UNIVERSITÀ DI PISA
CORSO DI DOTTORATO IN NEUROBIOLOGIA E CLINICA DEI
DISTURBI AFFETTIVI
PhD Thesis
Genetic association between bipolar disorder and 524A>C (Leu133Ile)
polymorphism of CNR2gene, encoding for CB2 cannabinoid receptor.
Candidate PhD Tutor Minocci Daiana Prof. Paola Nieri
Interaction between Pum2 and Stau1 RNA-binding proteins and their role
in behavior.
Candidate PhD Tutor Minocci Daiana Prof. Michael Kiebler
2
UNIVERSITÀ DI PISA
CORSO DI DOTTORATO IN NEUROBIOLOGIA E CLINICA DEI DISTURBI
AFFETTIVI
Interaction between Pum2 and Stau1 RNA-binding proteins
and their role in behavior.
PhD Thesis
Candidate PhD Tutor
3 Ho sempre rimandato questa dedica perché…. ho sempre saputo che avrei avuto il tempo per farla…
4
Summary
Introduction ... 3
Chapter 1: The Endocannabinoid system ... 4
The endocannabinoids... 4
Biosynthesis and metabolism of endocannabinoids ... 5
Metabolism of AEA ... 5 Metabolism of 2-AG ... 6 Cannabinoid receptors ... 7 CBR1 and CBR2 ... 7 CBR1 genetics ... 7 Biochemical structure of CBR1 ... 8 CBR2 genetics ... 9 Biochemical structure of CBR2 ... 11
Mechanism of action of CBR1 and CBR2 ... 12
Non-CB1 and non- CB2 receptors ... 13
Chapter 2: The Endocannabinoid system in Central Nervous System ... 16
The Endocannabinoid system in memory processes ... 16
The Endocannabinoid system and regulation of emotions ... 17
The Endocannabinoid system and mood regulation ... 18
The Endocannabinoid system in psychiatric disorders ... 20
The Endocannabinoid system in Bipolar Disorder and Schizophrenia ... 22
Polymorphisms in Endocannabinoid system genes and their role in psychiatric disorders ... 23
Chapter 3: Neuroinflammation, CBR2 and Bipolar Disorder ... 27
Role of the Endocannabinoid system in the Central Nervous System inflammation ... 27
Neuroinflammatory hypothesis of Bipolar disorder ... 29
Aim of the study ... 30
Materials and methods... 32
Criteria to choose the samples ... 32
DNA samples analyses ... 33
... RFLP genotyping method33 ASO-PCR genotyping method ... 34
5 Statistical analyses ... 35 Results ... 37 Discussion ... 39 References ... 40 Abbreviations ... 62 Aknowledgement ... 63
6
Introduction
Cannabis sativa, or Cannabis, is one of the oldest drugs of abuse in the world, but it is also the drug with the longest history of medicinal value (Fig.1). The major psychoactive constituent of marijuana, 9-tetrahydrocannabinol (THC), was identified by Ganoi and Mechoulam (1964), who subsequently revealed its chemical structure and activity (Mechoulam and Gaoni, 1967; Mechoulam, 1970; Mechoulam et al., 1970). Furthermore, studies on the biological effects of 9-THC and its synthetic analogues have shown a precise structural selectivity (Hollister, 1974) as well as stereo-selectivity (Jones et al., 1974), foreshadowing the existence of a drug-receptor interaction. In addition to the known euphoric effect, marijuana use also induces changes in learning and memory, anxiety, analgesia, hypothermia, stimulation of food intake, anti-emetic effects, and vasorelaxation in humans (Jones, 1977; Maykut, 1985; Taylor, 1998; Hall and Degenhardt, 2009). In rodents, Cannabis and its related compounds have been shown to produce a characteristic combination of up to four of the following symptoms: hypothermia, analgesia, hypoactivity and catalepsy (Grunfeld and Edery, 1969; Compton et al., 1993;Adams and Martin, 1996; Chaperon and Thiébot, 1999).
For many years the activity of THC was believed to be mediated by an aspecific interaction with cell membrane, while in the early 1990 years, two membrane proteins interacting with THC were discovered. In 1992, the discovery of the first endogenous mediator linking these receptors opened the way to the recognition of the so-called endocannabinoid system (ECS). Today, different endogenous mediators (endocannabinoids) as well as proteins involved in their metabolism have been clearly recognized and many other targets other than CBRs seem to be used by endocannabinoids for their actions. Actually, a very high number of studies have investigated the ECS physiology and many evidences were obtained suggesting a role of this system in different pathologies. Parallely, many progresses have been obtained in the medicinal chemistry of the ECS which have introduced many new pharmacological entities under preclinical and also clinical evaluation.
7
Chapter 1
The endocannabinoid system
The endocannabinoids
The first endogenous CBR agonist to be identified was the arachidonoyl ethanolamine (anadamide) (Devane et al., 1992). Anandamide is a partial agonist of CBR1 and a weak agonist of CBR2 receptors. The second to be identified was the 2-arachidonylglycerol (2-AG) (Sugiura et al., 1995), which activates CBR1 and CBR2 even if with different pharmacological profiles. Its levels in the body are one hundred times higher than anandamide. Other endocannabinoids are Virodhamine (OAE) (Porter et al., 2002), 2-arachidonyl glyceryl ether (noladin ether) (Hanus et al. 2001) and N-arachidonoyl-dopamine (NADA) (Bisogno et al., 2000) (Fig2). Endocannabinoids are produced on demand and then released from the cells where they are synthesized; in the brain they acts like retrograde signaling molecules being released from postsynaptic neurons and acting on presynaptic neurons where they inhibit the excitatory neurotransmitters release. After this process they are transported again inside the cell through re-uptake mechanism or diffusion. More recently, the non peptide known as hemopressin was identified as the first potent endogenous antagonist/inverse agonist of CBR1 (Di Marzo, 2009).
8 Biosynthesis and metabolism of endocannabinoids
The catabolic pathways and enzymes for AEA and 2-AG have been largely investigated and partly identified (Fig3).
Metabolism of AEA
AEA is biosinthesized from a precursor of membrane phospholipid, N-arachidonoyl-phosphatidyl-ethanolamine (NAPE). This is synthesized by an N-acyl-transferase, an enzyme dependent on cAMP and Ca2+, from phosphatidyl ethanolamine and from a phospholipid containing an acylic group of arachidonic acid. AEA is synthesized by a phospholipase D (PLD). This enzyme is a β-hydrolase belonging to the family of zinc-metal hydrolases, whose activity is linked to membrane depolarization or activation of ionotropic/metabotropic glutamate receptors or stimulation of neuronal nicotinic receptors or dopaminergic receptors (Giuffrida et al., 1999; Varma et al., 2001; Kim et al., 2002).
The signal of AEA, but also of other endocannabinoids, mediated by membrane receptors, is ended by its uptake into the cell and its hydrolisis (Beltramo et al., 1997). Anandamide is hydrolyzed by the enzyme fatty acid amide hydrolase (FAAH) in arachidonic acid and ethanolamide. FAAH (Tie et al., 1996) is a serine hydrolase typically distributed throughout the body but particularly abundant in the brain and liver. The FAAH triad Ser-Ser-Lys is involved in the catalytic mechanism; it is an integral membrane hydrolase with a single N-terminal transmembrane domain (Fig. 4). In vitro, FAAH has esterase and amidase activity. . The FAAH gene consists of 15 exons and is mapped to chromosome 1p35-p34 (Wan et al., 1998). It has been cloned for the first time by Ties in 1996. Since then, many studies have been conducted in order to find a correlation between some of the enzyme gene variants and susceptibility to certain pathological conditions. In particular, according to the Pubmed database, in the FAAH gene are present 19 Single Nucleotide Polymorphisms (SNP) synonymous, 23 SNPs missense and 2 SNPs frameshift. The relationship of missense polymorphism rs324420 of FAAH gene on obesity, cardiovascular risk factors and adipocytokines has been reported by de Luis et al. (de Luis et al., 2010) while an association of three SNPs in FAAH (rs324420 rs324419 and rs873978) with childhood obesity has been observed by Muller et al. (Muller et al., 2010). Moreover the
9 FAAH SNP rs324420 has been found associated to drug abuse (Sipe et al., 2002) and to BD and major depression (Monteleone et al., 2010).
