynaptic membranes of

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neuronal cells. They are classified into two major groups: ionotropic (iGluGs) and metabotropic glutamate receptors (mGluRs). In addition to glutamate, specific subtypes of iGluGs can be activated more selectively by N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), or kainate (KA; HUETTNER, 1990). The mGluRs can also be subdivided into group I, II, and III.

Nevertheless, they are all coupled with a G protein to activate ion channels and give rise to a postsynaptic current. Glutamate receptors are important for neural communication, memory formation, learning, and regulation of synaptic plasticity (MILLER et al., 2011). They also influence several CNS diseases including stroke, epilepsy, ALS, Huntington’s chorea, hyperalgesia, and psychosis (MELDRUM, 2000).

Synaptophysin (SYP) or P38 is a 38 kDa major calcium-binding glycoprotein of the synaptic vesicle membrane, which is expressed in neurons and neuroendocrine cells.

It plays an essential role in synaptic plasticity without being required for neurotransmitter release itself (REHM et al., 1986; MCMAHON et al., 1996; JANZ et al., 1999; EVANS and COUSIN, 2005). SYP has four membrane spanning domains and accounts for 7% of the total vesicle proteins (JAHN et al., 1985). Due to its presence in all presynaptic boutons in nervous tissue SYP immunostaining represents a standard method to quantify synapses (FLETCHER et al., 1991; CALHOUN et al., 1996). It is also used as a marker in a wide spectrum of neuroendocrine tumors including neuroblastomas phaeochromocytomas, medullary thyroid carcinomas, ganglioneuroblastomas, chromaffin and non-chromaffin paragangliomas (WIEDENMANN et al., 1986). SYP is believed to modulate the efficiency of the synaptic vesicle cycle and SYP knockout mice show deficits in learning and memory (SCHMITT et al., 2009). Despite the fact that SYP is not a major cause of schizophrenia, SYP protein expression is significantly decreased in the hippocampus and prefrontal cortex of patients with schizophrenia (VAWTER et al., 1999; SHEN et al., 2012b).

Neurotrophins (NT) are deeply involved in the differentiation, development, plasticity, and maintenance of the vertebrate nervous systems (BRUINING et al., 2009a). In vertebrate animals four neurotrophins have been identified: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). NT-6 and NT-7 have been only isolated in fish. NGF causes axonal growth and

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Introduction prevents neuronal apoptosis. It is important for maintaining homeostasis (VERHEYEN et al., 2009a). BDNF, NT-3, and NT-4 support the survival of neurons and stimulate the growth and differentiation of new neurons and synapses (BRUINING et al., 2009a).

Interestingly, proneurotrophins can have biological effects opposite to those of mature neurotrophins and induce apoptosis (VERHEYEN et al., 2009b).

These proteins activate two different classes of transmembrane receptors: the high affinity NT receptors (TrkA, TrkB, and TrkC) belonging to the Trk family of receptor tyrosine kinases and the low affinity neurotrophin receptor p75 (p75^NTR), which is a distant member of the tumor necrosis factor receptor family (AUBERT et al., 2009;

SWINNEN et al., 2009), and was initially believed to be a low affinity receptor specific for NGF. However, p75^NTR has been identified to bind to all neurotrophins with a similar affinity and transmits signals important for neuronal survival during development (SWINNEN et al., 2009). The three members of the tyrosin kinase family were shown to bind preferentially to different neurotrophins: NGF specifically binds to TrkA, BDNF and NT-4 is specific for TrkB, and NT-3 activates predominantly TrkC and less efficiently each of the other Trk receptors. Trk receptors represent functional, survival promoting receptors for neurotrophins and also activate many of the same intracellular signaling pathways regulated by mitogen receptors (BRUINING et al., 2009a). The p75^NTR receptor plays an important role in Schwann cell myelination by its interaction with the polarity protein Par-3 (CHAN et al., 2006). Different studies using p75NTR knockout mice showed that the absence of p75 decreases the sensitivity of DRG neurons to NGF at embryonic day 15 and postnatal day 3 (LEE et al., 1994) and impedes the development of all types of DRG sensory neurons (BERGMANN et al., 1997).

