Reevaluation of the dopamine D₂ receptor in the treatment of schizophrenia: Novel intracellular mechanisms as predictors of antipsychotic efficacy
Since the advent of antipsychotic medications, there have been many speculations about their precise mechanisms of therapeutic action. Although it is apparent that blockade of dopamine D₂ receptors (D₂R) is crucial to the efficacy of all current antipsychotic medications, it is not clear which signaling events downstream of the D₂R must be blocked for the therapeutic actions of antipsychotics and which events, when blocked, lead instead to side effects.

The best characterized D₂R-mediated signaling pathways involve coupling of the receptor to pertussis toxin-sensitive G proteins of the G_i and G_o subfamilies (Sidhu and Niznik, 2000), through which D₂R activation results in a decrease in cyclic AMP (cAMP). D₂R activation can also have a number of other effects, including...  Read more

Reevaluation of the dopamine D₂ receptor in the treatment of schizophrenia: Novel intracellular mechanisms as predictors of antipsychotic efficacy
Since the advent of antipsychotic medications, there have been many speculations about their precise mechanisms of therapeutic action. Although it is apparent that blockade of dopamine D₂ receptors (D₂R) is crucial to the efficacy of all current antipsychotic medications, it is not clear which signaling events downstream of the D₂R must be blocked for the therapeutic actions of antipsychotics and which events, when blocked, lead instead to side effects.

The best characterized D₂R-mediated signaling pathways involve coupling of the receptor to pertussis toxin-sensitive G proteins of the G_i and G_o subfamilies (Sidhu and Niznik, 2000), through which D₂R activation results in a decrease in cyclic AMP (cAMP). D₂R activation can also have a number of other effects, including enhancement of specific potassium currents, inhibition of L-type calcium currents, mediation of extracellular signal-regulated kinase 1 (ERK1) and potentiation of arachidonic acid release (Beom et al., 2004; Missale et al., 1998; Perez et al., 2006; Hernández-López et al., 2000). There is growing evidence that D₂Rs can interact with a number of membrane-bound or intracellular proteins, which may further modulate signaling specificity (reviewed in Terrillon and Bouvier, 2004; Ferré et al., 2007a). In particular, D₂R heteromerization may result in a switch from G_i/_o coupling to G_s (i.e., through D₂R and cannabinoid 1 receptor interaction) (Kearn et al., 2005) or to coupling with G_q (as suggested in D₂R and D₁R interactions) (Rashid et al., 2007). Moreover, heteromerization between D₂R and other receptors such as the adenosine A_2A receptor may allow for reciprocal modulation of D₂R function (Ferré et al., 2007a; Ferré et al., 2007b). It also has been suggested that calcium signaling mechanisms may modulate D₂R’s signaling efficacy; interaction between D₂R and calcium-binding protein S100B results in enhanced D₂R intracellular signaling (Liu et al., 2008; Stanwood, 2008).

The interaction between D₂R and arrestin has received increasing attention. Following D₂R activation, D₂R signaling is attenuated by recruitment of arrestin 3 to the cell surface where it binds to the receptor (Klewe et al., 2008; Lan et al., 2008a ; Lan et al., 2008b), leading to inactivation and internalization of the D₂R. Arrestin 3 also binds Akt—a serine/threonine kinase involved in multiple cellular functions and implicated clinically in schizophrenia (Arguello and Gogos; 2008; Beaulieu et al., 2005; Brazil and Hemmings, 2001; Brazil et al., 2004; Emamian et al., 2004; Kalkman, 2006). Following D₂R activation by dopamine, the signaling scaffold formed by arrestin 3, while facilitating receptor desensitization and internalization, also recruits Akt into a complex with the phosphatase PP2A, which dephosphorylates and consequently inactivates Akt (Beaulieu et al., 2007a ). Thus, D₂R activation inhibits Akt activity through an arrestin-dependent but G protein-independent pathway (Beaulieu et al., 2007a ; Beaulieu et al., 2007b). Curiously, the mood stabilizer, lithium, has been shown to disrupt the arrestin 3-Akt-PP2A complex, thereby preventing dopamine-induced dephosphorylation of Akt and blocking amphetamine-induced locomotion (Beaulieu et al., 2008). Moreover, amphetamine-induced locomotion is greatly diminished in arrestin 3 knockout mice, suggesting that this pathway is critical to at least some psychostimulant effects (Beaulieu et al., 2005).

