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Schizophrenia is a mental condition characterised by abnormalities in the perception or expression of reality, and its manifestations include chronic psychosis and social, occupational, behavioural, and cognitive impairment (Lewis and Lieberman, 2000).
There are several theories of the biological origin of schizophrenia, most of them involving aberrant neurotransmission systems. Reinforcement for the dopamine hypothesis of schizophrenia is derived from over 40 years of molecular, pharmacological and clinical evidence. This hypothesis attributes the symptoms of the disease to an altered and hyperactive production of dopamine in the brain of schizophrenic patients compared to a healthy brain (Sayed and Garrison, 1983).
For instance, neuroleptic drugs that are antagonists for dopamine receptors have a beneficial effect on schizophrenia; imaging studies, using single photon computed tomography (SPECT) and positron emission tomography (PET) techniques, showed an elevation of D2 receptor density parameters in schizophrenia compared to healthy controls; and studies of presynaptic neurons revealed a greater increase in dopamine transmission response and DOPA decarboxylase activity compared to healthy controls, when administered amphetamine (Laruelle, 1998; Laruelle et al., 1999; Kennedy, et al., 2001). Thus, the dopaminergic system is an important target for analysis in regard to disease identification and treatment.
This essay aims to review the signalling pathways involved in the dopamine system and the methodologies used to identify aberrant components present in schizophrenia. Numerous research techniques are currently used to study protein-protein interactions associated with dopamine-dependant signalling. While each strategy has significant strengths and limitations, the techniques discussed in the following are all commonly performed and result in meaningful data in the study of the pathophysiology of psychiatric illnesses.
Dopamine Signalling Pathways
The dopamine is important for the modulation of many brain functions, such as cognition, attention, reward, working memory (Roesch-Ely, et al., 2005) and locomotor regulation (Rajput et al., 2008). Dopamine belongs to the family of slow acting neurotransmitters, and acts as a neuromodulator, capable of altering the responses of target neurons to other neurotransmitters depending on the functional state of these neurons. Abnormalities in dopamine transmission are associated with assorted neurological and psychiatric conditions, such as Parkinson's disease, drug addiction, compulsive behaviour, attention-deficit hyperactivity disorder and schizophrenia (Ref 88).
Dopamine exerts its effect on neurons through five different seven-transmembrane G-protein-coupled receptors (GPCRs) known as D1, D2, D3, D4 and D5. These receptors are subdivided into two groups: D1-like (D1 and D5) and D2-like (D2, D3 and D4).
The G protein Î± subunit (GÎ±) binds guanine nucleotides and cycles between an inactive GDP-bound state and an active GTP-bound state (Oldham et al., 2008). It appears that the binding to different GÎ± subunits mediates differential activities; when dopamine binds to GÎ±s-coupled receptors, the enzyme adenylate cyclase is activated and the secondary messenger cAMP is produced. Contrary to this, when dopamine binds to the GÎ±i/o-coupled D2 receptors, adenylate cyclase activity is blocked and cAMP production is reduced (Neve et al., 2004). Together with the increase of calcium produced by glutamate, cAMP activates a number of signalling pathways involved in the regulation of excitability and responsiveness of striatal neurons. Receptor subtypes also differ in their affinity for dopamine and coupling to downstream effectors, such as G proteins in those signalling pathways (Callier et al., 2003).
Dopamine activation of its receptors activates a variety of signalling pathways, including the PKA, PLC, ERK1/2, and Akt/GSK-3Î² pathways. Figure 1 illustrates the complexity of the downstream signalling machinery of the D2 dopamine receptor.
Figure 1: The D2 receptor and its downstream signalling pathways (from Bibb, 2005)
Dopamine receptor subtypes are expressed differentially throughout the brain. From both families, D1 and D2 receptors are the most abundant, with mRNA of the former expressed in the neostriatum, nucleus accumbens, and olfactory tubercle and, at lower levels, in the cerebral cortex, hypothalamus, and thalamus. D2 receptors, however, are predominantly expressed in the neostriatum, nucleus accumbens, and olfactory tubercle, as well as the midbrain and the pituitary (Meador-Woodruff et al., 1991).
Dopamine Receptors in Schizophrenia
Several studies have shown the involvement of D1 receptors in schizophrenia by the ability of D1 receptor-acting drugs to alter working memory performance. In addition, Lidow and colleagues demonstrated the therapeutic activity-such as reduction of psychotic symptoms-of dopamine D1 (Lidow et al., 1998) and D2 (Lidow, 2000) receptor antagonists.
