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Genetic inactivation of GSK3α rescues spine deficits in Disc1-L100P mutant mice
Schizophrenia Research, 1, 129, pages 74 - 79
Disrupted-in-Schizophrenia 1 (DISC1), a strong candidate gene for schizophrenia and other mental disorders, regulates neurodevelopmental processes including neurogenesis, neuronal migration, neurite outgrowth and spine development. Glycogen synthase kinase-3 (GSK3) directly interacts with DISC1 and also plays a role in neurodevelopment. Recently, our group showed that theDisc1-L100P mutant protein has reduced interaction with both GSK3α and β. Genetic and pharmacological inhibition of GSK3 activity rescued behavioral abnormalities inDisc1-L100P mutant mice. However, the cellular mechanisms mediating these effects of GSK3 inhibition inDisc1mutant mice remain unclear. We sought to investigate the effects of genetic inactivation ofGSK3αon frontal cortical neuron morphology inDisc1L100P mutant mice using Golgi staining. We found a significant decrease in dendritic length and surface area inDisc1-L100P,GSK3αnull and L100P/GSK3αdouble mutants. Dendritic spine density was significantly reduced only inDisc1-L100P and L100P/GSK3α+/− mice when compared to wild-type littermates. There was no difference in dendritic arborization between the various genotypes. No significant rescue in dendritic length and surface area was observed in L100P/GSK3αmutants versus L100P mice, but spine density in L100P/GSK3αmice was comparable to wild-type. Neurite outgrowth and spine development abnormalities induced byDisc1mutation may be partially corrected throughGSK3αinactivation, which also normalizes behavior. However, many of the other dendritic abnormalities in theDisc1-L100P mutant mice were not corrected byGSK3αinactivation, suggesting that only some of the anatomical defects have observable behavioral effects. These findings suggest novel treatment approaches for schizophrenia, and identify a histological read-out for testing other therapeutic interventions.
Keywords: Disrupted-in-schizophrenia 1 (DISC1), Glycogen synthase kinase 3 (GSK3), Mutant mice, Neuronal morphology, Spine density.
Schizophrenia (SZ) is a severe and chronic psychiatric disorder characterized by psychotic, negative and cognitive symptoms (Wong and Van Tol, 2003 and Tamminga and Holcomb, 2005). There is substantial evidence that SZ is a neurodevelopmental disorder with many genes affecting susceptibility (Wong and Van Tol, 2003 and Ross et al, 2006). Disrupted-in-Schizophrenia 1 (DISC1) is a prominent risk gene, first identified in a large Scottish family with a balanced chromosomal translocation (1q42.1:11q14.3) co-segregating with major mental illnesses including SZ, bipolar disorder and major depression (Millar et al, 2000 and Blackwood et al, 2001). TheDISC1locus shows genetic linkage with SZ and polymorphic variants in theDISC1gene are associated with SZ (Nakata et al, 2009, Rastogi et al, 2009, and Schumacher et al, 2009). DISC1 acts as a scaffold protein that interacts with and regulates many other proteins that are involved in cytoskeletal structure and signaling. Thus, DISC1 is involved in brain development and functions such as neurogenesis, neuron migration, neurite outgrowth, spine development and neurotransmitter signaling (Camargo et al, 2007 and Brandon et al, 2009).
Glycogen synthase kinase 3β (GSK3β) has been shown to regulate neuronal proliferation ( Kim et al., 2009 ), and it also interacts with DISC1 (Mao et al, 2009 and Lipina et al, 2010). GSK3 is a highly-conserved serine/threonine kinase expressed in two paralogous proteins (GSK3α and β) that share similar DNA sequence ( Woodgett, 1990 ). GSK3 activity is inhibited by several mechanisms, such as phosphorylation, complex formation and cellular translocation (Frame and Cohen, 2001, Jope and Roh, 2006, and Kockeritz et al, 2006). GSK3 is widely expressed in the brain ( Perez-Costas et al., 2010 ) and as part of a large molecular network has key roles in many neurodevelopmental processes including neurogenesis, neuron growth and synaptic plasticity (Zhou et al, 2004, Kim and Kimmel, 2006, Peineau et al, 2008, Kim et al, 2009, Mao et al, 2009, Hur and Zhou, 2010, and Lange et al, 2011).
