SfN Atlanta: Paul Greengard on DARPP-32 and p11
Editor's Note: We put out a call for correspondents from the Society for Neuroscience meeting, and brave Susannah F. Locke of the University of Pennsylvania stepped up to the line. In this installment, she reports from one of the sessions she attended. We encourage you to contact us whenever you’re headed to a meeting. Your meeting reports can help speed information to your colleagues stuck at home.
31 October 2006. Paul Greengard, of The Rockefeller University in New York City, presented a special lecture at the Society for Neuroscience meeting in Atlanta, highlighting some essential findings from his fruitful career. His talk, entitled “Signal Transduction Pathways Used by Therapeutic Agents and Drugs of Abuse,” covered both old and recent data. He shared a summary of his continuing project on DARPP-32, a protein that inhibits PP-1 in response to a variety of neurotransmitters and drugs, and presented newer research on the regulation of the 5-HT1B receptor by p11.
DARPP-32 and schizophrenia
Greengard’s work on DARPP-32 was part of the signal transduction research for which he was awarded the Nobel Prize in Physiology or Medicine in 2000. He has been studying DARPP-32 for over 2 decades and is still adding to the DARPP-32 story today.
DARPP-32 (dopamine and cAMP regulated phosphoprotein of 32 kD) amplifies PKA signaling and acts as a potent inhibitor of protein phosphatase PP-1 in postsynaptic locations. By regulating phosphorylation cascades, DARPP-32 affects many key proteins, including ion channels, neurotransmitter receptors, and transcription factors. What is remarkable about DARPP-32 is its presence in different signaling cascades caused by a wide variety of neural events. Lying downstream of the dopamine, glutamate, and serotonin pathways, DARPP-32 acts as a signaling hub that elucidates the relationships between these various neurotransmitters. Greengard’s in vivo work has established DARPP-32’s roles in several psychiatric disorders, including schizophrenia.
DARPP-32 provides clues to why decreasing the ratio of D1 to D2 receptor activity has been a feature of many antipsychotic agents. D1 and D2 receptors have opposite effects on DARPP-32. D1 signaling activates PKA, which directly phosphorylates and activates DARPP-32. DARPP-32 can then inhibit PP-1, shifting the environment in favor of the phosphorylation of PKA targets. D2 signaling, however, activates PP2B, which dephosphorylates and inactivates DARPP-32. The phosphorylation of DARPP-32, and its consequences on other PKA substrates, can be viewed as a tug-of-war between D1 and D2 signaling cascades.
DARPP-32’s role in glutamatergic signaling lends support to the hypoglutamine hypothesis of schizophrenia. NMDA receptor activation results in the inactivation of DARPP-32 through PP2B. Similar to D1 signaling, a hypoglutamatergic environment would lead to active DARPP-32.
Greengard’s in vivo work has also linked DARPP-32 function with schizophrenia via psychotomimetic drugs. D-amphetamine, PCP, and LSD, all of which mimic aspects of psychosis, have common behavioral effects even though they target different receptors. Greengard’s data show that all three drugs activate a common substrate: DARPP-32. Mice lacking DARPP-32 function are less responsive to these drugs. In DARPP-32 knockout mice or mice expressing a nonfunctional DARPP-32 variant, D-amphetamine, PCP, and LSD cease to alter sensorimotor gating (such as prepulse inhibition of the startle reflex) and repetitive movements. Mice with nonfunctional DARPP-32 also have lower levels of c-fos mRNA after drug treatment than their wild-type counterparts.
Although not addressed in Greengard’s talk, a postmortem study has shown lower DARPP-32 in the prefrontal cortices of schizophrenic subjects than in non-schizophrenic subjects (Albert et al., 2002). It is not yet known whether this difference is a factor in the development of schizophrenia or a consequence of the disease or its treatment.
p11 regulation of the 5-HT1B receptor
Whereas the saga of DARPP-32 has been unfolding for over 2 decades, the story of p11’s regulation of the 5-HT1B receptor is a new tale, which was published this year in Science (Svenningsson et al., 2006; see also SRF related news story). The paper shows that p11 regulates the localization of the 5-HT1B receptor and that p11 is related to depression. In addition, it suggests that less p11 function could lead to depression and that increasing p11 activity could be a promising direction for antidepressant therapy.
p11 (annexin II light chain) is a member of the S100 protein family. It translocates annexin II’s heavy chain, as well as ion channels, to the cell membrane. p11 was identified as a subject of interest when it was picked up in a yeast two-hybrid screen of the intracellular portion of the 5-HT1B receptor. Greengard presented his in vitro data demonstrating that p11 and the 5-HT1B receptor interact specifically in co-immunoprecipitations and that they colocalize to the cell membrane. In addition, p11 cotransfection increases 5-HT1B receptor levels at the cell surface.