Anandamide is also a substrate of cyclooxygenase 2 (COX2). The products of these reactions are the prostaglandin-ethanolammides (prostamides). These compounds are not able to activate the CBRs but they seem, however, able to activate the vanilloid receptor TRPV1, although only at high concentrations (5-30 M).
Finally AEA may also be the substrate of lipoxygenase as assessed by Hampson et al. that characterised the brain metabolite 12-hydroxyanandamide (Hampson et al., 1995). In 2003 Veldhuis et al. (Veldhuis et al., 2003) showed the involvement of lipoxygenase metabolites of AEA in conveying neuroprotection. Using HPLC and gas chromatography/mass spectroscopy, they demonstrated that rat brain and blood cells converted AEA into 12-hydroxy-N-arachidoylethanolamine (12-HAEA) and 15-hydroxy-N-arachidonoylethanolamine (15-HAEA) and that this conversion is blocked by addition of the lipoxygenase inhibitor nordihydroguaiaretic acid.
Metabolism of 2-AG
2-AG is biosinthesized mainly from diacylglycerol (DAG), which is formed by a a phospholipase C (PLC) which hydrolyzes a membrane phosphoinositide upon activation of metabotropic receptors coupled to it. The DAG hydrolysis is catalyzed by a DAG lipase, an enzyme Ca2+ dependent, localized on the plasma membrane to form the 2-AG. The DAG lipase is found in postsynaptic neurons of the adult brain and in presynaptic ones during brain development. Instead, degradation process of 2-AG is mediated prevalently by monoacylglycerol lipase (MAGL) (Dinh et al., 2002) although, a poor metabolism may be realized also by FAAH and cyclooxygenase (Dinh et al., 2002). MAGL is a serine hydrolase and cleaves the 2-AG in arachidonic acid and glycerol. Presynaptic MAGL not only hydrolyzes 2-AG released from activated postsynaptic neurons but also contributes to degradation of constitutively produced 2-AG and prevention of its accumulation around presynaptic terminals (Hashimotodani et al., 2007).
10 Fig3: Endocannabinoids biosynthesis and metabolism (Nature Review; Di Marzo et al. 2004)
Cannabinoid receptors
CBR1 and CBR2 CBR1 genetics
CBR1 is coded by CNR1 gene mapped on human chromosome 6 q14.15 and the mRNA sequence and transcript size is known (Gerard et al., 1991). This receptor is one of the most abundant G-protein-coupled receptors expressed in brain and it can be detected in the olfactory bulb, cortical regions (neocortex, pyriform cortex, hippocampus and amygdala), several parts of basal ganglia, thalamic and hypothalamic nuclei and other subcortical regions, like the septal region, cerebellar cortex, and brainstem nuclei. Considering the cellular expression, we can find CBR1 in several cell types of the pituitary gland, in the thyroid gland, and most likely in the adrenal gland.
However this receptor is not present only at brain level but it is also expressed in several cells with relevant role in metabolism, such as fat, muscle and liver cells (and also in the endothelial cells, Kupffer cells). It is also expressed in the lungs, kidney and in the digestive and reproducitive tract. Then CBR1 is present also on Leydig cells and human sperms as well as in ovaries, oviducts myometrium, decidua and placenta (Pagotto et al., 2006).
11 A spliced variant of human CBR1, named CB1-A, was isolated and characterized appearing derived from the excision of an intron at 5‘-extremity of the coding region of the human receptor mRNA (Shire et al., 1995; Rinaldi-Carmona et al., 1996). A study of the distribution of the human CB1A mRNA by reverse transcripase PCR showed the presence of minor quantities of this isozyme in brain and in all peripheral tissues examined (Shire et al., 1995).
Biochemical structure of CBR1
CBR1 is formed by 460 aminoacids and as CBR2, presents seven transmembrane helices and is coupled to G-proteins. Many studies have investigated domains of the CBR1 receptor that are important for activating selective Gi/o proteins, using strategies that include use of peptides that mimic intracellular domains and co-immunoprecipitation of G proteins to determine selectivity of protein–protein associations. The domains of the CBR1 receptor that interact with G proteins were studied using peptides representing the juxtamembrane C-terminal region or a series of peptide analogs (Howlett et al., 1998; Mukhopadhyay et al., 1999). Palmitoylation of a cys residue anchors the C-terminal domain to the plasma membrane distal to the putative helical intracellular domain (Mukhopadhyay et al., 2002). Thus, this region is also referred to as the fourth intracellular loop (IC4). The peptide mimicking the juxtamembrane C-terminal domain promoted G protein activation in rat brain membranes and the inhibition of Gs-stimulated or forskolin-activated adenylyl cyclase in N18TG2 membranes (Howlett et al,. 1999; Mukhopadhyay et al., 1999; Howlett et al., 1998). In solubilized brain or N18TG2 membrane preparations, the juxtamembrane C-terminal peptide competed for the protein–protein association of the CBR1 receptor with Gαo or Gαi3 (Mukhopadhyay et al., 2000; Mukhopadhyay and Howlett 2001). Because this peptide failed to disrupt the CBR1 receptor interaction with Gαi1 or Gαi2, it is believed that the C-terminal IC4 domain interacts primarily with Gαo or Gαi3 proteins. The IC4 peptide was able to form a helical structure only in a negatively charged environment (Mukhopadhyay et al., 1999), suggesting that changes in the seventh transmembrane helix (TM7) that would alter the positions of critical amino acids could promote activation of Gαo or Gαi3. CBR1 receptor mutants that are truncated two residues distal to the palmitoylated cys showed perturbed regulation of Ca2+ currents (Nie and Lewis, 2001). However, mutants truncated such that the entire IC4 region was deleted were devoid of Ca2+ channel regulation (Nie and Lewis, 2001), as would be expected if this region were critical for
12 interaction with Go as the transducer of this response. Three peptides comprising the third intracellular loop (IC3) of the CB1 receptor were able to disrupt the CBR1 receptor association with Gαi1 or Gαi2 in solubilized preparations of rat brain or N18TG2 membranes (Mukhopadhyay et al., 2000). The C-terminal side of IC3 was considered to be most important for the activation of G proteins, presumed to be Gαi1 or Gαi2 (Howlett et al., 1998). In support of this, a nine-amino acid peptide, mimicking the C-terminal side of IC3 at the membrane-cytosol interface, promoted GTPase activity of a pure preparation of Gαi1 (Ulfers et al., 2002). The structure of a larger peptide comprising the entire IC3 loop was shown by nuclear magnetic resonance (NMR) analysis to be helical at the N-terminal side distal to TM5 (Ulfers et al., 2002). The peptide appeared to be amorphous at the middle third except for a turn occurring at an intracellular Gly residue, and exhibited helical structure beginning within the C-terminal third approximately two turns proximal to TM6 (Ulfers et al., 2002). NMR analysis of a peptide representing this C-terminal region indicated that this peptide was also helical in the presence of Gαi1 (Ulfers et al., 2002). A Leu-Ala-Lys-Thr sequence at the membrane interface may be critical to Gi interaction because reversal of this Leu-Ala sequence to Ala-Leu in a mutated CBR1 receptor resulted in a loss of coupling to Gi, thereby attenuating inhibition of cyclic AMP production (Abadji et al., 1999).
