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Unfinished Business: Understanding Drug Effects on Dopamine Pathways

25 July 2008. Since its discovery 25 years ago by Nobel prize winner Paul Greengard, the phosphatase and kinase inhibitor DARPP-32 (dopamine- and cAMP-regulated protein of 32 kilodaltons) has proven to be a central signaling molecule in the dopamine pathway, and in the action of other neurotransmitters (see SRF related news story and also SRF news story for a perspective on Greengard’s work on the protein). Dopamine signaling is altered in schizophrenia, as are DARPP-32 levels (Albert et al., 2002), and genetic variation in DARPP-32 was recently tied to schizophrenia risk (see SRF related news story). Just how DARPP-32 might figure in producing the symptoms of schizophrenia, however, is unclear.

In a paper in the July 11 issue of Nature Neuroscience, Greengard and colleagues at the Rockefeller University in New York take another step toward unraveling the tangled web of DARPP-32’s actions. They report how two drugs—cocaine, a psychostimulant, and haloperidol, a sedative antipsychotic still being used to treat schizophrenia—that on the surface appear to similarly alter the phosphorylation DARPP-32 in the same brain region, actually target the protein in distinct neuronal subpopulations. The work provides a possible explanation for the diverse behavioral effects of the two agents. Along the way, first author Helen Bateup and colleagues present a powerful approach to studying cell type-specific phosphorylation of proteins and provide a cautionary tale about making biochemical observations on mixed populations of cells.

In other news concerning the mechanism of action of a different type of drug, Randy Blakely and Aurelio Galli of the Vanderbilt University Medical Center in Nashville, Tennessee, report in the July 9 Journal of Neuroscience that a rare, human dopamine transporter (DAT) variant associated with attention-deficit hyperactivity disorder (ADHD) triggers a reversal of the transporter action to cause dopamine efflux from cells. This is intriguing because the “reversal of function” mutant mimics the action of amphetamine, a common ADHD treatment that is thought to raise synaptic dopamine in part by stimulating DAT-mediated dopamine efflux. Surprisingly, amphetamine actually blocks efflux through the mutant proteins, the investigators show. The results suggest that abnormal dopamine efflux could contribute to dopamine signaling disorders, and that blockade of inappropriate efflux could somehow contribute to the therapeutic actions of DAT-targeted stimulants.

The ups and downs of DARPP-32 phosphorylation In the first study, the researchers set out to investigate a paradox: both the psychostimulant cocaine and the sedation-producing antipsychotic haloperidol act on striatal neurons to modulate DARPP-32 phosphorylation, but their behavioral effects are vastly different. It was known that the drugs act on different subpopulations of striatal neurons that make up distinct output pathways (cocaine acts on striatonigral neurons, while haloperidol acts on striatopallidal neurons). To test the hypothesis that the drugs might differentially affect DARPP-32 phosphorylation in the two cell types, the researchers expressed different epitope-tagged versions of DARPP-32 in either striatonigral neurons (using a dopamine type 1 receptor [D1R] promoter) or in striatopallidal neurons (using a dopamine type 2 receptor [D2R]) promoter) in transgenic mice. Then, they used tag-specific immunoprecipitation after treatment of animals with the medications to follow the phosphorylation of DARPP-32 at two critical residues, threonine 34 and 75 (T34 or T75), in both cell types. The method, they write, “allowed a quantitative side by side comparison of phosphorylation events occurring selectively in two distinct neuronal populations in vivo.” Phosphorylation at T34 is important because it activates the phosphatase inhibitory activity of DARPP-32, while modification at T75 makes the protein an activator of protein kinase A.

In this way, the researchers tested the effects of cocaine and haloperidol on DARPP-32 phosphorylation in vivo. Previous work showed that both cocaine and haloperidol increase DARPP-32 phosphorylation at the critical regulatory residue T34 in whole striatum. The transgenic mice confirmed this result, but revealed that the increase actually occurred in different neurons for the two agents. After cocaine treatment, it was the striatonigral neurons that displayed an increase in T34 phosphorylation, while striatopallidal neurons showed a decrease. Phosphorylation at T75 showed reciprocal changes in the opposite direction in each cell type. On the other hand, haloperidol induced an increase in T34 only in striatopallidal neurons, and caused no change at T75.