Myelin 2’, 3’-cyclic nucleotide 3’-phosphodiesterase (CNPase) is a myelin-associated enzyme, which makes up 4% of total myelin protein in the CNS (TRAPP et al., 1988). CNPase plays an important role in the assembly and formation of myelin membranes, axonal support, and interactions of axons and surrounding glial cells at nodes of Ranvier in the CNS (ANGELIS et al., 1994; GRAVEL et al., 1996;

LAPPE-SIEFKE et al., 2003; RASBAND et al., 2005). CNPase is also involved in RNA trafficking, splicing, and metabolism and regulates the expression of myelin genes (GOBERT et al., 2009). CNPase is widely considered as a marker protein of

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myelin-forming glial cells such as oligodendrocytes (VOGEL and THOMPSON, 1988;

CHANDROSS et al., 1999). In the PNS, CNPase is expressed by myelinating Schwann cells, terminal Schwann cells at neuromuscular junctions, and SGCs, which upregulate CNPase expression after peripheral nerve injury. In addition, CNPase immunoreactivity was described in Remak bundles in mixed nerves and in sympathetic ganglia and grey rami of the sympathetic nervous system (TOMA et al., 2007).

CNPase can bind the retroviral Gap protein and block viral particle assembly, thereby inhibiting the replication of some lentiviruses such as HIV-1 and the genesis of nascent viral particles (WILSON et al., 2012). The age-related accumulation of CNPase in lipid rafts was associated with myelin and axonal pathology (HINMAN et al., 2008). In addition, immunodominant epitope clusters in the CNPase molecule (343-373, 356-388) might act as targets for an autoimmune T cell response in MS (MURARO et al., 2002).

S100 proteins belong to a big family of low molecular weight, acidic proteins characterized by two Ca²⁺-binding EF-hand motifs, which are formed by characteristic helix-loop-helix structures (MARENHOLZ et al., 2004). The name EF-hand was devised as a graphical description of the six α-helixes (A-F) forming the calcium-binding motif (LEWIT-BENTLEY and RÉTY, 2000). S100 was identified in a fraction from bovine brain, which was 100% soluble in ammonium sulfate at neutral pH (MOORE, 1965). According to the HUGO nomenclature committee, until now 21 different S100 genes have been identified in the human genome, 17 of which are located in region 1q21 of chromosome 1 (SCHAFER et al., 1995; MARENHOLZ and HEIZMANN, 2004). Members of this protein family have a broad range of intracellular and extracellular regulatory activities as multifunctional signaling proteins and are involved in the regulation of diverse cellular processes, such as protein phosphorylation, cell growth, differentiation, transcription, and Ca²⁺ homeostasis (DONATO, 2003; MARENHOLZ et al., 2004).

Altered S100 protein levels are associated with various diseases of the nervous system, cancer, and inflammatory disorders (SALAMA et al., 2008; YARDAN et al., 2011). Elevated levels of S100B seem to enhance or amplify neurodegeneration and induce apoptosis, which might explain its detection in traumatic brain injuries, AD and

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Introduction MS (SHENG et al., 1994; HUTTUNEN et al., 2000). The family of S100 proteins acts in some cancers as tumor suppressors, whereas in other cancers as tumor promoters.

These proteins also play a role in tumor metastasis by interacting with different proteins, such as p53, matrix metalloproteinases, cytoskeletal proteins and BRCA1.

Although the exact role of the different S100 proteins in cancer is still unclear, the specific expression patterns of these proteins can be used as clinical biomarkers (MUELLER et al., 2005).

2.2.3 Transcription factors EGR2/Krox20

The early growth response 2 (EGR2), also known as Krox20, belongs to the family of early growth response (EGR) proteins, which includes EGR1, EGR2, EGR3 (JOSEPH et al., 1988). The ERG2 protein is encoded by the ERG2 gene and contains a DNA-binding domain composed of three Cys2-His2 zinc fingers, which bind to the consensus sequence GCGGGGGCG (SWIRNOFF and MILBRANDT, 1995). The ERG2 gene contains a serum response element, which can induce its transcription as immediate early gene. The EGR protein participates in transcriptional regulation and is also implicated in the PTEN-induced apoptotic pathway in various cancer cell lines (RANGNEKAR et al., 1990; UNOKI and NAKAMURA, 2003). EGR2 has been detected in the hindbrain in the developing nervous system, early neural crest cells, neural crest-derived boundary cap cells, and glial cells of the cranial and spinal ganglia (MARO et al., 2004; WILKINSON et al., 1989). Consequently, EGR2 plays a critical role during the development of the hind brain and associated cranial sensory ganglia in mice (SWIATEK and GRIDLEY, 1993). In addition, EGR2 plays an essential role in peripheral nerve myelination and is required for the onset of myelination and peripheral myelin maintenance. Its expression in myelinating Schwann cells is controlled by OCT6 and Sox10 (GHISLAIN et al., 2002; DECKER et al., 2006;