CBR2 genetics
The CBR2 is coded by the CNR2 gene which is mapped on human chromosome 1p36.11 (Fig4). The human CBR2 mRNA is present in two isoforms: the first, called hCB2A, was discovered and characterized by Munro et al. in 1993 (Munro et al., 1993) and then by Schatz et al. in 1997 (Schatz et al., 1997) while the second one, called hCB2B, was discovered by Liu et al. in 2009
13 Fig4. Human CBR2 (CNR2, 1p36.11) genomic structure and neighboring genes. The gene size is in bp; black vertical bars represent exons; triangles represent splicing patterns, arrows represent neighboring gene transcription directions; red squares represent CpG islands; blue rectangle represents copy number variant (Onaivi et al., 2011).
Through analyses via quantitative RT-PCR a tissue-dependent expression of the two CBR2 mRNA isoforms has been discovered. CB2A mRNA has been found expressed in the human brain regions of caudate, amygdala, hippocampus, cerebellum, nucleus accumbens, putamen, and cortex, with similar levels in comparison to peripheral tissue expression, such as muscle, spleen, intestine, leukocytes, and kidney, except testis expression, which is more than 100 times that of other tissues. In contrast, CB2B mRNA resulted expressed in spleen and to a lesser extent in leukocytes, muscle, intestine, liver, testis and heart (Liu et al., 2009).
The isoform CB2A presents a gene promoter with starting exon located about 45 Kb upstream from the identified promoter transcribing the spleen isoform CB2B. The exons 1a and 1b of hCB2 are located at 85478 bp and 75971 bp, respectively, upstream from the major coding exon 3. Both exons 1a and 1b are 5‘UTR sequences because they are not in frame with the initiation methionine of hCB2 of exon 3 (Liu et al., 2009). In contrast, the previously identified upstream 5‘UTR exon is located 39769 bp upstream from the major exon 3 (Munro et al., 1993). Therefore, the size of the newly identified hCB2A isoform spans a genomic region of 90,8 Kb, about twice as large as the previously identified human hCB2B of 45,7 Kb (Liu et al., 2009). The promoter region of CB2A contains CpG islands and several CCAAT boxes with a transcription factor binding site for stress response such as AP-1 (activator protein 1), HSF (heat shock factor), and STRE (stress response element). In contrast, the promoter region of CB2B contains neither CpG islands nor CCAAT boxes, but has transcription factor binding sites of GATA (GATA-binding factor), HSF, Ntx2.5 (homeo domain factor, tinman homolog) and AP-4 (activator protein 4). It is therefore likely
14 that human CB2A target CBR2 to specific tissues and neuron or glia cell components in response to physiological stressors (Onaivi et al., 2011).
The presence of CpG islands in the promoter regions of the four neighboring genes to CBR2 suggest an epigenetic regulation of CNR2 gene locus. In fact the close proximity of PNRC2 and BTBD6P genes with CNR gene indicates that CB2A gene transcription might be co-regulated. In addition, there is also a copy number variant (CNV) of 2.4 kb (Zhang et al., 2006) located in intron 2 whose genetic significance awaits further investigation (Onaivi et al., 2011).
There are four probable poly A signals and poly A sites in the extended 3‘UTR sequence of hCB2 cDNA; the first poly A site is the previously identified cDNA clone (HCX5.36) with imperfect polyadenilation signal sequence of GATAAA (Munro et al., 1993). There are three additional poly A sites with perfect polyadenilation signal sequence AATAAA; the second poly A site is located at 2799 bp of hCBR2A (EU517121) with poly A containing 3‘EST alignments (AW515513, AI864752 and AI475798). The third is located at 4632 bp with poly A containing 3‘EST alignments (AW967543, AW966929, AV715015 and AA250845) and the fourth is located at 5349 bp with poly A containing 3‘EST alignments (AW405600, AW294847, AA909480,AI218261 and AI424994). All these 3‘EST clones were derived from cDNA libraries of B cells or lymphocytes.
Biochemical structure of CBR2
The biochemical structure of CBR2 is formed by 294 amino acids fold into seven transmembrane helices, extra and intra cellular loops, as well as a cytoplasmatic helix (helix VIII) (Xie et al., 2003) (Fig5). To determine helix angles, the helix VIII domain was assumed to lie relatively parallel to the membrane with a tilt angle of 90°. Xie et al. results show that helix I is tilted from the plane of the membrane by 22°, and helix II is off 24°. Helix III is the longest transmembrane segment, extending from the residue Lys103 to Arg136 and it has the largest tilt angle, 26° from the plane of membrane. This results in helix III being in close contact with the rest of helices except helix I through inter-helix hydrogen bonding and other non-bonded intermolecular interactions (Xie et al., 2003). Helix IV is the shortest transmembrane segment consisting of 25 residues and runs almost perpendicular to the membrane with a tilt angle of 5°. Helix V has a length of 29 residues and is tilted with a 5° angle. Helix VI was measured showing two tilt angles, 32° and 10°, correspondent to the extra and intra cellular helical segment with respect to the membrane plane. Helix VII‘s tilt angle is 25° (Xie et al., 2003). An analysis of inter-helix hydrogen
15 bond and non-bonded interactions shows that in addition to hydrogen-bond network formed among these helices. This contributes to stabilize the seven transmembrane domains of CBR2. Helix III positions itself in close contact with the rest of the helices except helix I through inter-helix hydrogen boding (e.g Phe87-Ser112, Leu126-Arg149, Leu125-Thr208, Phe117-Trp258, Ser120-Asn295) and other non-bonded inter-helix interactions, such as the hydrophobic interactions between non-polar side chains (e-g- Ala83-Met115, Leu76-Ala119, Ile110-Pro168, Leu133-Leu145, Val121-Leu201, Leu125-Ile205, Leu124-Leu251, Leu124-Leu254, Val113-Leu289). Helix IV has inter-helix hydrogen bonds (e.g. Ser75-Trp158, Asn188-Leu167, Ser165-Ala111) and hydrophobic interactions (e.g. Leu154-Leu71, Ala150-Leu75, Leu167-Leu192, Val164-Phe197, Leu160-Phe197, Leu145-Leu133, Pro168-Ile100, Leu169-Leu101) with helix II, III and V. Other hydrogen bond interactions were also detected among helix I and helix II and VII. Helix VI only has inter-helix hydrogen bond interactions with helix III, whereas it interacts with helices II, V and VII through van der Waals interactions.
Fig6. 3D structure model of CBR2 (Xie et al., 2003).
The high lipophilicity of cannabinoids brought to think that their mechanism of action was based on the ability to establish nonspecific interactions with membrane lipids, altering their fluidity but numerous observations about cannabinoids stereoselectivity brought to the discovery of the CBRs (Fig6).
16 Fig6. CBR1 and CBR2 structures
Mechanism of action of CBR1 and CBR2
Both cannabinoid receptors CBR1 and CBR2 are typical G protein-coupled receptors. They are activated by plant and synthetic cannabinoids and of course by endocannabinoids even if the AEA behaves as a partial agonist at both CBR1 and CBR2 receptors with an highest affinity for CBR1, while 2-AG is a complete agonist at both of them but with less affinity than AEA. Their aminoacid sequence presents about the 44% of similarity (68% if we consider only the transmembrane region).