Accumulating evidence implicates disruption of GSK3 signaling in psychiatric disorders. Genetic studies have demonstrated thatGSK3polymorphisms are associated with SZ (Souza et al, 2008 and Benedetti et al, 2010). Multiple susceptibility genes for SZ includingDISC1, neuregulin-1 (NRG1) andERBb4have been shown to affect GSK3 regulators such as Akt, or pathways downstream from GSK3 such as β-catenin and the canonical Wnt signaling pathway (Koros and Dorner-Ciossek, 2007, Lovestone et al, 2007, and Freyberg et al, 2010). Post-mortem brain studies of patients with SZ show reduced GSK3β phosphorylation, decreased Akt protein, and β- and γ-catenin expression (Cotter et al, 1998 and Emamian et al, 2004). The GSK3 pathway is also targeted by a number of important drugs used in psychiatry, such as lithium, valproic acid and antipsychotics such as haloperidol and clozapine, which provides further evidence that GSK3 is important for understanding mental illnesses (Kang et al, 2004, Bibb, 2005, Li et al, 2007, Rosenberg, 2007, Beaulieu et al, 2008, and Li and Jope, 2010).
Since the N-terminus of DISC1 can directly interact with and suppress GSK3β activity and also reduce signaling downstream through the Wnt/β-catenin pathway, a change in DISC1 expression may affect neurodevelopment through GSK3. Indeed, recent studies have reported a direct interaction between DISC1, GSK3β ( Mao et al., 2009 ) and the Akt-binding partner Girdin ( Enomoto et al., 2009 ) affecting neurogenesis. Furthermore, administration of a GSK3 inhibitor, SB216763 rescued the behavioral deficits in mice with reduced DISC1 expression in the dentate gyrus of the hippocampus of adult animals ( Mao et al., 2009 ). These data support a role for DISC1 in modulating the multiple cellular functions of the GSK3 signaling pathway.
Our group previously described a mouse carrying a single point mutation inDisc1(Disc1-L100P) with abnormalities related to SZ, including pronounced deficits in prepulse inhibition (PPI), latent inhibition (LI) and working memory. Their behavioral phenotype was reversed by using antipsychotic drugs ( Clapcote et al., 2007 ), as well as by administration of TDZD-8 (a GSK3 inhibitor) ( Lipina et al., 2010 ). The morphology of frontal cortical pyramidal neurons from these mice is also abnormal, with reduced dendritic length, dendrite surface area and spine density ( Lee et al., 2011 ). Moreover, genetic deletion of one allele ofGSK3αwas equally effective in rescuing abnormalDisc1-L100P behaviors ( Lipina et al., 2010 ). Therefore, we sought to investigate whether the beneficial effect ofGSK3αgene deletion onDisc1-L100P behaviors is associated with changes in neuronal morphology. Our findings confirmed the morphological deficits previously reported inDisc1-L100P mutants and we also found similar abnormalities inGSK3αnull mice. Genetic inactivation ofGSK3αinDisc1-L100P mice significantly rescued spine density, but had no effect on dendritic length, surface area and arborization. These results show that GSK3α is involved in neurite outgrowth and spine development, since inactivation ofGSK3αcan partially restore some deleterious effects of theDisc1mutation. However, the precise relationship between these two molecules in regulating dendritic development and morphology remains unclear. Further studies are required to understand how specific components of the complex GSK3 signaling cascade may interact with other important disease genes to mediate neural development.
2. Materials and methods
ENU-mutagenizedDisc1-L100P homozygous (−/−), GSK3α null (−/−) mutant mice and wild-type (WT) littermates on a C57BL/6 background were generated as previously described (Clapcote et al, 2007 and Kaidanovich-Beilin et al, 2009). Similarly,GSK3αheterozygous (+/−) mice were backcrossed to C57BL/6 mice and bred withDisc1-L100P mutants. The resultant offspring were then intercrossed to obtain WT andDisc1-L100P −/− with either none or one copy of theGSK3αgene ( Lipina et al., 2010 ). Mice of both sexes for each group were used for all analyses. All animal protocols were approved by the TCP Animal Care Committee.