TOPILKO et al., 1994; REIPRICH et al., 2010). The important role of EGR2 in PNS myelination is underlined by different human myelinopathies associated with mutations of the EGR2 gene including congenital hypomyelinating neuropathy, Charcot-Marie-Tooth type 1, and Dejerine-Sottas syndrome (WARNER et al., 1999).

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Sox2

SRY (sex determining region Y)-box 2 (Sox2) is a member of the Sox family of transcription factors, which belongs to the High Mobility Group (HMG) box superfamily of DNA-binding proteins (WEGNER, 1999). Sox2 is required for the maintenance of neural stem cells and the differentiation of specific neuron sub-types in the brain and the eye, where its action is markedly dose-dependent (PEVNY and NICOLIS, 2010). It is expressed in embryonic early neural precursors of the ventricular zone and, in the adult, in ependyma. In addition, few differentiated neurons, particularly in the thalamus, striatum and septum, express Sox2 (FERRI et al., 2004; FAVARO et al., 2009). In addition to the physiological role of Sox2 in stem cell maintenance and CNS especially neocortex development, its overexpression or gene amplification has been related to tumorigenesis (BANI-YAGHOUB et al., 2006; LIU et al., 2013).

Furthermore, in combination with three other transcription factors (Oct3/4, c-Myc, Klf4) Sox2 can generate pluripotent stem cells from fibroblasts (TAKAHASHI and YAMANAKA, 2006; SARKAR and HOCHEDLINGER, 2013). In the PNS, a strong Sox2 expression has been detected in rat SGCs, whereas a weak Sox2 expression was found in non-myelinating Schwann cells (KIOKE et al., 2013). Sox2 blocks myelination and plays a key role in the differentiation of DRG sensory neurons from neural crest cells (CIMADAMORE et al., 2011; KIOKE et al., 2013).

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3. In vivo and in vitro characterization of canine dorsal root ganglia neurons and satellite glial cells reveal the presence of a unique glial precursor cell

K. Hahn^1,2,4, A. Lehmbecker^1,4, Y. Wang^1,2,4, A. Habierski¹, K. Kegler¹, W. Tongtako¹, I.

Spitzbarth¹, K. Schughart³, W. Baumgärtner^1,5,6, I. Gerhauser^1,5

1Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany

2Center of Systems Neuroscience Hannover, Germany

3Department of Infection Genetics, Helmholtz Centre for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany; University of Veterinary Medicine Hannover, Hannover, Germany; University of Tennessee Health Science Center, Memphis, USA

4Authors contributed equally to the manuscript and are considered as first authors;

authors are in alphabetical order

5Authors contributed equally to the manuscript and are considered as last authors;

authors are in alphabetical order

6Corresponding author:

Prof. Dr. Wolfgang Baumgärtner, Ph. D., Dipl. ECVP Department of Pathology

University of Veterinary Medicine Hannover Bünteweg 17, D-30559 Hannover, Germany Tel.: +49 (0) 511 953 8620

Fax: +49 (0) 511 953 8675

E-mail: Wolfgang.Baumgaertner@tiho-hannover.de

Submitted for publication

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In vivo and in vitro characterization of canine dorsal root ganglia

neurons and their satellite glial cells reveals the presence of a unique glial precursor cell population

K. Hahn^1,2,4, A. Lehmbecker^1,4, Y. Wang^1,2,4, A. Habierski¹, K. Kegler^1,2, W. Tongtako¹, I. Spitzbarth¹, K. Schughart³, W. Baumgärtner^1,2,5,6, I. Gerhauser^1,5

1 Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany

2 Center of Systems Neuroscience Hannover, Hannover, Germany

3 Department of Infection Genetics, Helmholtz Centre for Infection Research, Inhoffenstr. 7, D-38124 Braunschweig, Germany; University of Veterinary Medicine Hannover, Hannover, Germany; University of Tennessee Health Science Center, Memphis, USA

Running title: Canine DRG neurons and satellite glial cells

Words: Abstract: 250, Introduction: 805, Material and Methods: 1444, Results: 894, Discussion: 1348, Acknowledgments: 59, References: 2046, Tables: 564, Figure legends: 597, Total: 8063