The most relevant property is their preservation throughout evolution, for example mice and humans have 97-99% amino acid sequence identity. This aspect reflects the important functions played by ECS in cell and system physiology (De Fonseca et al. 2005).
Both CBRs are coupled to similar transduction systems. CBR activation was initially reported to inhibit cAMP formation through its coupling to Gi/0 proteins (Devane et al., 1988), through the subunits of which they can inhibit adenylate cyclase and stimulate mitogen-activated protein kinases (MAPK) (Howlett, 2005). However, additional studies found that the CBRs were also coupled to ion channels through the Golf protein resulting in the inhibition of Ca2+ influx through N (Mackie et al. 1992), P/Q (Twitchell et al., 1997) and L (Gebremedhin et al., 1999) type calcium channels, as well as, the activation of inwardly rectifying potassium conductance and A current (Mackie et al., 1995; Childers et al., 1996). Other studies showed how, in certain cell types, CBR1 and CBR2 agonists, including endocannabinoids like 2-AG and anandamide, can directly stimulate:
17 -the hydrolysis of PIP2 by PLC-with subsequent release of inositol-1,4,5-phosphate (IP3) and Ca2+ mobilization from endoplasmatic reticulum via either Gq/11-mediated or Gi/0 -mediated mechanisms by CBR1 (De Petrocellis et al., 2007);
- the modulation of the PI3K-mediated signaling cascade via Gi/0 thereby affecting the downstream Akt/protein kinase B pathway by CBR1 (Esposito et al., 2008);
- the release of nitric oxide (NO) (Lépicier et al., 2007) and the subsequent increase of cGMP levels by CBR2;
- increased release of ceramide by CBR2 (Howlett et al., 2005).
Non-CBR1 non-CBR2 receptors linking cannabinoids
Many evidences suggested that cannabinoids may also act throught other receptors than classical CBR1/CBR2. The best established non-CBR1 non-CBR2 receptor for AEA and NADA, but not for 2-AG, is the transient receptor potential vanilloid type-1 (TRPV1) receptor. It can be activated by nociceptive physical (heat, low pH) or chemicals stimuli. TVRP1 receptors belong to the large family of TRP receptor (transient receptor potential) which are cation channels with sixtransmembrane domains (Fig7). The activation of TRPV1 with vanilloid compounds causes a rapid increase in intracellular Ca2+ levels in neurons that leads to a depolarization threshold value which determines the onset of an action potential. The activation of the receptor, however, is immediately followed by a state of desensitization in which neurons do not respond to subsequent additions of vanilloids. At the level of the nervous system where this receptor is widely distributed, it is located in the dopaminergic neurons in the substantia nigra, in hippocampus, hypothalamus, locus coeruleus and in various layers of the cortex (van der Stelt 2004); moreover it is abundantly expressed in root ganglia of dorsal spinal cord. In brain it is involved in very important functions as neuronal plasticity, control of body temperature and food but also in psychiatric disorder aging like anxiolytic (Chalh et al., 2011). Studies in mice lacking TRPV1 receptor function have shown that TRPV1 is involved in the perception of neuronal terminal sensitivity and inflammation (Caterina et al., 2000). TRPV1 is also present in cells outside the nervous system such as epidermal keratinocytes, bladder, smooth muscle, liver, polymorphonuclear granulocytes, macrophages and spermatozoa (Gervasi et al., 2000).
18 Fig7: TRPV1 signaling (Binshtok et al. 2007)
A recent evidence, limited to in vitro experiments, suggests that some plant and synthetic cannabinoids as well as endocannabinoids might bind to the orphan receptor GPR55 (Ryberg et al., 2007). The role of GPR55 is object of discussion but recently a possible role of this receptor in the regulation of cannabinoid 2 receptor-mediated responses in human neutrophils has been suggested by Belenga and colleagues (Belenga et al., 2011). Another aspect of the interaction between GPR55 and ECS is reported in numerous studies which show that bone cells express CBR1 and CBR2 and the orphan receptor GPR55 and pharmacological and genetic inactivation of all the three receptors singularly, CBR1, CBR2 and GPR55 in adult mice suppress bone resorption, increase bone mass and protect against bone loss, suggesting that inverse agonists/antagonists of these receptors may serve as anti-resorptive agents (Idirs et al., 2010).
Another feature of this receptor has been demonstrated by Hu and colleagues: in their studies emerged that GPR55 is expressed in various cancer types in an aggressiveness-related manner, suggesting to be a potential novel cancer biomarker and therapeutic target (Hu et al., 2011).
Other cannabinoid targets, quite recently discovered, are the intracellular peroxisome proliferator-activated receptors (PPARs) (Fig8). The PPARs function as lipid-sensing receptors and, through the activation or repression of large sets of particular genes, they are intimately involved in the regulation of crucial metabolic events (Berger et al., 2005) .
The first evidence to propose PPARs as a cannabinoid target has been revealed by O‘Sullivan and colleagues who demonstrated that some of the longer term vascular effects of the plant cannabinoid 9-THC are mediated by this entity (O‘Sullivan et al., 2005). Moreover some effects of endogenous cannabinoid-like molecules (ECLs) appear to be due to activation of PPARs, for example the regulation of appetite and lipolysis by
N-19 oleoylethanolamine (OEA) and the anti-inflammatory effects of N-palmitoylethanolamine (Fu et al., 2003; Guzman et al., 2004; Lo Verme et al., 2005). The results presented by Sun and colleagues (Sun et al., 2007) demonstrated that a number of cannabinoid compounds are PPAR agonists and that OEA has neuroprotective properties via PPAR activation.
20
Chapter 2
The endocannabinoid system in CNS
The ECS and memory processes
In contrast to the classical signaling for the information travel from presynaptic to postsynaptic neurons, the endocannabinoids use retrograde signaling in glutammatergic and GABAergic terminals. The membrane depolarization in presynaptic neuron causes the glutamate release which bonds to postsynaptic neuron receptors inducing calcium channel to open. Calcium ions are accumulated in post-synaptic neurons and this causes the release of ECs from membrane lipids. The ECs binding to CBR1 receptor on presynaptic neurons induce potassium channels to open and the results is the suppression of presynaptic neurotransmitter release. Then the ECs are taken inside the cell and are degraded.
Using this type of signaling the ECS has a key role in all brain processes mediated by glutamatergic and GABAergic terminals and in particular it is largely involved in learning and memory.
In memory extinction CBR1, with L-Type-Voltage-gated-calcium channel (LTVGCC), are required to regulate the stability of the original memory trace (Suzuki et al., 2008) while in spatial memory the inhibition of CBR1 and LVGCC in the hippocampus blocks the disruption of the reactivated spatial memory by the inhibition of protein synthesis. The reactivated spatial memory is destabilized through the activation of CBR1 and LVGCC and then reestablished through the activation of NMDA receptor and CREB (cAMP response element-binding) mediated transcript (Kim et al., 2011) (Fig9).
Moreover it has been demonstrated that agonists of CBR1 impair memory formation while antagonists reverse this effect or act as memory enhancers: WIN55,212-2, a full agonist of CBR1 affects thigmotaxis and hinders memory consolidation (Jamali-Raeufy et al., 2009) while SR141716A, a CBR1 antagonist, provokes memory improvements and reverse scopolamine-induced amnesia (Takahashi et al., 2005).
In neuronal plasticity chronic activation or inhibition of CBR1 leads to continuous suppression of this process in hippocampus and in other brain regions, suggesting that ECs may have a functional role in synaptic processes which produce state dependent transient modulation of hippocampal activity (Puighermanal et al., 2009).