The researchers tested other psychoactive agents, and found that they, too, had cell type-specific actions. The atypical antipsychotic clozapine acted like haloperidol to increase T34 phosphorylation specifically in striatopallidal neurons, but it had a different effect on T75, where it increased phosphorylation at that residue in both cell types. In addition, caffeine had an opposite effect to haloperidol, acting to increase T75 phosphorylation, consistent with the opposing stimulant and sedative effects of the two agents.

The differential regulation of striatonigral and striatopallidal neurons could explain the drugs’ different behavioral effects, the authors conclude, as the two types of neurons couple to opposing output pathways. “We have resolved the paradox that psychostimulants and antipsychotics cause the same biochemical change in the striatum, but have opposing behavioral and clinical effects,” they write.

The basis for the cell-specific effects are not completely clear. Differential expression of type 1 versus type 2 dopamine receptors on the two cell populations may explain why, for example, the type 2 antagonist haloperidol acts selectively on striatopallidal neurons. The whole story is likely to be more complex, however. In other experiments, the researchers showed that the effects of specific D1R and D2R receptor agonists on DARPP-32 phosphorylation in vivo differed from their effects in isolated striatal slices. Thus, in vivo effects of drugs are likely affected by the presence of intact physiological inputs into the striatum, which allow for a host of secondary actions of dopamine receptor blockade. (The slice experiments also raise the concern that isolated in vitro preparations, often used to study signaling pathways, do not give an accurate readout of drug effects.)

From their results, the authors argue that the regulation of T34 phosphorylation in striatopallidal, dopamine type 2-expressing neurons may represent a common mechanism of action of antipsychotics. In support of this idea, they show that the levels of phosphorylation achieved with haloperidol are much higher than with clozapine, which they speculate may be associated with locomotor side effects. The levels of T34 achieved with clozapine, coupled with its additional effects on T75, may make it more tolerable, they speculate.

“The importance of selectively analyzing responses in these two neuronal populations is becoming increasingly clear, as recent studies have shown substantial difference between D1R- and D2R-expressing striatal neurons with regard to their synaptic properties and regulation of dendritic spine number,” the authors conclude. The investigators show that since the tagged DARPP-32 protein only represents a few percent of total DARPP-32, its presence does not appear to disrupt physiological regulation of phosphorylation, or DARPP-32’s ability to regulate downstream kinase and phosphatase pathways. Thus, the method of expressing cell-targeted epitope-tagged proteins may be generally useful for probing the biochemistry of other cell types, and other protein modifications.

The ins and outs of dopamine The second study focuses on an aberration in dopamine signaling induced by a rare human dopamine transporter (DAT) variant found in two boys with ADHD and a woman with bipolar disorder. The variant, an Ala559Val substitution, has normal dopamine uptake activity, but the researchers, led by first authors Michelle Mazei-Robison and Erica Bowton, found that the mutant leaks dopamine from depolarized cells. This behavior is similar to the effects of amphetamine on dopamine transporters, where the drug not only inhibits dopamine uptake, but also stimulates efflux. The clinical benefits of amphetamine are thought to be due to its ability to increase synaptic dopamine levels and overcome dopamine deficiencies.

Surprisingly, both boys with the effluxing mutant had been treated successfully with amphetamines. This led the researchers to test amphetamine and the other most common ADHD medication, methylphenidate, on the A559V transporter. In both cases, they found the drugs blocked the abnormal efflux. From these results, they speculate that the therapeutic actions of transporter antagonists, “currently attributed to blockage of neurotransmitter reuptake, may in some cases arise from cessation of transporter-mediated neurotransmitter efflux. Our findings argue for further inspection of anomalous transporter-mediated neurotransmitter efflux as an unappreciated source of risk for brain disorders.” Among those diseases could be “disorders that benefit from blockade of DA receptors, such as schizophrenia,” they speculate.—Pat McCaffrey.