Tables: 3 Figures: 7

4 Authors contributed equally to the manuscript and are considered as first authors

5 Authors contributed equally to the manuscript and are considered as last authors

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6 Corresponding author:

Prof. Dr. Wolfgang Baumgärtner, Dipl. ECVP Department of Pathology

University of Veterinary Medicine Hannover Bünteweg 17, D-30559 Hannover, Germany Tel.: +49 (0) 511 953 8620

Fax: +49 (0) 511 953 8675

E-mail: Wolfgang.Baumgaertner@tiho-hannover.de

Main points: Cultured canine dorsal root ganglia neurons are a suitable translational large animal model system to study human neurological diseases. Satellite glial cells have stem cell-like properties and might be used in future cell transplantation strategies.

Key words: dog, immunohistochemistry, immunofluorescence, light microscopy, primary cell culture, transmission electron microscopy

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Abstract

Cell culture of rodent dorsal root ganglia (DRGs) has been extensively used in fundamental neuroscience, pain, virus, and prion protein research. Recent advantages in detailed characterization of species-specific properties of canine glia revealed their close resemblance to its human counterpart. Furthermore, dogs represent a suitable species for a highly needed translational large animal model in research as a bridging species to close the gap between basic discoveries in rodents and clinical trials in humans. However, more species-specific differences remain to be detailed. In the present study canine DRGs and the expression of structural and functional proteins by their satellite glial cells (SGCs) cells have been investigated by light microscopy, electron microscopy, and immunohistochemistry both in vivo and in vitro. Neuronal class III β-tubulin and non-phosphorylated neurofilaments represent reliable markers of canine DRG neurons, which can be maintained over 18 days in vitro. Over 80% of canine SGCs expressed CNPase, GFAP, and vimentin. Furthermore, Sox2 expression in over 80% of cells indicates that they exhibited characteristics of pluripotent stem cells. In contrast to rodents, only a minority of canine SGCs was positive for glutamine synthetase. Moreover, canine SGCs demonstrated a neurite promoting intrinsic capacity in co-culture with DRG neurons. These findings indicate that SGCs represent a unique cell population with phenotypical characteristics of an astro- and oligodendroglia-like cell population. Whether these cells possess intrinsic regenerative capabilities in vivo and are a useful candidate for cell transplantation studies or targets for endogenous regeneration represents an exciting field for future research.

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Introduction

Since the discovery of glial cells over a century ago, substantial progress has been made in understanding the origin, development, and function of the different types of glial cells in the central and peripheral nervous system (Webster and Aström, 2009).

Similar to neurons, astrocytes and oligodendrocytes are of neuroectodermal origin, whereas peripheral Schwann cells and olfactory ensheathing cells arise from the neural crest (Ulrich et al., 2014). Microglial cells are derived from mesenchymal precursors, which invade the nervous system in the fetal period (Gomez Perdiguero et al., 2013). In addition, the central nervous system contains Schwann cell-like glia (Synonyms: Aldynoglia, Schwann cell-like brain glia, central nervous system Schwann cells) that emerge in response to axonal damage in demyelinating diseases (Imbschweiler et al., 2012; Gudiño-Cabrera and Nieto-Sampedro, 2000). Initially regarded as non-excitable cells scaffolding and feeding neurons, glial cells have turned out to actively participate in brain function modulating neuronal communication by multiple mechanisms such as the production of glial neurotransmitters (Tasker et al., 2012). In addition to their physiological function, glial cells play an important role in the pathogenesis of various diseases and disorders of the human central nervous system (CNS) including Alzheimer disease, multiple sclerosis, stroke, epilepsy, and spinal cord injury, which cause severe and often progressive disabilities in millions of patients worldwide (Alonso and Hernán, 2008; Lee et al., 2013; Mukherjee and Patil, 2011; Reitz et al., 2011). Similarly, glial cells are deeply involved in the immune pathogenesis of several idiopathic, infectious, and traumatic canine CNS diseases (Spitzbarth et al., 2012).