21 Fig9. Modulation of synaptic strength in the brain by anandamide
The ECS and regulation of emotions
The ECS has emerged as a major neuromodulatory system critically involved in the control of emotional homeostasis and stress responsiveness (Marco et al., 2011). It playes an important role during all the lifespan especially in critical phases like adolescence; during this life period dramatic hormonal and physical changes occur in the major neurotransmitter systems. In this regard, auto-radiographic studies in forebrain areas of adolescent non-human primates reported an overproduction followed by a subsequent pruning among GABAergic, adrenergic, cholinergic, serotoninergic and dopaminergic receptors (Lidow et al., 1991). Similarly the ECS continues rearrangement and maturational process during adolescence. Brain AEA levels gradually increase during development, reaching maximal levels during adolescence, while CBR1 appears maximal among adolescents and drops in adults (Belue et al., 1995). For this reason discontinuities in endocannabinoids signaling during adolescence might be of great relevance for understanding the neurobiological mechanisms underlying the characteristic behavioral repertoire of youth, including sensation-seeking and proneness for risk behaviours (Laviola et al., 2003). The participation of CBR2 in emotional-related behavior has been rarely investigated, although recently the overexpression of CBR2 in mutant mice has been reported to reduce anxiety-like behaviors (Garci et al., 2011).
22 The ECS and mood regulation
For its important role in different brain processes the dysregulation of the ECS has been associated with various pathophysiological states, including psychiatric disorders. The high level of expression of CBR1 receptors in brain areas involved in the regulation of cognition and mood functions implies in fact that the ECS is probably involved in emotional processing, mood and anxiety regulation and in the pathophysiology of depression (Parolaro et al., 2010).
To better understand the role of CBR1 in the brain many studies have been conducted using CBR1 knock-out mice in different ways: Martin and colleagues showed that these mice were more vulnerable to the anhedonic effect of chronic stress (Martin et al., 2002) while Steiner et al. observed an increase in passive coping behavior in the forced swim test (FST) (Steiner et al., 2008). In line with these findings it has been observed that long-lasting impairment of CBR1 function led to the development of a depressive phenotype characterized by anhedonic state, passive coping behavior in FST and cognitive dificits (Rubino et al., 2009).
In CBR1 ko mice also CREB levels, another parameter of depression, changes as the levels of other markers of neuroplasticity, which are lower than in wt mice, and as the synapses activation in prefrontal cortex (Pfc) (Rubino et al., 2008). All these data point to the idea that stable impairment of CBR1 leads to depression-like symptoms (Parolaro et al., 2010). This conclusion appears to be questioned by some reports that blocking the CBR by cannabinoid antagonists induced an antidepressive-like response in FST or tail suspension test (TST) (Griebel et al., 2005). However the discrepancy is only apparent because of differences in the experimental conditions: on one hand, a lasting deficit in the CBR1, on the other hand an acutely blockade of CBR1 (Parolaro et al., 2010).
Anyway, changes in CBR1 expression have been found in different models of depression; in the chronic mild stress paradigm there is a significant increase in CBR1 density or mRNA in Pfc (Bertolato et al., 2007; Hill et al., 2008) together with a significant decrease in the hippocampus (Hill et al., 2005), hypothalamus and ventral striatum (Hill et al., 2008). A significant decrease in CBR1 -mediated control of GABA transmission in the striatum was observed after a social defeat stress paradigm (Rossi et al., 2008). Clinical studies by Hill et
23 al. described how the basal serum concentrations of AEA and 2-AG were significantly lower in women with major depression then in matched controls (Hill et al., 2004).
Starting from these results different studies have tried to verify if enhancing the ECS tone it is possible to obtain an antidepressant effect and it has been possible to discover how acute intraperitoneal injection of different direct or indirect CBR1 receptor agonists reversed behavioral despair in the FST (Bambico et al., 2007; Gobbi et al., 2005; Hill and Gorzalka, 2005). This effect was also evident when cannabinoid agonists were injected in specific brain areas with key roles in emotion, such as hippocampus (McLaughlin et al., 2007) and the Pfc (Bambico et al., 2007).
Interestingly, chronic treatment with URB597, an inhibitor of FAAH exerted antidepressant-like effects in rats exposed to chronic mild stress, a highly specific, predictive animal model of depression. To better understand the mechanism used by cannabinoid to induce an antidepressant effect, biochemical alterations, thought to underline the depressive syndrome or at least the major neurobiological alterations induced by current antidepressant treatments, has been used in many studies. For example, all compounds that possess effective antidepressant properties act increasing the monoamine neurotransmitters, serotonin or norepinephrine (Berton et al., 2006). Both CBR1 agonists and inhibitors of AEA hydrolysis increase the firing activity of neurons in the dorsal raphe, the major source of serotonin neurons, enhancing serotonin neurotransmission and exhibiting antidepressant activity (Bambico et al., 2007; Gobbi et al., 2005). Similarly, stimulation of CBR1 activity has been shown to increase firing activity of noradrenergic neurons in the locus coeruleus as well as the release of norepinephrine in the prefrontal cortex (Gobbi et al., 2005; Oropeza et al., 2005). The involvement of the noradrenergic system in the antidepressive effect of a chronic CBR1 agonist was further demonstrated by Morrish et al. who reported that co-treatment with both alpha and beta-adrenoreceptor antagonists attenuated the reduction in immobility in the FST induced by the CBR1 agonist HU210 (Morrish et al., 2009). Also changes in the hypothalamic–pituitary–adrenal (HPA) axis are characteristic of major depression and antidepressant agents normalize the hyperactivity of the HPA axis. Agents which act facilitating EC neurotransmission (such as uptake inhibitors or metabolic enzyme inhibitors), facilitate adaptive stress coping behaviors and attenuates the neuroendocrine response to psychological stressors (Patel et al, 2004; Hill et al., 2008). It is well known that factors predisposing to depression, such as stress, suppress neurogenesis, whereas interventions reducing depression stimulate neuron formation. These observations led to the
24 hypothesis that modulation of hippocampal neurogenesis is crucial to both the onset and treatment of depression (Perera et al., 2008; Drew and Hen, 2007). In adult rats, administration of cannabinoid agonists as well as FAAH and re-uptake inhibitors has been shown to increase hippocampal neurogenesis (Marchalant et al., 2009; Goncalves et al., 2008; Hill et al., 2006; Jiang et al., 2005). Finally, according to the neurotrophic hypothesis of depression, decreased BDNF expression in the hippocampus seems to be associated with depression, whereas increased BDNF expression with antidepressant action (Duman and Monteggia, 2006). THC treatment significantly increased the level of BDNF in the hippocampus (Rubino et al., 2006; Derkinderen et al., 2003).
Cannabis sativa for example have been used since a long time for its anxiolytic –like effect even if its real effect depends by the dose; after low doses, in fact, it‘s possible to observe an anxiolytic effect while after high doses an anxiogenic response is observed. The reason for this dose-dependence is not clear but it‘s possible that receptors with different sensitiveness to the cannabinoid action might be involved with different activities on anxiety response. We should also consider that, for example, AEA at high concetration could activate not only CBR1 but also TRPV1 receptors which could affect anxiety in opposite ways (Rubino et al., 2008). If it is true, blocking CBR1 through genetic or pharmacological strategies should lead to anxiogenic response. In different studies CBR1 ko mice have been analyzed with different behavioral tests such as elevated-plus maze, the light-dark box, the open-field and the social interaction test and anxiogenic effects have been observed only in the test with stressful unfamiliar environment as elevated plus-maze (Haller et al., 2002) and light-dark box (Maccarone et al., 2002). An explanation for this data could be find considering that endocannabinoids, produced on demand and stress or aversive situations can trigger their production to induce the physiological coping mechanism involved in the stress response, mice with genetically disrupted cannabinoid signaling can be expected to show increased anxiety only in aversive conditions (Parolaro et al., 2010).