Greengard then shared in vivo work that correlates depression with lower p11 expression in both rodents and humans. The helpless H/Rouen mouse strain (an animal model of depression) has lower levels of p11 mRNA than non-helpless mice. A similar pattern of mRNA expression exists between the postmortem brains of depressed and non-depressed patients. In addition, various forms of antidepressant therapy, including imipramine treatment and electroconvulsive therapy, increase both p11 mRNA and protein in rodents. Overexpression of p11 in a transgenic mouse can mimic the effects of antidepressants, yielding mice that exhibit less depression-like and anxiety-like activity in behavioral tests. p11 knockout mice, on the other hand, have increased depression-like behavior.
The depression-like phenotype of the p11 knockout mouse could easily be due to the decrease in 5-HT1B receptor function observed in these animals. The mice have fewer functional 5-HT1B receptors on the cell surface. Primary cultures from the knockouts have reduced 5-HT1B receptor activity as shown by decreased inactivation of ERK1/2, more serotonin turnover (5-HT1B receptors act as autoreceptors), and less 5-HT1B-mediated inhibition of excitatory transmission.
Although Greengard did not address other neurotransmitter systems in the p11 portion of his talk, his Science paper suggests that p11 regulation of dopaminergic signaling is unlikely. p11 did not interact with D1 or D2 receptors through a yeast two-hybrid screen. In addition, long-term treatment with neither haloperidol nor risperidone increased p11 mRNA in mice.—Susannah F. Locke.
Albert KA, Hemmings HC Jr, Adamo AI, Potkin SG, Akbarian S, Sandman CA, Cotman CW, Bunney WE Jr, Greengard P. Evidence for decreased DARPP-32 in the prefrontal cortex of patients with schizophrenia. Arch Gen Psychiatry. 2002 Aug; 59(8):705-12. Abstract
Svenningsson P, Chergui K, Rachleff I, Flajolet M, Zhang X, El Yacoubi M, Vaugeois JM, Nomikos GG, Greengard P. Alterations in 5-HT1B receptor function by p11 in depression-like states. Science. 2006 Jan 6; 311(5757):77-80. Abstract
Comments on News and Primary Papers
Comment by: Karl-Ludvig Reichelt
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.
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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Comments on Related News
Related News: A New Link between Serotonin and DepressionComment by: Todd Gould
Submitted 13 January 2006
Posted 14 January 2006
I recommend the Primary Papers
Proof that model organisms can “suffer” from psychiatric illness is at very best modest. However, the use of animal models gets around this either through symptom modeling or studying endophenotypes (see recent SRF endophenotype discussion and Gould and Gottesman, 2005). Thus, only facets, whether they be face valid “re-creations” of symptoms or models of inherent and quantifiable measures of brain functions, are utilized.
The recent paper by Svennignsson, Greengard, and colleagues takes advantage of these approaches to describe a novel function of p11, namely, the modulation of depression-like states. This includes increased tail suspension test (TST) immobility in mice where p11 has been removed (knockout; KO mice), and decreased TST immobility in mice that overexpress p11. Further, p11 KO mice spent more time along the “safer” sides of an open field, while mice overexpressing p11 tended to move away from the sides. These data are consistent with evidence that p11 is involved in modulating cellular pathways involved in depression-like and anxiety-like behavior. There are a number of additional behavioral tasks related to depression-like (e.g., forced swim test, learned helplessness) and anxiety-like (e.g., the elevated plus maze and black/white box) behavior, which could be studied in these mice. Additionally, the researchers used modern molecular and cellular biology, in addition to electrophysiological techniques, to strongly make the case that the behavioral changes likely involve interactions with the 5-HT1B receptor.
The authors “link” their basic science and rodent behavior data to humans by showing that both mRNA and protein levels of p11 are decreased in the postmortem brain of depressed patients compared to control subjects. Their finding that both ECT and imipramine increase p11 levels in the mouse brain suggests that similar effects could occur in humans. However, caution is warranted: The present field of psychiatric genetics was initiated with the understanding that human diseases are complex in nature—needing multiple genes working in disharmony with nongenetic contributors for the human syndrome. Thus, while a single gene disruption (e.g., p11) in the mouse may result in “human-related” psychiatric phenotypes, in humans, any involvement of p11 in the pathophysiology or treatment of mood disorders undoubtedly requires complex interactions with other susceptibility genes.
Gould TD, Gottesman II (in press): Psychiatric endophenotypes and the development of valid animal models. Genes, Brain, and Behavior. (full text courtesy of Genes, Brain, and Behavior)
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Related News: A New Link between Serotonin and Depression
Comment by: Mary Reid
Submitted 21 January 2006
Posted 23 January 2006
It's most interesting that Paul Greengard and colleagues report lower levels of p11 in brain samples from depressed patients. Renegunta et al. report that knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Hopwood et al. find that present data suggest that the excitatory effects of 5-HT on DVN are mediated in part by inhibition of a TASK-like, pH-sensitive K+ conductance, and the Perrier group reports that 5-HT1A receptors inhibit TASK-1-like K+ current in the adult turtle. Might we suspect that a specific inhibitor of TASK-1 conductance would be beneficial in depression, and might this in part explain the benefit reported by SSRIs and agents with 5-HT1A receptor agonist activity in the treatment of depression?