Using newly developed BRET (bioluminescent resonance energy transfer) biosensors in assays that measure direct protein-protein interactions within the living cell, recent studies have demonstrated that antipsychotic medications prevent arrestin 3 recruitment by blocking D₂R activation (Klewe et al., 2008; Masri et al., 2008). Masri et al. (2008) hypothesized that antipsychotic drugs achieve their therapeutic effect through a common mechanism involving blockade of arrestin-mediated signaling (Masri et al., 2008). Masri et al. (2008) also demonstrated that nearly all antipsychotics tested (including haloperidol, clozapine, olanzapine, desmethylclozapine, chlorpromazine, quetiapine, risperidone and ziprasidone) behave as inverse agonists to decrease constitutive G protein signaling as well as to prevent the agonist quinpirole from inhibiting cAMP synthesis (via D₂R-mediated G_i/_o signaling). The lone exception, aripiprazole, behaved as a partial agonist instead of as an inverse agonist of the G protein mediated effects. The latter finding is consistent with previous studies highlighting aripiprazole’s ability to differentially modulate various G protein-mediated effector pathways, a property termed “functional selectivity” (Mailman, 2007; Urban et al., 2007). Using the BRET assay, Klewe et al. (2008) and more recently Masri et al. (2008) demonstrated that all antipsychotics, including aripiprazole, block arrestin 3 recruitment. This finding has led Masri et al. (2008) to suggest that blockade of arrestin 3 recruitment to the D₂R, and not modulation of G-protein-mediated pathways, is a common and specific property of all current antipsychotics and may be used to predict the antipsychotic efficacy of drugs in development (Masri et al., 2008). This hypothesis remains to be tested and at present appears to lean heavily on the evidence for aripiprazole’s atypical effects on constitutive (non-agonist-dependent) D₂R-mediated G-protein signaling. Indeed, the fact that lithium acts to prevent arrestin-mediated signaling in response to amphetamine but is not an effective antipsychotic in monotherapy suggests that antipsychotic action may be more complex than simple blockade of D₂R-mediated arrestin signaling. In addition, the ability of antipsychotics, including aripiprazole, to block agonist binding to the D₂R and thus activation of the receptor, makes it likely that agonist-induced activity in multiple signaling pathways will also be blocked by these drugs.

Despite the paucity of direct evidence for D₂R-arrestin coupling as the mechanism underlying the antipsychotic effects of drugs, the hypothesis remains quite intriguing Given that Akt and its downstream target GSK-3 (glycogen synthase kinase-3) have been implicated in schizophrenia in a number of genetic and postmortem studies, and the Akt/GSK-3 pathway might represent an opening into alternative therapeutics of schizophrenia. Akt is a serine/threonine kinase that may have significant roles in synaptic physiology and neurodegeneration (Brazil et al., 2004). Recruited to the cell surface by binding to phosphatidylinositol 3,4,5 trisphosphate, Akt is activated via phosphorylation of 3-phosphoinoitide-dependent protein kinase 1 (PDK1) and the rictor-mTOR complex (Brazil and Hemmings, 2001; Sarbassov et al., 2005). Once active, Akt phosphorylates GSK-3, thereby inactivating it. Since D₂R activation leads to inactivation of Akt, this also results in increased GSK-3 activity (Beaulieu et al., 2004; Lovestone et al., 2007). GSK-3 activity also plays an important role in modulating the dopaminergic response to amphetamine. Amphetamine’s stimulation of DAT-mediated dopamine efflux and subsequent D₂R stimulation likely results in Akt inactivation and increased GSK-3 activity. Rats treated with the specific GSK-3 inhibitor, AR-A014418, failed to display amphetamine-induced hyperactivity (Gould et al., 2004). Similarly, heterozygous GSK-3β knockout mice (expressing approximately half of wildtype levels of GSK-3β) displayed significantly reduced levels of locomotor activity following amphetamine treatment (Beaulieu et al., 2004). Additionally, treatment of dopamine transporter (DAT) knockout mice with multiple GSK-3 inhibitory drugs inhibited the ordinarily hyperactive behavior of the non-treated DAT knockout mice (Beaulieu et al., 2004).

In a mouse model, acute and chronic haloperidol treatment was shown to increase levels of active, phosphorylated Akt isoform Akt1 and increased phosphorylation and inactivation of GSK-3β (Emamian et al., 2004). Thus, it was suggested that haloperidol treatment may compensate for the decreased levels of endogenous Akt1 in the frontal cortex of people with schizophrenia (Emamian et al., 2004). Atypical antipsychotics also impact on the regulation of Akt and GSK-3β activities. For example, treatment with clozapine results in increased levels of phosphorylated GSK-3β (Kang et al., 2004; Sutton et al., 2007). Interestingly, however, differences between haloperidol and atypical antipsychotics have emerged in the kinetics of Akt/GSK-3 phosphorylation, the levels of proteins expressed following drug exposure, and the signaling pathways that are preferentially activated (Roh et al., 2007).