The ECS and psychiatric disorders
If we focus on psychosis, the disorder where the role of ECS has been better studied is schizophrenia. In the past, a lot of studies that showed as the consumption of Cannabis expecially during adolescence could be a risk factor for schizophrenia (Henquet et al., 2005; Moore et al., 2007). A possible explanation could be found considering the CBR1 expression in the brain that is more abundant in Pfc, basal ganglia, hippocampus and
25 anterior cingulate cortex which are all brain regions implicated in schizophrenia. In different studies that long term cannabis abusers presented deficits in cognitive functions, such as working memory (Solowij et al., 2002) that are also impaired in schizophrenia (Elvevag et
al., 2000).
Zavitsanou et al. (2004) showed that the binding of the CBR1 antagonist SR141716A was significantly elevated (64%) in the anterior cingulate cortex (ACC) of schizophrenic patients compared to control. Considering that the ACC plays an important role in normal cognition, particularly in relation to attention and motivation Solowij et al. (2002) analyzed the cognitive functions in this area in long term cannabis users and in schizophrenic patients discovering impairments in both groups. Then Newell et al. (2006) found a significant increase in CBR1 binding in the posterior cingulate cortex (PCC), another area relevant for schizophrenia, where the CBR1 density was increased of 25% in schizophrenic subjects compared to controls, none of whom had recently used cannabis (Newell et al., 2006). These results support the hypothesis of the presence of a dysfunction in CBR1 in selected brain areas of schizophrenic patients, specifically localized in cortical regions (ACC, the dorso-lateral PfC, and PCC) involved in cognition and memory, two functions highly compromised in schizophrenia (Parolaro et al., 2010).
In animal model of schizophrenia induced by N-mathyl-D-aspartate (NMDA) antagonists as phencyclidine (PCP) has been reported how the CNR1 gene disruption dramatically alters the behavioral effects of PCP, suggesting that the CBR1 are involved in schizophrenia (Haller et al., 2005).
Another very interesting aspect of ECS in schizophrenia is the AEA alteration in the cerebrospinal-fluid (CSF). A negative correlation of CSF AEA levels with psychopathological symptoms has been found in acute schizophrenic patients suggesting that AEA might play an adaptive role, counteracting the neurotransmitter abnormalities in acute schizophrenia, and confirms the existence of an ―anandamid-ergic‖ dysregulation in schizophrenia (Parolaro et al., 2010). This result is in apparent contrast with the association between cannabis use and precipitation of psychotic symptoms which has been shown in numerous clinical studies (Henquet et al., 2005; Zammit et al 2002). For this reason, Leweke et al. examined how cannabis use might alter the adaptive role played by AEA in schizophrenia (Leweke et al., 2007) showing that frequent cannabis use by schizophrenic patients down-regulated CSF AEA levels but it had no such effect in healthy volunteers. So it is possible that cannabis consumption, at a certain level, may provoke a down-regulation
26 of AEA signaling in the CNS through a decrease in AEA biosynthesis or an increase in its degradation (Piomelli, 2003).
According to this, the explanation for the cannabis-induced psychotic symptoms may depend to AEA changes in signaling (Parolaro et al., 2010).
Even if the most studied CBR in psychiatric disorder is CBR1 it is important to note that also CBR2 seems to play an important role in several psychiatric diseases like alcoholism, depression, schizophrenia, drug abuse, bipolar disorder, eating disorder, obesity but also osteoporosis, multiple sclerosis and cancer (Ishiguro e tal., 2007; Onaivi et al., 2008; Onaivi et al., 2010).
In alcoholism CBR2 might work as a modulator of reward and its expression appears to be altered in response to substances abuse (cocaine and heroin) (Onaivi et al., 2008). During Water T Maze test on mice treated with alcohol it was found that the CBR2 agonist, JWH015, contributed to increase the consumption of the substance, if associated with a situation of chronic stress. This increase, however, did not occur when the agonist is administered alone. By contrast in these animals, using a CBR2 antagonist, such as AM630, it was possible to observe a reduction in the consumption of alcohol, associated with particular stress conditions. In this case, the antagonist alone has no effect.
The ECS in Bipolar Disorder and schizophrenia
There has been considerable debate regarding the causal relationship between chronic cannabis abuse and psychiatric disorder especially for mood disease as the BD and schizophrenia.
The classical Kraepelinian distinction between schizophrenia (dementia praecox) and manic depression (unipolar and bipolar disorders) is under question by present-day psychiatrists, with mixed states, as typified by schizoaffective disorder, being increasingly recognized. However, clinical observations suggest that the ECS is dysfunctional in schizophrenia and in BD and fails to control the level of cortical excitation and inhibition in the brain. Thus, excessively high EC tone (positive schizophrenic symptoms, mania) or excessively low EC tone (negative schizophrenic symptoms, depression) may be manifest in these disorders. The role of high ECS stimulation in schizophrenia and mania appears to be supported by the effects of cannabis and THC (Ashton and Moore, 2011): high intake of cannabis can cause acute psychosis, sometimes with marked hypomanic features (Johns, 2001), probably mediated by CBR1 (Morrison et al., 2009).
27 Moreover cannabis has been observed to aggravate positive symptoms in schizophrenia and its prolonged or heavy use in childhood or adolescence increases the risks of later schizophrenia, suicide, depression and other psychiatric disorders (Mcgee et al., 2000). Only a few studies have been focused also on the effect of this drug in BD. It has been observed that cannabis abuse in this patients can allay the racing thoughts, flight of ideas and hyperactivity associated with hypomanic ⁄manic phases. This may be partly because of anxiolytic and sedative effects of low ⁄moderate doses of THC, but the major contribution is probably provided by the antipsychotic, sedative and anxiolytic effects of CBD and its antagonism of the psychomimetic effects of THC (Zuardi, 2008). For this reason CBD has been proposed as a possible treatment for both BD and schizophrenia (Ashton et al., 2005). Clinical trials of CBD alone are few but small studies suggest a positive effect in schizophrenia (Zuardi et al., 1995; Morgan et al., 2008) while CBD was ineffective for a manic episode of BD in two patients as reported by Zuardi et al. (2010).
Several studies have been focused also on the alteration in CBR1 expression in the anterior cingulate gyrus (Brodman area 24), an important area of study for psychiatric diseases. Based on its rich interconnections with prefrontal and limbic as well as dopaminergic brain areas, it plays an important role in cognition, particularly in relation to motivation and attention. The ACC represents the rostral part of the cingulated gyrus on the medial surface of each hemisphere, overlaying the corpus callosum. Neuropathological changes in the ACC have been reported in BD, major depression, and schizophrenia (Knable et al., 2002). Using quantitative autoradiography, Zavitsanou et al. (2004) examined the distribution and density of CBR1 in postmortem ACC from schizophrenic patients by radioligand binding of SR141716A, a CBR1 antagonist. The CBR1 displayed a homogeneous distribution among the layers of the ACC; a significant increase of 64% of SR141716A specific binding to CBR1 was found only in schizophrenic patients, but not in bipolar patients, compared to controls. Previously, Dean et al. (Dean et al., 2001) reported increased binding of CP-55940 in the dorsolateral prefrontal cortex of schizophrenic patients as compared to controls. In the prefrontal cortex of postmortem brains of depressed suicide victims, a significant up-regulation of CBR1 density and CBR1-stimulated G-protein activation were observed (Hungund et al., 2004). The role of the ECS in the pathophysiology of depression is further supported from animal studies, were it was shown that CBR1 antagonism leads to increased levels of serotonin (Tzavara et al., 2003). Although the alteration of the ECS might be related to suicidal behavior, as elevated levels of endocannabinoids, CBR1 and its G-protein coupling system were evident in the prefrontal cortex of alcoholic suicide victims as well all
28 these the findings suggest that hyperactivity of CBR1 function might be connected to depression (Vinod and Hungund, 2006).