2.2. Golgi-Cox staining
Golgi-Cox staining was performed as previously described ( Gibb and Kolb, 1998 ). In summary, adult mice (age 6–8 weeks) were anesthetized with xylazene/ketamine (10ml/kg) and intracardially perfused with 0.9% saline. Brains were removed and immersed in Golgi-Cox solution in the dark for 14 days before transferring to 30% sucrose solution for 5 days. Sections of 200 μm were sliced using a microtome (Leica VT1000S, Germany) and were placed on 2% gelatinized microscope slides. The slides were stored in a humidified chamber for 3 days prior to further staining and fixation.
2.3. Neuron morphology and dendritic spines
For morphometric analyses of individual neurons, Golgi images at 40× magnification were captured under bright-field illumination with a Nikon Eclipse E600 microscope. Neurons were chosen based on the following criteria: (i) fully visible and showing clear, distinct morphology, (ii) all dendrites visible within the 40× magnification field, and (iii) pyramidal neurons in layers III and V of the frontal cortex, as demarcated in the Golgi Atlas Of The Postnatal Mouse Brain ( Valverde, 1998 ). 15 neurons from 4 to 6 mice per group were randomly selected for analysis of dendritic length, surface area and arborization. A z-stack of different focal lengths was generated for each neuron to capture the three-dimensional dendritic branching tree in different planes. Acquisition parameters were kept the same for all images. The neurites of each neuron were traced, and the length and surface area were estimated using Neuromantic software ( http://www.rdg.ac.uk/neuromantic ). All parameters were then normalized to soma surface area for comparison.
Sholl analysis provides a quantitative measure of the radial distribution of neuronal dendritic arborization ( Sholl, 1953 ). Using ImageJ, we created 15 concentric circles (each with 8 μm larger radius than the previous circle) centered at the perikaryon and then counted the number of dendritic intersections with each. The log of the number of intersections per circle area versus the circle radius was plotted (the semi-log Sholl method). The slope of the regression line (κ = Sholl regression coefficient) is a measure of the decay rate of the number of branches with increasing distance from the soma ( Sholl, 1953 ). The Schoenen ramification index, RI(maximum number of intersections/number of primary dendrites), a measure of the ramification richness for each neuron ( Schoenen, 1982 ) also provides an indicator of the degree of dendritic branching complexity.
Dendritic spine density was measured with Golgi-stained images captured at 100× magnification (Nikon Eclipse E600). 45 neurons from 4 to 6 mice per group were selected for spine analysis. Spines were counted only on the apical dendrites of pyramidal neurons in layers III and V of frontal cortex. Spine density was expressed as the number of spines per dendritic length (μm). All images for quantification were blinded prior to analysis.
2.4. Statistical analysis
All parameters were analyzed by comparing the average values for each group of mice sharing the same genotype. Statistical differences among WT,Disc1-L100P, L100P/GSK3α+/− and L100P/GSK3α−/− were assessed using one-way ANOVA (SPSS 13.0), followed by the Fisher's Least Significant Difference (LSD) test as a post-hoc comparison. The Student's two-tailedt-test was used to compare WT andGSK3αnull mice. Data are expressed as mean ± standard error of mean. A significance level ofp < 0.05 was used for all analyses.
3.1. GSK3α is involved in neurodevelopment
Most studies have focused on the role of GSK3β in neurodevelopment, but knowledge of GSK3α function is relatively sparse.GSK3β−/− mice die before birth, but mice lacking both alleles ofGSK3αare viable, illustrating the distinct function of the two GSK isoenzymes (Hoeflich et al, 2000, MacAulay et al, 2007, and Kerkela et al, 2008). Nonetheless, some neurodevelopmental effects of deletion of either gene are similar, such as suppressed neurogenesis and disruption of cell polarity ( Kim et al., 2009 ). Our group previously characterized the behaviors ofGSK3α−/− mice, and found abnormalities such as social interaction deficits, increased stress response, facilitated PPI, and disrupted associative (LI) and fear-conditioned memory. These animals also showed less depression-related behaviors than WT control mice ( Kaidanovich-Beilin et al., 2009 ).
