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

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

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

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

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