Koethe et al. (2007) studied CBR1 densities in the ACC of patients suffering from BD or major depression and showed how in bipolar patients the intake of first generation antipsychotics significantly reduced the numerical density of CBR1 in glial cells. In their study it was observed no change in schizophrenic but in bipolar patients, in the form of a decrease in CBR1 in glial cells following antipsychotic medication of the first generation. Furthermore, they found an even stronger reduced numerical density of CBR1 in neurons after intake of SSRI in major depression (Koethe et al., 2007).
Polymorphisms in ECS genes and their role in psychiatric disorders
The genetic analyses of psychiatric disorder have the scope first of all to discover all the genes which could interfer with the pathology and secondly to identify which allele of these genes could be associated to the disorder object of investigation. A classical method of genetic study on candidate genes of a certain disorder is the analyses of polymorphisms of them, alterations of the gene sequence which are present in a certain population with a percentage at least of 1%. The most studied polymorphisms are the single nucleotide polymorphisms (SNP) which are DNA sequence variation occurring when a single nucleotide in the genome differs between members of a biological species or paired chromosomes in an individual.
The SNPs study showed a lot of candidate genes and alleles correlated to psychiatric pathologies and interesting results have been obtained about the analyses of correlation between CBR and FAAH polymorphisms and psychiatric disorder.
About the polymorphic aspect of CNR1, 10 polymorphisms have been described on Pubmed website for CNR1 gene, in particular 5 synonymous single nucleotide polymorphisms (SNPs) and 5 missense ones and all of them have been studied to verify possible effects on different diseases. The most studied polymorphism of CNR1 is rs1049353 which has been associated with with anorexia and bulimia nervosa (Monteleone et al,. 2009) and with BD (Monteleone et al., 2010).
29 The silent polymorphism rs1049353 presents a positive association with alcoholism (Schmidt et al. 2002) while the SNP rs806379 has been found associated to polysubstance dependence (Zhang et al.2004).
About the polymorphism aspect of CNR2, 15 SNPs are described on Pubmed website, 8 synonymous and 7 missense. The most studied missense SNP of CNR2 is the rs2501432 which is resulted associated with different psychiatric disease and which results in the substitution of a glutamine with an arginine in position 63 of aminoacid chain (Q63R). Onaivi and Ishiguro studied the association between the rs2501432 CBR2 SNP and drug abuse and depression in Japanese population finding a significant difference in allelic frequency between depressive and drug abuser subjects respect a control population. In 2009 Ishiguro et al. conduced a similar study using as case group schizophrenic patients and they showed a significant difference about the distributions in case and control groups for the rs2501432 and for the C allele of rs12744386 which altered the CBR2 mRNA expression (Ishiguro et al., 2009). To understand better the role of CBR2 in schizophrenia Ishiguro and
colleagues tested CBR2 ligands responses in
CHO (Chinese Hamster Ovary) transfected with the gene containing the SNP rs2501432 observing a quite strong reduction in CBR2 signaling (Ishiguro et al., 2009).
Pharmacological studies performed in animal models and in vitro supported these evidences showing that the receptor transcribed from the gene with R63 allele appeared to have reduced responses to three CBR ligands 2-AG, AM630, a CBR2 receptor inverse agonist and JWH-015, a CBR2 agonist, observed in cAMP activity in cultured cells (Ishiguro et al., 2010). Low function of the receptor seemed to have impact on several other physical disorders, such as autoimmune disorders as well as neuropsychiatric disorders. In fact rs2501432 was reported to be associated with autoimmune disease and with human osteoporosis(Karsak et al., 2005; Sipe et al., 2005). Sipe et al. reported its functional change from the polymorphism in the immune system in vitro (Sipe et al., 2005). Schizophrenic patients have lower bone mass than the community population since they are young, while aging and menopausal transition effect on bone mass in the general female population cannot be seen in the schizophrenic patient group (Renn et al., 2009). Abnormalities in peripheral immune cells have been indicated in schizophrenia and numerous epidemiological studies have associated schizophrenia with autoimmunity and allergies (Muller and Schwarz, 2006; Patterson, 2009; Strous and Shoenfeld, 2006).
30 In Carrasquer et al. (2009) study the CBR2 wild type and polymorphic receptors, for the SNPs rs2501432 and rs2229579, expressed in HEK293 cells, bound a structurally diverse group of cannabinoid ligands and maintained both ligand-stimulated and constitutive activities. In general, binding affinities of the CBR2 polymorphic receptors compared with CBR2 wild type response were similar because rs2501432 and rs2229579 are located on the cytoplasmic side of the plasma membrane; however the authors showed a reduced affinity of 2-AG for the rs2229579 polymorphic receptor. The same group of research showed how in ligand-induced inhibition of phosphokolin-stimulated cyclic AMP accumulation assays, WIN55212-2 and 2-AG were less efficacious in cells expressing polymorphic sites than in cell expressing wild type CBR2 (Carrasquer et al., 2009). These results suggest that the activation of the receptor has been modified. It is possible that these SNPs might cause a shift in the conformation of the receptor; the detailed molecular mechanisms regarding the differences between wild type CBR2 and polymorphic receptors include the possibilities of receptor of desensitization, receptor cycling and reduction in receptor-G protein coupling (Carrasquer et al., 2009).
Regarding the rs41311993 SNP of CBR2 is it possible that the polymorphic substitution could modify the inter-helix hydrogen bonds between the helix I, II, III and V of CBR2 structure and this may result in an altered signaling response after cannabinoid or EC binding.
FAAH polymorphisms have been studied in psychiatric disorder too. Associations were observed between the SNP rs324420 of FAAH and eating disorders such as obesity (Lieb et al., 2009), anorexia and bulimia nervosa (Monteleone et al., 2009) and also with mood disorders like major depression and BD (Monteleone et al., 2010), drug addiction and alcoholism (Sipe et al., 2002).
The rs324420 SNP is missense polymorphism which determines the translation of the amino acid codon to a threonine instead of a proline in 129 position a amino acids chain (Sipe et al., 2002). Although this genetic variant presents normal catalytic properties, an increased sensitivity to proteolitic degradation has been suggested. This altered turnover of the enzyme might determine as consequence an altered metasbolism of AEA (Sipe et al., 2002).
31
Chapter 3
Neuroinflammation, CBR2 and Bipolar Disorder
Role of ECS in Central Nervous System inflammation
Chronic activation of microglia plays a major role in disorders characterized by nervous tissue inflammation (Matsumoto et al., 1992). CBR1 is expressed constitutively in microglia cells while CBR2 expression is not present in resting cells and is related to the cell activation state (Cabral et al., 2008). Microglia, activated by main injury and other stimuli, including cytokines such as TNF-IL-1 and IL-12, secretes mediators that regulate phenotype of T helper cells and inflammation inside the central nervous system (CNS) (Aloisi et al., 2000). Its activation may amplify damage by releasing toxic cytokines and mediators above described and for this reason cannabinoids may help the survival of neurons and axons by interfering this process and several experiments have shown the key importance of CBR2 in the inhibition of pro-inflammatory cytokines by cannabinoids (Ashton et al., 2007; Ehrhart et al.,2005) (Fig 11).