To gain further insights into the cellular mechanisms that might underlie the observed behavioral abnormalities, we performed a detailed morphological analysis of dendritic trees on frontal cortical neurons inGSK3α−/− mutants ( Fig. 1 A). Golgi staining provides a clear and complete image of dendritic arbors for a subset of neurons, without interference from neighboring neurons. Apical dendritic length (ADL) (176.4 ± 53.1 μm), basal dendritic length (BDL) (701.5 ± 173.1 μm) and total dendritic surface area (DSA) (4564.3 ± 992.6 μm2) were all significantly lower inGSK3α−/− mutants when compared to WT (ADL: 227.7 ± 72.9 μm; BDL: 1053.6 ± 262.7 μm; DSA: 6674.5 ± 940.1 μm2) ( Fig. 1 B). Cell soma size has been reported to correlate with dendritic structure ( Somogyi and Klausberger, 2005 ). Thus we normalized ADL, BDL and DSA to soma surface area. BDL and DSA showed similar results with or without normalization while ADL was not different betweenGSK3α−/− mutants and WT after normalization ( Fig. 1 C). Furthermore, we evaluated dendritic arbor complexity inGSK3α−/− micevs.WT via Sholl analysis. There was no significant change in dendritic branching pattern as measured by RIor κ ( Fig. 1 D). Thus, our results suggest that GSK3α may be involved in the regulation of dendritic outgrowth.
Cognitive deficits have been attributed to altered synaptic transmission and plasticity, in which dendritic spines play a critical role ( Calabrese et al., 2006 ). The involvement of GSK3 in long-term potentiation and depression has been extensively studied as well ( Peineau et al., 2007 ). Although GSK3 is expressed within dendrites and dendritic spines, its exact role in spine development and synaptic transmission remains unknown ( Peineau et al., 2008 ). Intriguingly, we found that spine density withinGSK3α−/− mice was comparable to WT controls ( Fig. 1 E) suggesting that GSK3α is not required for normal dendritic spine growth and development.
3.2. Genetic inactivation of GSK3α rescues spine density in Disc1-L100P mutants
Since alterations in the activation state of GSK3 can influence axonal/dendritic growth (Zhou et al, 2004 and Kim and Kimmel, 2006), and since genetic inactivation of one allele ofGSK3αrescuedDisc1-L100P mutant mouse behaviors, we hypothesized that genetic inactivation ofGSK3αactivity might also reverse some of the dendritic abnormalities seen inDisc1-L100P mice. To test this hypothesis, we examined the morphological features of frontal cortical neurons inDisc1-L100P, L100P/GSK3α+/−, L100P/GSK3α−/− mutants and WT mice ( Fig. 2 A). We observed a significantly shorter ADL inDisc1-L100P (167.3 ± 38 μm), L100P/GSK3α+/− (159.6 ± 51.3 μm) and L100P/GSK3α−/− mutants (179.6 ± 55.9 μm) when compared to WT (227.7 ± 72.9 μm). BDL showed a similar pattern, with significant reductions inDisc1-L100P (709.4 ± 221.8 μm), L100P/GSK3α+/− (784.4 ± 137.4 μm) and L100P/GSK3α−/− (787.3 ± 232 μm)vs.WT (1033.6 ± 262.7 μm) ( Fig. 2 B). We also found a significantly lower DSA inDisc1-L100P (4800.8 ± 957.3 μm2), L100P/GSK3α+/− (4955.7 ± 721.3 μm2) and L100P/GSK3α−/− mutants (5318.9 ± 1076.1 μm2) when compared to WT (6674.5 ± 940.1 μm2) ( Fig. 2 B). Similar results were observed with parameters normalized to soma surface area ( Fig. 2 C). Sholl analysis revealed no significant change in dendritic branching pattern amongDisc1-L100P, L100P/GSK3α+/−, L100P/GSK3α−/− mutants and WT controls ( Fig. 2 D).
To further determine if GSK3α interacts with DISC1 in modulating spine development, we measured dendritic spine density in our mutant mice. BothDisc1L100P (0.533 ± 0.102) and L100P/GSK3α+/− mutants (0.544 ± 0.079) had reduced spine density when compared to WT (0.588 ± 0.095) ( Fig. 2 E). However, the spine density of L100P/GSK3α−/− mice (0.61 ± 0.096) was significantly higher thanDisc1L100P and L100P/GSK3α+/− mice and comparable to WT (p < 0.01) ( Fig. 2 E), suggesting the possibility that the behavioral effects ofGSK3αinactivation inDisc1L100P mice might be mediated in part through rescue of dendritic spine deficits. Nonetheless, further experiments are required to fully elucidate the molecular mechanisms involved and the relationship between DISC1 and GSK3α.