References:
Bateup HS, Svenningsson P, Kuroiwa M, Gong S, Nishi A, Heintz N, Greengard P. Cell type-specific regulation of DARPP-32 phosphorylation by psychostimulant and antipsychotic drugs. Nat Neurosci. 2008 Jul 11. Abstract

Mazei-Robison MS, Bowton E, Holy M, Schmudermaier M, Freissmuth M, Sitte HH, Galli A, Blakely RD. Anomalous dopamine release associated with a human dopamine transporter coding variant. J Neurosci. 2008 Jul 9;28(28):7040-6. Abstract

Comments on Related News


Related News: SfN Atlanta: Paul Greengard on DARPP-32 and p11

Comment by:  Karl-Ludvig Reichelt (Disclosure)
Submitted 7 November 2006
Posted 7 November 2006

Serotonin Transmission in Mental Disorders
As always, Greengard makes outstanding contributions. Very, very interesting.

We, as well as several other groups, have demonstrated peptide increases in schizophrenia (Hole et al., 1979; Drysdale et al., 1982; Idei et al., 1982; Cade et al., 2000) and also in several other disorders (e.g., Cade et al., 2000; Reichelt and Knivsberg, 2003). This confirms older data from Sweden (Lindstrom et al., 1986), where opioids were found, but measured as receptor binding total level. Unfortunately they named these endorphins, too, while we find that these are probably exorphins.

Opioids affect uptake and release of monoamines, and long ago we could demonstrate uptake inhibition of dopamine and serotonin (Hole et al., 1979), and later a serotonin uptake stimulating tripeptide, which in oocytes from frog stimulate the transport protein in a bell-shaped dose response (hormetic) manner (Pedersen et al., 1999; Keller, 1997). There has been some dispute about the structure of the tripeptide, and we are re-running mass spectrometry as soon as possible to see if we made any mistake. The structure we arrived at was pyroglu-trp- glyNH2 and in depression (in press) pyroglu-trp-gly.

Peptides are a bit tricky because of their considerable tendency to aggregate (Reichelt, in press), which might explain some of the problems. We use tri-fluoroacetic acid (TFA) on HPLC, therefore, and offline mass spectrometry in methanol formic acid (10mM). Formic acid 10mM is not electrometrically as strong and dissociating as TFA. (The mass spectrometry does not tolerate TFA well). In our hands, formic acid 10 mM does not deaggregate all peptide complexes.

Be that as it may, peptides regulating uptake and release of transmitters have been neglected too long. Also, the immune data on peptides in brain should by and large have been confirmed by independent methods such as HPLC and also, preferably, mass spectrometry. Immuno-like does not really ensure identity. For an overview of schizophrenia in this regard, see Reichelt et al, 1996.

We do not know what percentage of the schizophrenics show peptide increases, but a fairly large untreated cohort does. (We have great problems in getting untreated patient urine, 10 ml of the first morning urine (frozen) of carefully diagnosed cases). Our data seem able to explain the onset and suggest reasonable treatment, as shown for autism (Knivsberg et al., 1995; Knivsberg et al., 2002). It does not apply to all, of course, but a large percentage. The curse of medicine is that diagnosis is usually based on symptoms and not aetiology, almost like Morbus febris once was a diagnosis, but with a thousand different causes. We have suffered considerable opposition as would be expected, but now seem to get support from many experiments carried out properly.

References:

Cade RJ, Privette M, Fregly M, Rowland N, Sun Z, Zele V, Wagemaker H and Edelstein C (2000) Autism and schizophrenia: intestinal disorders. 3: 57-72.

Keller J. (1997) Impact of autism-related peptides and 5-HT system manipulations on cortical development and plasticity -Ist Ann report for EU proj. BMH4-CT96-0730 pp 1-10.

Knivsberg A-M, Reichelt KL, Nödland M and Höien T. (1995) Autistic syndromes and diet :a follow up study. Scand J Educat. Res 39: 225-236.

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Related News: DARPP-32 Haplotype Affects Frontostriatal Cognition and Schizophrenia Risk

Comment by:  Jonathan Burns
Submitted 14 February 2007
Posted 14 February 2007

This study provides hard empirical evidence for the hypothesis that psychosis (and schizophrenia in particular) represents a costly "byproduct" of complex human (social) brain evolution. Interestingly, the activation paradigms in the fMRI study (N-back and emotional face-matching tasks) are both testing social cognition. And the demonstrated changes in frontostriatal connectivity support the hypothesis that schizophrenia is a disorder of evolved intrahemispheric circuits comprising the Social Brain in our species.