Cannabinoids capability to decrease oxidative injury by acting as scavengers of ROS or by enhancing endogenous antioxidant defenses and their anti-inflammatory activity, that is exerted predominantly through CBR2 on glial cells, in which cannabinoids would enhance neuronal survival by inhibiting microglia-mediated cytotoxic influencs and/or by increasing astroglia-mediated positive effects, are the two most important properties of these molecules.
A number of studies from mouse to human subjects, using a variety of techniques, show the functional presence of CBR2 in neural progenitor cells, neurons, glial and endothelial cells (Brusco et al., 2008; Chin et al., 2008; Garcia-Gutierrez et al., 2010; Palazuelos et al., 2006; Viscomi et al., 2009). The role of CBR2 in these cells involves neuroinflammation and neuropathic pain but future studies should be focused on the neurobiological basis, physiological activity of this receptor and its interaction with CBR1.
However, research on the involvement of CBR2 in neuroinflammatory conditions and in neuropathic pain has advanced more than other areas in neuropsychiatry and drug addiction (Onaivi et al., 2011). Therefore, improved information about CNR2 gene and its human variants might add to our understanding not only of the role of CBR2 during
32 neuroinflammatory conditions in the CNS but also beyond neuro-immuno-cannabinoid activity (Onaivi et al., 2011).
33 Neuroinflammatory hypothesis of BD
The macrophage theory of depression was articulated about 20 years ago, also before the discovery of CBRs, correlated to some observations such as pro-inflammatory cytokines pathway in depressive symptoms and that depression usually occurs with symptoms related to inflammatory disease (Smith, 1991). Since that time accumulating evidence indicates that alteration in inflammation is crucial to pathogenesis and treatment of major depression disorder (MDD) (Schiepers et al., 2005). Several studies have investigated the role of inflammation in different neuropsychiatric disorder like schizophrenia and Alzheimer disease and they have showed how in health volunteers a provocation of pro-inflammatory response is accompanied by increased levels of affective symptoms and decrease of neurocognitive performance (Reichenberg et al., 2001). These studies suggest that pro-inflammatory cytokines may subserve depressive symptomatology probably by activation of HPA axis and by affecting central monoaminergic system though several factors (Barkhundaryan et al., 1999; Muller et al., 1998).
The role of pro-inflammatory markers (PIM) such as cytokines, TNF- and ILs has been well studied by Goldstein and colleagues (Goldstein et al., 2009) first of all showing how these markers increase in mania phase of BD. Genetic foundings have suggested that BD is associated to IL11 and IL6 gene, and maybe also with TNF- polymorphisms. Moreover the high prevalence of comorbid medical illnesses, smoking and alcohol abuse, combined with decrements of physical activity and sleep parameters, contribute to a pro-inflammatory hypothesis of BD.
Finally, conventional anti-inflammatory therapies may possess symptoms-alleviating effects among acutely ill patients at early stages of their illness (Leonard, 2001) .
34
Aim of the study
BP, an illness that affects an estimated 2.3 million American adults, has been characterized in many different ways. The original diagnosis of ―manic-depressive insanity,‖ described by Emil Kraepelin in the 1899 edition of Clinical Psychiatry, has evolved through the years to the current classification system of four subtypes: Bipolar I Disorder, Bipolar II Disorder, Cyclothymic Disorder and Bipolar Disorder Not Otherwise Specified. The primary mood disturbance in Bipolar I Disorder is either mania or a mixed episode and it is usually accompanied by episodes of depression while the primary mood disturbance in Bipolar II disorder is depression and it is accompanied by at least one episode of a mild form of mania called hypomania. Instead, an individual with Cyclothymic Disorder cycles between periods of hypomanic symptoms and periods of depressive Symptoms. The hypomanic symptoms are never severe enough to be considered a manic episode and the depressive symptoms are never severe enough to be considered a depressive episode. BD Not Otherwise Specified captures all of the other variants of the disease that do not fit nearly into one of the above categories.
The etiology of BD is very complex. A lot of various biological system are involved in neurobiology of this disorder impairing function or just changing in morphology.
The best characterized neurotransmitter systems involved in pathophysiology of BD are dopaminergic and serotononinergic, but also GABAergic and glutamatergic (Manji et al., 2003).
More recently also the ECS has been identified as an important target of several psychiatric disorders including the bipolar one.
In the past a lot of studies have investigated more the role of CBR1 in CNS than CBR2 because the expression of this latter receptor was considered only at microglia levels; recently the discovery of mRNA of the isoforms CBR2A in very important regions of human brain such as caudate, amygdale, hippocampus, cerebellum, nucleus accumbens, putamen and cortex (Liu et al., 2009) could permit to understand better the role of CBR2 not only in nuroinflammation process but also in neurotransmission and so also in depression and related disorders, as is BD..
Then, the important role of CBR2 as mediator of PIM and the evidences that a lot of bipolar patients suffer of obesity, insulin resistance, arthritis, pain and cardiovascular illness, all pathologies correlated to alteration of CBR2, suggest that this CBR could really interfere with the pathology of BD.
35 So, in this study we tested the hypothesis that genetic variants of CNR2 might be associated with BD. In particular, by means of a case–control study the frequencies of three missense SNPs of CNR2, namely rs2501432 (315A>G; Arg63Gln), rs41311993 (524C>A; Leu133Ile) and rs2229579 (1073C>T; Tyr316His) (Fig.12) have been assessed in a sample of 80 BD patients and 160 healthy control.
Fig12. Amminoacid changes in the CBR2 sequence corresponding to the three polymorphisms under investigation: rs2501432 (315A>G; Arg63Gln), rs41311993 (524C>A, Leu133Ile) and rs2229579(103C>T; Tyr316His).
The comparison between patients and healthy group shows that allele frequencies were significantly different for CNR2 524C>A (p(χ2) =0.001) while there was no significant difference for CNR2 315A>G (p(χ2) =0.15) or1073C>T (p(χ2) =0.21).
The CNR2 524C>A substitution leads to the aminoacid change Leu133Ile which has been suggested to influence the stability and/or functionality of the CBR2. In fact, Leu133 provides a hydrophobic residue involved in an intrachain bond in the transmembrane domains and its presence is thought to be crucial for the stability of the receptor and may influence the receptor-G protein coupling (Xie et al., 2003).
36
Materials and methods
Bipolar patients were recruited from adult outpatients in treatment at the Psychiatric Clinic of the University of Pisa; the recruitment phase was carried out between September 2009 and November 2010. Aims of the study were thoroughly explained to potential participants and an appointment was arranged for the clinical interview, after obtaining their written consent to participate. Thefinal sample included 80 subjects (age 40± 2,5, 17 males and 53 females). The diagnostic assessment was carried out by clinicians, using the Structured Clinical Interview for DSM-IV axis-I disorders (SCID-I/ P) (First et al., 1995). Socio-demographic variables were also collected.
366 control subjects were recruited among students of University of Pisa and healthy volunteers and we selected 160 age and gender matched subjects for statistical analysis. Study procedures were approved by the Ethical Committee of the Azienda Ospedaliero- Universitaria di Pisa, according to the code of Ethics of the World Medical Association (Declaration of Helsinki).
Criteria to choose the samples
Criteria to be included in the control group
1. Being Italian
2. Age range 18-60 years
3. Signing the authorization form to analyze the own DNA
Criteria to be included in the case group
1. Being Italian
2. Age rang 18-60 years 3. Being Bipolar
4. Signing the authorization form to analyze the own DNA
Criteria to be excluded from the control group
1. Use of drugs
2. Being unable to understand the authorization form 3. Poor cooperation