Figure 2. Two signalling pathways downstream of the dopamine receptor
Dopamine Receptor-Interacting Proteins and Their Involvement In Schizophrenia
To achieve a comprehensive understanding of the mechanisms of dopamine receptor signalling, the effects of cellular protein interactions must also be taken into account. The recent identification of a group of dopamine receptor-interacting proteins (DRIPs) suggests that the intracellular activity of individual dopamine receptor subtypes is regulated by the combined actions of a group of cytoskeletal, adaptor and signalling proteins. DRIPs regulate many aspects of dopamine receptor biology through protein-protein interaction, they such as trafficking and desensitisation (Bergson et al., 2003). This way, the dopamine receptor exists as part of macromolecular protein complexes, also known as signalplexes or signalosomes. These protein complexes illustrate how a single receptor is capable of activating different G-proteins and associated effector systems or different intracellular pathways by interacting with and activating other proteins (Beaulieu et al, 2005). The purpose of the signalplex, therefore, is to enable the dopamine receptor to adapt and perform optimally and rapidly within a changing cellular environment, based on factors such as receptor maturity, cytosolic pH, calcium concentration, and ligand binding. As a result, signalosomes provides a sharp level of organisation within the cell for spatial and temporal control of signalling, facilitating the many functions mediated by dopamine in different organs and even different cell types (Hazelwood et al., 2008.).
The key role of DRIPs in dopamine signalling has raised the possibility that they may play a key role in the abnormalities observed in schizophrenia. We shall review some of the recently discovered DRIPs involved in dopamine signalling in what follows.
Figure 3. A list of DRIPs showing which dopamine receptor subtype they interact with. The DRIPs are grouped into classes based on cellular functions. (from Bergson et al., 2005)
In both primates and rodents, treatment with antipsychotic drugs is shown to promote an alteration in the expression of D1-like and D2-like receptors in the cortex and striatum . In a recent report, chronic treatment of mice with two classes of antipsychotic drugs was found to promote a change in the expression of DRIPs within the cortex . In light of additional findings that indicate that levels of the D1-like DRIP calcyon and the D2-like DRIP NCS-1 are elevated in the prefrontal cortex of schizophrenic patients [95,96], it is suggested that DRIPs represent a class of molecules underlying adaptations of affected neuronal networks in schizophrenia [97,98].
Yeast two hybrid system and DRIPS
Many of the known DRIPs have been identified using the yeast two-hybrid (Y2H) system. This system uses segments of a dopamine receptor to identify interacting proteins from brain cDNA libraries. Using this technique, it was found that D1 and D2 receptor subtypes interact with two independent sets of DRIPs, suggesting that both receptor subfamilies may transduce different signalling complexes. Using Y2H systems, Bermak et al (2001) showed that a carboxy-terminal hydrophobic motif (FxxxFxxxF) conserved amongst GPCRs, functions independently as an endoplasmic reticulum (ER)-export signal for the D1 receptor. Further, an ER-membrane-associated protein termed DRiP78 was found to bind the D1 receptor within this motif, and to sequester the receptor in the ER. The interaction between DRiP78 and the D1 receptor is suggested to play an important role in regulating the trafficking of D1 receptors to the cell surface (Dupre et al., 2007).
Another study using Y2H, employed the third intracellular loop of the D2S and D3 dopamine receptors in order to screen a human brain cDNA library; this way, they identified the protein filamin A (FLN-A), which interacts with both receptors. Furthermore, they showed that expression of D2 receptors in FLN-A-deficient cells was mainly localised intracellularly, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localised at the plasma membrane, suggesting that the identified protein may be required for correct cell surface expression of the D2 receptor (Lin et al., 2001).
Proteomics and DRIPs
Proteomic techniques have also been shown to be valuable in the identification of novel DRIPs. One of the first examples of the use of proteomics techniques was that of Edgar and colleagues (1999). Using 2 dimensional gel electrophoresis (2D-GE), they identified four different proteins differentially expressed in schizophrenia: diazepam binding protein (DBP) and manganese superoxide dismutase (MnSOD), both of them shown to be under-expressed, and collapsing response mediator protein 2 (CRMP-2) and t-complex protein 1 (TCP-1), which were over-expressed. DBP is a regulator of gamma-aminobutyric acid (GABA) actions, while MnSOD is involved in the protection and survival of cell membranes. On the other hand, CRMP-2 is a regulator of axonal growth and polarity, and TCP-1 aides proper protein folding and arrangement, thus protecting neurons against apoptotic signals (reviewed by Lakhan, 2006). The role of these proteins in the correct functioning of neuronal systems is crucial. Consequently, to identify their abnormal expression in schizophrenia could lead to new therapies.
Surface enhanced laser/desorption ionisation (SELDI) technology, is a variation of mass spectrometry, which is based on ProteinChip arrays. Each chip offers a distinctive chromatographic surface for selective protein capture. A recent study using ProteinChip technology has identified 21 protein biomarker peaks with lower levels in schizophrenia group as compared to the controls. The proteins constituting these biomarker peaks were recognised via matrix-assisted laser desorption time of flight/postsource decay mass spectrometry (MALDI-TOF-PSD-MS), and represented a wide range of functional groups implicated in cell metabolism, signalling cascades, regulation of gene transcription, protein and RNA chaperoning, and other aspects of cellular homeostasis (Novikova et al., 2006).