The abilities of antipsychotic drugs to activate distinct signaling pathways to mediate their ostensible differential pharmacologic effects would suggest clinical variation in their therapeutic effects. However, meaningful differences in the clinical effects of these compounds have not been clearly or consistently evident. The initial reports of superior efficacy of the so-called second generation or atypical antipsychotics on measures of psychosis (Kane et al., 1988), negative symptoms (Tollefson et al., 1997), cognitive deficits (Keefe et al., 1999), relapse prevention (Csernansky et al., 2002), adherence (Wahlbeck et al., 2001) and illness progression (Lieberman et al., 2005a), have not been borne out by more recent studies (Geddes et al., 2000; Lieberman et al., 2005b; Jones et al., 2006; Leucht et al., 2008). Indeed, the differences between antipsychotic drugs are most evident in the types, frequency and severity of side effects rather than in their therapeutic actions (Leucht et al., 1999; Allison et al., 1999; Henderson et al., 2005). In this regard the emerging pattern of variation in the molecular mechanisms of antipsychotic drugs in the face of their common clinical profiles resembles what was previously observed with the variability in neuroreceptor binding profiles (Kinon and Lieberman, 1996). The marked differences in the affinities and selectivity of the various antipsychotics for the receptors of different neurotransmitters were thought to underlie a rich pattern of clinical variation in the therapeutic actions of this group of drugs (Miyamoto et al., 2005). However, this hypothesis has not been supported by clinical studies (Lieberman, 2006; Lewis and Lieberman, 2008).

Nevertheless, there is reason to be hopeful that through functional selectivity, or other potential actions, the abilities of drugs to engage different signaling pathways will confer novel therapeutic effects that will improve the efficacy of treatments. In this context, the studies of Masri et al. (2008) and Klewe et al. (2008) highlight the plausibility that D₂R/arrestin 3 modulation of Akt and GSK-3 activity is an important mechanism underlying psychosis and a potential target for future antipsychotic drugs. Further study of this pathway, including studies designed to reverse the effects of D₂R antagonists or partial agonists (antipsychotic drugs) with systematic differential manipulation of the signaling pathways induced by D₂R activation, is likely to be a fruitful path toward the development of novel treatments for schizophrenia-related disorders.

Acknowledgements: The authors would like to acknowledge the generous support of the Lieber Center for Schizophrenia Research at Columbia University

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Implicit in the findings of Schmid et al. is the idea that the relationship among ligand, receptor signaling, and cellular context is an extremely complex one that will take a great deal more work to tease out. Thus, Dr. Bryan Roth has proposed on a number of occasions (see, for example, Gray and Roth, 2007; Abbas and Roth, 2005) that novel approaches for drug discovery may prove more effective in producing schizophrenia drugs that have greater therapeutic efficacy with lower side effect liability. Since it will likely be many years before the field has a detailed understanding of the "nitty-gritty" of the receptor signaling and trafficking relevant to schizophrenia and its treatment, we have suggested a number of approaches that are less reliant on such information.

For example, approaches based on screening for drugs that either mimic the gene expression profiles of gold standard drugs such as clozapine or normalize schizophrenia-associated changes in gene expression are being...  Read more

Implicit in the findings of Schmid et al. is the idea that the relationship among ligand, receptor signaling, and cellular context is an extremely complex one that will take a great deal more work to tease out. Thus, Dr. Bryan Roth has proposed on a number of occasions (see, for example, Gray and Roth, 2007; Abbas and Roth, 2005) that novel approaches for drug discovery may prove more effective in producing schizophrenia drugs that have greater therapeutic efficacy with lower side effect liability. Since it will likely be many years before the field has a detailed understanding of the "nitty-gritty" of the receptor signaling and trafficking relevant to schizophrenia and its treatment, we have suggested a number of approaches that are less reliant on such information.

For example, approaches based on screening for drugs that either mimic the gene expression profiles of gold standard drugs such as clozapine or normalize schizophrenia-associated changes in gene expression are being explored. Another approach is behavior-based screening, in which targeted screens are performed with drugs to find those that have efficacy in animal disease models. A further related approach, exemplified by Psychogenics' Smartcube(TM) (the associated database is called Smartbase[TM]) involves injecting drugs and monitoring the resulting behavior using computer-based machine learning to generate a multidimensional behavioral signature for gold standard drugs. Drugs can then be screened to look for those that mimic gold standard drugs in terms of their signatures. Though Psychogenics does not appear to have done much (at least publicly) with this approach, it represents the sort of innovative thinking that may prove fruitful in future behavior-based drug discovery efforts since it is not dependent on knowing anything about the mechanism. In the end, at least in the near future, we believe such approaches may prove extremely useful in drug discovery efforts since they do not rely on extensive mechanistic knowledge of the processes underlying schizophrenia.