I would suggest that further candidates (conferring vulnerability to psychosis) should be sought from amongst those genes known to have played a significant role in human brain evolution.

References:

Burns J. (2007) The Descent of Madness: Evolutionary Origins of Psychosis and the Social Brain. Routledge Press: Hove, Sussex.

Burns J. The social brain hypothesis of schizophrenia. World Psychiatry. 2006 Jun;5(2):77-81. Abstract

Burns JK. Psychosis: a costly by-product of social brain evolution in Homo sapiens. Prog Neuropsychopharmacol Biol Psychiatry. 2006 Jul;30(5):797-814. Epub 2006 Mar 3. Review. Abstract

Burns JK. An evolutionary theory of schizophrenia: cortical connectivity, metarepresentation, and the social brain. Behav Brain Sci. 2004 Dec;27(6):831-55; discussion 855-85. Review. Abstract

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Related News: DARPP-32 Haplotype Affects Frontostriatal Cognition and Schizophrenia Risk

Comment by:  Daniel Durstewitz
Submitted 8 June 2007
Posted 8 June 2007
  I recommend the Primary Papers

The phosphoprotein DARPP-32 occupies a central position in the dopamine-regulated intracellular cascades of cortical and striatal neurons (Greengard et al., 1999). It is a point of convergence for multiple signaling pathways, is differentially affected by D1- vs. D2-class receptor activation, and mainly through inhibition of protein-phosphotase-1 mediates or contributes to a number of the dopaminergic effects on voltage- and ligand-gated ion channels. These, in turn, by regulating intracellular Ca2+ levels, themselves influence phosphorylation of DARPP-32 and thereby interact with dopamine-induced processes.

Given its central, vital role in dopamine-regulated signaling pathways, it is quite surprising that (to my knowledge) only a few studies exist on the implications of DARPP-32 variations for cognitive functions and brain activity. Therefore, this comprehensive series of studies by Meyer-Lindenberg et al. combining human genetics, structural and functional MRI, and behavioral testing represents an important milestone. Meyer-Lindenberg et al. identified different functionally relevant DARPP haplotypes, associated with differential DARPP mRNA activity in postmortem studies, and found that these were linked to significant differences on a number of cognitive tests probing “executive functions,” as well as to differences in putamen volume and activity, and structural and functional covariation between striatal and prefrontal cortical areas. Thereby, they paved the way for detailed investigations of the role of DARPP-32 in human cognition.

Since DARPP-32 is so intricately interwoven into so many intracellular and physiological feedback loops, as with dopamine itself (Durstewitz and Seamans, 2002), mechanistic accounts for the functional involvement of DARPP-32 variations in neural network dynamics may be hard to obtain. “Linear” causal thinking usually breaks down in such complex functional networks constituted of so many interacting positive and negative feedback loops on different time scales. Thus it may still be a while until we gain a deeper, biophysically based understanding of the neural processes that mediate the influence of DARPP variations on cognition, and integrative computational approaches may be required to help resolving these issues. Given the complexity of DARPP-regulated networks, I also would expect that fine-grained behavioral testing and analysis of error types of human subjects on different cognitive tasks may ultimately reveal quite subtle and differential effects of DARPP polymorphisms. Moreover, the effects on neural network dynamics may be such (e.g., changing the temporal organization of spiking patterns) that they may not always be detectable by current neuroimaging methods, meaning that while the most dramatic effects were found on activation and volume of striatum, where DARPP-32 is most abundantly expressed, a significant contribution of other brain areas in DARPP-associated cognitive differences may not be ruled out. Regardless of these difficulties in unraveling the underlying neural mechanisms, the work by Meyer-Lindenberg et al. allows us to tackle the question of how the balance in dopamine-regulated intracellular networks relates to cognition in humans, and points toward the neural structures and interactions most interesting to look at.

View all comments by Daniel Durstewitz

Related News: Biology of Reinforcement—Dopamine Linked to Three Separate Reward Paths

Comment by:  Patricia Estani
Submitted 16 November 2007
Posted 16 November 2007
  I recommend the Primary Papers