Calcyon, Par-4 and NCS-1
Calcyon is a single transmembrane protein largely expressed in the brain. Using receptor autoradiography techniques, Lezcano and colleagues (2000) localised this protein to membranous intracellular compartments within neuronal dendrites and dendritic spines. Studies in primates and mice have shown that calcyon is highly expressed in multiple brain regions, including the prefrontal cortex, which mediates cognitive-executive functions (Lezcano et al., 2000). Calcyon has been shown to bind to D1 and D5 receptors and activate the release of intracellular calcium via activation of the phosphoinositide second messenger cascade (Lezcano et al., 2000), a crucial process in dopamine signalling. A significant increase in the levels of calcyon has been observed in the dorsolateral prefrontal cortex of schizophrenic patients (Koh et al., 2003)
Park et al (2005) have identified a proapoptotic protein, prostate apoptosis response 4 (Par-4), essential for the D2-mediated inhibition of cAMP activity. Par-4 is a leucine zipper-containing protein that regulates cell survival during development and plays a role in neurodegeneration in the brain. They further demonstrated that Par-4 directly interacts with the dopamine D2 receptor within the calmodulin binding domain. Moreover, to examine the role of Par-4 in dopamine signalling and its interaction to the D2 receptor in vivo, they used mutant mice lacking the D2 receptor interacting leucine zipper domain. Interestingly, the mice displayed specific behavioural phenotypes reminiscent of depression, signifying a role for the association of Par-4 and the D2 receptor in the regulation of behaviour.
Nef et al (1995) reported the characterisation of a novel neuronal calcium sensor (NCS) named NCS-1 by Northern analysis and in situ hybridisation. NCS-1 is a calcium binding protein from the recoverin subfamily, which is present in neuronal cells all through the brain. This protein has been shown to form complexes with G-protein-coupled receptor kinase-2 (GRK2) and D2 receptor, and it is capable of preventing GRK2-mediated desensitisation of activated D2 receptor in a calcium-dependent manner (Kabbani et al., 2002), thereby enhancing the influence of D2 receptors on cell activity. This protein has also been observed to be increased in schizophrenic patients (Koh et al., 2003b). Bai et al (2004) used western blot and real time rtPCR to demonstrate that both NCS-1 and calcyon were elevated in the prefrontal cortex of schizophrenic patients. Increased NCS-1 expression may result in overactive D2 receptor signalling, and might help explain the efficacy of D2 receptor antagonists in treating the positive symptoms of schizophrenia.
Arresting dependent signalling
The scaffolding proteins Î²-arrestin 1 and 2 have also been associated with the termination of G-coupled receptor signalling and receptor desensitation and internalisation (Fergusson et al., 1996). In addition, they have been involved in the regulation of receptor signalling via the scaffolding of a variety of signalling molecules (Lefkowitz & Shenoy, 2005). Beaulieu et al (2005) used a functional in vivo approach in mice followed by protein identification by western blot to study phosphorilation of several proteins downstream of dopamine receptor activation. They showed that arrestin 2 is essential for inactivation of Akt by dopamine (see figure 2). The signalling process described by the authors involves the formation of protein complexes which include signalling proteins such as Akt, arrestin and protein phosphatases. Inhibition or modulation of any of the components of the complex will alter the functioning of the rest of the proteins.
This essay illustrates how many studies in the past 20 years have provided evidence for the existence of a number of proteins crucial in the dopamine signalling pathways and of their abnormalities in patients with schizophrenia. The complex interactions found in the dopamine pathway the wide variety of effects of this neurotransmitter, as well as the wide range of symptoms present in schizophrenia.
Many drug treatments available for schizophrenia target the D2 receptor, and D2 antagonists such as haloperidol or risperidone are effective reducing psychosis. However, the signalling pathways activated through D2 receptor are complex and not fully elucidated and, consequently the mechanism through which most of these drugs work are not totally understood, and therefore most of the currently available drug treatments produce adverse side effects that limit their therapeutic utility. The interaction of dopamine receptors with DRIPs, in the form of signalplexes, appears to regulate key aspects of receptor function. Consequently, further understanding of disruptions or modifications of these signalling complexes may help to unveil the biological basis of brain disorders, and the development of novel treatments for schizophrenia and other brain diseases. In fact, the potential therapeutic use in manipulating dopamine receptor interactions with DRIPs such as Par-4 has already been demonstrated (Park et al., 2005).
To date, no laboratory diagnosis for schizophrenia exists. Consequently the understanding of the defects found DRIPs and their link to a particular symptom of brain disease may help the diagnosis and prognosis, and find an appropriate and tailored treatment for each patient.