References:

Gray JA, Roth BL. The pipeline and future of drug development in schizophrenia. Mol Psychiatry. 2007 Oct ;12(10):904-22. Abstract

Abbas A, Roth B. Progress towards better understanding and treatment of major psychiatric illnesses. Drug Discov Today. 2005 Jul 15;10(14):960-2. Abstract

Some researchers in the field of psychiatric genetics have become somewhat pessimistic about the ability to detect robust genotype-phenotype correlations using the diagnostic criteria defined by DSM-IV. If we analyze tens of thousands of samples, the ensuing results may be statistically robust, but still the effect of common variant(s) of each gene will be modest. Recently, Tan et al. (2008) reported that the AKT1 gene SNP rs1130233 and its encompassing haplotypes are significantly associated with IQ/processing speed, activities that may reflect frontal cortex function. They also showed that performance in their psychological test battery is influenced not only by AKT1 genetic variants but also the well-known COMT gene non-synonymous polymorphism (SNP rs4680, Val158Met). By undertaking fMRI analysis, they intertwined the IQ/processing speed-frontal cortex-AKT1 signal-DA system, i.e., the. integration of multidimensional disciplines. In citing references (  Read more

Some researchers in the field of psychiatric genetics have become somewhat pessimistic about the ability to detect robust genotype-phenotype correlations using the diagnostic criteria defined by DSM-IV. If we analyze tens of thousands of samples, the ensuing results may be statistically robust, but still the effect of common variant(s) of each gene will be modest. Recently, Tan et al. (2008) reported that the AKT1 gene SNP rs1130233 and its encompassing haplotypes are significantly associated with IQ/processing speed, activities that may reflect frontal cortex function. They also showed that performance in their psychological test battery is influenced not only by AKT1 genetic variants but also the well-known COMT gene non-synonymous polymorphism (SNP rs4680, Val158Met). By undertaking fMRI analysis, they intertwined the IQ/processing speed-frontal cortex-AKT1 signal-DA system, i.e., the. integration of multidimensional disciplines. In citing references (Meyer-Lindenberg and Weinberger, 2006; Weinberger et al., 2001), they state that “there is a growing body of data showing that genes weakly associated with complex constellations of behavioral symptoms are much more strongly associated with in vivo brain measures.” Indeed, they have succeeded in explaining a possible role for AKT1 in brain execution capability, but have not provided convincing evidence for genetic associations between AKT1 and schizophrenia.

Their current results are elegantly derived from “a complex set of experiments addressing association of multiple variants in a gene with many phenotypic measures.” However, from a genetic perspective, we may still ask the following questions, irrelevant of the current study:

1. What is the genetic component (or heritability) of each psychological and imaging trait? Can variations in some of the psychological/cognitive/intellectual performances be fully captured by a single gene in an experimental set that examines, at the most, a hundred samples? We have learned the hard way from genetic association studies done in the 1990s, which examined a small number of samples, that we simply cannot trust those results. With regard to this point, the heritability calculations of so-called “endophenotypes” as reported by Greenwood et al. (2007) can give helpful information [also see Watanabe et al., 2007, supplementary Table S2]. There is the possibility that the genetic architecture of neurocognitive functions and imaging measures may not be simpler than the current disease category (entity).

2. Given the rapid advances in genotyping technology, we may be able to generate genome-wide genetic test results for every neuropsychiatric trait in the near future.

3. Because of the functional significance of AKT1 and the divergence in the signaling cascade downstream of AKT1, it would be wise to confine analysis to this gene. However, it is frustrating that we still do not know the functionally important SNP(s) of AKT1 in spite of numerous association studies.

4. Nackley et al. (2006) have convincingly demonstrated that the haplotype of the COMT gene constructed by synonymous SNPs has much more functional impact than the Val158Met polymorphism. Therefore, we would like to see the association studies examining this haplotype in future neuropsychiatric studies.

From a biochemical perspective, the following issues would be interesting and future targets for clarification:

1. The authors suggest that the coding synonymous variation of AKT1 affects protein expression, leading to the alteration of frontostriatal function and gray matter volume. The activity of AKT1 is regulated by its phosphorylation status. Therefore, readers would want to know whether the reduction of AKT1 expression levels actually affect the AKT signaling pathway. Behavioral analysis and an MRI study of Akt1 heterozygote knockout mice may provide relevant information.