The increasing knowledge of glial cell capacities in CNS homeostasis and disease prompted the idea of using peripheral glial cells including Schwann cells and olfactory

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ensheathing cells in transplantation-based therapies of spinal cord trauma (Skinner et al., 2005; Ulrich et al., 2014). However, therapeutic success compared to promising preclinical data based on studies in rodent models remains limited (Kieseier et al., 2009; Wewetzer et al., 2011; Löscher, 2011; Raddatz et al., 2014). The reasons for this frequent observation are generally unknown, but morphological and physiological differences between the rodent and human CNS might partly account for difficulties in their extrapolation. In contrast, structure and organization of the canine and human CNS is similar to a large extent and recent studies demonstrated that species-specific properties of human glia are closer related to dogs than rodents (Omar et al., 2011;

Techangamsuwan et al., 2008; Wewetzer et al., 2011). In addition, some human CNS diseases including spinal cord injury and multiple sclerosis, have spontaneously occurring counterparts in dogs with comparable pathogenetic mechanisms, lesion appearance, and clinic (Bock et al., 2013; Spitzbarth et al., 2011; Beineke et al., 2009;

Spitzbarth et al., 2012). Consequently, the dog represents a valuable translational large animal model to study the pathogenesis of human inflammatory and degenerative CNS diseases (Jeffery et al., 2011; Spitzbarth et al., 2012; Wewetzer et al., 2011; Potschka et al., 2013; Bock et al., 2013).

The dorsal roots of the spinal cord contain sensory ganglia. Such dorsal root ganglia (DRGs) are composed of afferent neurons, ensheathing satellite glial cells (SGCs), and connective tissue cells (Hanani, 2005). DRG neurons and SGCs form a unique structural unit (Pannese, 2010), representing the structural basis for their intense bidirectional communication (Thippeswamy and Morris, 1997). Similar to astrocytes in the CNS, SGCs control the microenvironment of DRG neurons and functionally substitute the lacking blood-brain barrier in sensory ganglia (Hanani, 2005). DRG neurons transmit sensory signals from the body to the CNS and can be divided into two

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main morphological subtypes namely “large light” (LL) and “small dark” neurons (SD;

Lawson, 1992). LL neurons give rise to A fibers (myelinated, fast conducting), whereas C type fibers (non-myelinated, slowly conducting) originate from SD neurons. DRG neurons, even from adult animals, can easily be accessed in order to cultivate them in vitro. They have been used to investigate the physiologic formation of neuronal processes and synapses as well as specific interactions of neurons with different types of glial cells (Gerhauser et al., 2012 a; Grothe and Unsicker, 1987). This in vitro system allows the evaluation of the mode of action and the efficacy of various pharmacological substances designed to influence the maturation and physiological function of neuro-glial interactions. In addition, cell cultures of DRG neurons have been used to study the underlying molecular mechanisms following virus and prion agent infection as well as in pain sensation (Santos et al., 2013; Arthur et al., 2001; Owen and Egerton, 2012; Kao et al., 2012). However, despite the potential wide application spectrum the composition and phenotypical characteristics of cells cultured from canine dorsal root ganglia are largely unknown. Therefore, the present study aimed to investigate light microscopic and ultrastructural characteristics of DRGs and to characterize the physiological expression of specific transport, structural, and functional proteins of canine DRG neurons and their SGCs in vivo and in vitro. Results of these experiments will provide a basis to design novel therapeutic concepts of various common canine and human CNS diseases.

Material and Methods

Tissues used

Antigen-specific immunoreaction was evaluated in DRGs of five healthy Beagle dogs (dogs 1-5; 1 year old; from each dog one DRG of the 3^rd cervical nerve). DRGs of

Characterization of canine dorsal root ganglion neurons and growth promoting effects of GM1-gangliosides

ISBN 978-3-86345-225-4

Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH 35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375

E-Mail: info@dvg.de · Internet: www.dvg.de Y imin W ang Hannover 2014

University of Veterinary Medicine Hannover Department of Pathology

Center for Systems Neuroscience Hannover

Characterization of canine dorsal root ganglion neurons and growth promoting effects of GM

-gangliosides

THESIS

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD)

awarded by the University of Veterinary Medicine Hannover by

Yimin Wang Born in Jilin province/China

Hannover, Germany 2014

To my family

Contents

1. Aims of study

2. Introduction

3. In vivo and in vitro characterization of canine dorsal root ganglia neurons and satellite glial cells reveal the presence of a unique glial precursor cell

In vivo and in vitro characterization of canine dorsal root ganglia

neurons and their satellite glial cells reveals the presence of a unique glial precursor cell population

Abstract

Introduction

Material and Methods