Renigunta V, Yuan H, Zuzarte M, Rinne S, Koch A, Wischmeyer E, Schlichthorl G, Gao Y, Karschin A, Jacob R, Schwappach B, Daut J, Preisig-Muller R. The Retention Factor p11 Confers an Endoplasmic Reticulum-Localization Signal to the Potassium Channel TASK-1.
Traffic. 2006 Feb;7(2):168-81.
Hopwood SE, Trapp S. TASK-like K+ channels mediate effects of 5-HT and extracellular pH in rat dorsal vagal neurones in vitro.
J Physiol. 2005 Oct 1;568(Pt 1):145-54. Epub 2005 Jul 14.
Perrier JF, Alaburda A, Hounsgaard J. 5-HT1A receptors increase excitability of spinal motoneurons by inhibiting a TASK-1-like K+ current in the adult turtle.
J Physiol. 2003 Apr 15;548(Pt 2):485-92. Epub 2003 Mar 7.
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Related News: DARPP-32 Isoform Elevated in Psychiatric Disorders
Comment by: Jamal Nasir, Nirmal Vadgama
Submitted 8 March 2013
Posted 8 March 2013
Kunii et al. used two different TaqMan Assays (Applied Biosystems) to compare expression levels of full-length (FL) and truncated DARPP-32 (t-DARPP-32) in various regions of the brain, and detected increased expression of t-DARPP-32 in DLPFC in both schizophrenia and bipolar disorder samples compared to controls. Overexpression of genes can cause developmental abnormalities in the brain. For example, increased LIS1 expression can lead to significant brain abnormalities in humans and mice (Bi et al., 2009).
We previously showed increased expression of DARPP-32 in human DLPFC tissue from both schizophrenia (n = 33) and bipolar disorder (n = 32) samples using the same TaqMan assay (Hs00259967_ml) as above (this detects both FL-DARPP-32 and t-DARPP-32), after excluding brain weight, age of onset, postmortem interval, time in hospital, duration of illness and antipsychotics, gender, race, smoking, alcohol, drugs, suicide status, family history, insight and psychotic features as potential confounding factors (Zhan et al., 2011). After applying Bonferroni corrections to account for multiple comparisons, our findings remained significant, and after correcting for brain pH our p-values became much more significant (p <0.001 for both schizophrenia and bipolar disorder samples vs. controls [n = 34]).
Hierarchical clustering analysis of our data revealed a distinct pattern for DARPP-32 expression in comparison to other dopamine signaling genes and dopamine receptors D1-D5, although the expression of these genes appeared to be co-regulated with the exception of dopamine receptors and D2, in particular (Zhan et al., 2011). DARPP-32 expression in relation to D2 expression is strikingly different in controls but remarkably similar in schizophrenia and bipolar samples, suggesting aberrant D2-regulated expression of DARPP-32 may be an important trigger in pathogenesis.
We found increased DARPP-32 expression in DLPFC of schizophrenia and bipolar samples by using an assay that detects both FL-DARPP-32 and t-DARPP-32. We are, therefore, unable to say whether this is attributable to increased expression of FL-DARPP-32, t-DARPP-32, or both. Kunii et al. failed to find any differences in expression levels using this assay, but since they found increased expression of t-DARPP-32, this would indicate that FL-DARPP-32 expression levels have gone down in schizophrenia and bipolar samples. However, there is inevitably considerable variability between postmortem brain samples as shown in the data presented by Kunii et al. and in other studies, so this could also account for the results. Finally, it would be useful to compare the relative expression of both isoforms of the gene in the brain during various stages of development in the same patients. This might shed useful light on their respective functions.
Zhan L, Kerr JR, Lafuente M-J, Maclean A, Chibalina MV, Liu B, Burke B, Bevan S, Nasir J. (2011) Altered expression and coregulation of dopamine signalling genes in schizophrenia and bipolar disorder. Neuropathology and Applied Neurobiology 37, 206-219. Abstract
Bi W, Sapir T, Shchelochkov OA, Zhang F, Withers MA, Hunter JV, Levy T, Shinder V, Peiffer DA, Gunderson KL, Nezarati MM, Shotts VA, Amato SS, Savage SK, Harris DJ, Day-Salvatore DL, Horner M, Lu XY, Sahoo T, Yanagawa Y, Beaudet AL, Cheung SW, Martinez S, Lupski JR, Reiner O. (2009) Increased LIS1 expression affects human and mouse brain development. Nat Genet. 41:168-177. Abstract
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