2. Impairment of the AKT signal is known to result in tau hyperphosphorylation through activation of GSK3 as seen in Alzheimer disease brains. According to this idea, a reduction of AKT levels caused by SNP(s) should elicit hyperphosphorylation of tau and ultimately form neurofibrillary tangles (NFTs). In contrast, there are some reports suggesting the absence of NFTs and neuroinjury in elderly patients with schizophrenia (Arnold et al., 1998; Purohit et al., 1998). It is also reported that GSK3 is reduced in schizophrenia (Beasley et al., 2001). It would be interesting to know whether the genetic variation(s) of AKT1 that induce decreased protein expression affect tau accumulation.

3. Lithium inhibits the arrestin-Akt signal (Beaulieu et al., 2008). If so, it would be interesting to know whether lithium treatment can restore some of the effects of reduced AKT1 expression levels caused by the SNP(s) of interest.

References:

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Greenwood TA, Braff DL, Light GA, Cadenhead KS, Calkins ME, Dobie DJ, Freedman R, Green MF, Gur RE, Gur RC, Mintz J, Nuechterlein KH, Olincy A, Radant AD, Seidman LJ, Siever LJ, Silverman JM, Stone WS, Swerdlow NR, Tsuang DW, Tsuang MT, Turetsky BI, Schork NJ. Initial heritability analyses of endophenotypic measures for schizophrenia: the consortium on the genetics of schizophrenia. Arch Gen Psychiatry 2007 64:1242-1250. Abstract

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Several of the reports from the NYAS meeting describe the potential role of GSK-3β in DISC1 functions. This is one of two isoforms, and the other, GSK-3α, tends to get short shrift from researchers. This is problematic for several reasons. Firstly, the two isoforms, despite being derived from distinct genes, are essentially identical within their catalytic domains. Consequently, there are no small molecule inhibitors that that are isoform selective, and the two proteins are highly redundant (albeit not completely) in function. Secondly, in the case of DISC1, there are new data indicating a role for GSK-3α in DISC1 functions. Small molecule (isoform indiscriminate) inhibitors of GSK-3 restore behavioral deficits of DISC1 L100P animals, and this is also achieved by genetic inactivation of one allele of GSK-3α (Lipina et al., 2011). Examination of the brains of the DISC1 and DISC1/GSK-3α+/- animals revealed that dendritic spine density deficits observed in DISC1 L100P brains were restored upon deletion of one...  Read more

Several of the reports from the NYAS meeting describe the potential role of GSK-3β in DISC1 functions. This is one of two isoforms, and the other, GSK-3α, tends to get short shrift from researchers. This is problematic for several reasons. Firstly, the two isoforms, despite being derived from distinct genes, are essentially identical within their catalytic domains. Consequently, there are no small molecule inhibitors that that are isoform selective, and the two proteins are highly redundant (albeit not completely) in function. Secondly, in the case of DISC1, there are new data indicating a role for GSK-3α in DISC1 functions. Small molecule (isoform indiscriminate) inhibitors of GSK-3 restore behavioral deficits of DISC1 L100P animals, and this is also achieved by genetic inactivation of one allele of GSK-3α (Lipina et al., 2011). Examination of the brains of the DISC1 and DISC1/GSK-3α+/- animals revealed that dendritic spine density deficits observed in DISC1 L100P brains were restored upon deletion of one copy of GSK-3α (Lee et al., 2011).

From a therapeutic point of view, there appears to be no easy way to selectively inhibit only one isoform of GSK-3 (the only means is via RNA interference), so perhaps it is fortunate that both isoforms appear to play similar roles? Birds, on the other hand, appear to have selectively lost GSK-3α, though the consequences in terms of brain development and function are currently unclear (Alon et al., 2011).

References:

Lipina TV, Kaidanovich-Beilin O, Patel S, Wang M, Clapcote SJ, Liu F, Woodgett JR, Roder JC. (2011). Genetic and pharmacological evidence for schizophrenia-related Disc1 interaction with GSK-3. Synapse 65(3):234-48. Abstract

Lee FH, Kaidanovich-Beilin O, Roder JC, Woodgett JR, Wong AH. (2011) Genetic inactivation of GSK3α rescues spine deficits in Disc1-L100P mutant mice. Schizophr Res. Abstract

Alon LT, Pietrokovski S, Barkan S, Avrahami L, Kaidanovich-Beilin O, Woodgett JR, Barnea A, Eldar-Finkelman H. (2011) Selective loss of glycogen synthase kinase-3α in birds reveals distinct roles for GSK-3 isozymes in tau phosphorylation. FEBS Lett. 585(8):1158-62. Abstract