Since changes in GSK3β expression can influence neurodevelopment, it is possible that altered GSK3β levels in L100P/GSK3αdouble mutants could be responsible for the normalization of behavior and dendritic spine density. We used Western blots on brain lysates to determine GSK3β protein levels, but detected no significant differences between L100P/GSK3αdouble mutants and WT (data not shown). Thus it is unlikely that a compensatory up-regulation of GSK3β can account for our other results.
GSK3β has a well-established role in regulating various neurodevelopmental functions ( Hur and Zhou, 2010 ). Recently, DISC1 was shown to affect neuronal proliferation by directly inhibiting GSK3β and stabilizing β-catenin ( Mao et al., 2009 ). However, the function of GSK3α has not been extensively studied in the context of psychiatric disorders. Our group had previously shown thatGSK3αknockdown in mice affects behavior ( Kaidanovich-Beilin et al., 2009 ), and that pharmacological and geneticGSK3αinactivation could normalize abnormal behaviors inDisc1-L100P mutants ( Lipina et al., 2010 ). In this study, we attempt to extend our understanding of cellular mechanisms underlying the effect of GSK3α on DISC1 by examining the neuron morphology of these mice.
We first examined the effect ofGSK3αdeletion on neuronal growth, branching complexity and spine development. Behaviourally,GSK3α−/− mice showed decreased immobility time in the forced swim test, decreased social novelty, increased prepulse inhibition and impaired long-term memory ( Kaidanovich-Beilin et al., 2009 ). Involvement of GSK3α in behavior associated with depression, sociability and memory formation was supported by studies in lithium-treated and GSK3β +/− mice ( O'Brien et al., 2004 ) as well as in GSK3α and β knock-in mice (Mines et al, 2010 and Polter et al, 2010), and mice overexpressing GSK3β ( Prickaerts et al., 2006 ). In our study, we found a significant decrease in dendritic length and surface area inGSK3α−/− mice, but no change in dendritic arborization and spine density. Altered GSK3 signaling has a variety of effects on neurites. Inhibition of GSK3 has been reported to promote microtubule assembly at the growth cone or block axon growth ( Hur and Zhou, 2010 ). The outcome depends on the phosphorylation of specific downstream substrates. Suppression of GSK3 activity towards primed substrates such as adenomatosis polyposis coli (APC) and collapsing response mediator protein 2 (CRMP2) promotes axon growth, whereas unprimed substrates such as microtubule associated protein 1B (MAP1B) prevents axon growth ( Kim and Kimmel, 2006 ). In our study, GSK3α may be preferentially acting via MAP1B and hence its inactivation results in shorter dendrites. Further studies are necessary to determine the molecular mechanisms responsible for the different effects of GSK3 modulation on neurite growth and morphology.
A large number of upstream regulators and downstream substrates modulate the complex GSK3 signaling pathways (Jope and Roh, 2006, Kockeritz et al, 2006, and Hur and Zhou, 2010). We examined whether DISC1 and GSK3α interact in affecting neuron morphology and development by examining the dendrites ofDisc1-L100P/GSK3αdouble mutants. Since GSK3α and β are highly homologous, it is possible that the knockout ofGSK3αwill cause a compensatory increase in its β counterpart. However, we did not see a change in GSK3β protein levels with deletion of GSK3α in our double mutant mice. Our results are consistent with previous studies reporting no alterations in GSK3β levels in mouse P19 cells or embryonic stem cells with reduced expression of GSK3α (Yu et al, 2003 and Doble et al, 2007). Since GSK3 protein levels might not correlate directly with kinase activity, further experiments are required to determine the function of both GSK3 isoforms in our mutants.
Genetically inactivatingGSK3αinDisc1-L100P mice resulted in dendritic deficits comparable toDisc1-L100P mutants. Interestingly, no additive or synergistic effects were observed with the L100P/GSK3αdouble mutants. These findings suggest that the L100PDisc1mutation is affecting the same pathway as GSK3α deletion. This model is consistent with the work of others showing that DISC1 regulates GSK3β activity to control neurogenesis ( Mao et al., 2009 ). In addition, our group had previously shown that theDisc1-L100P mutant protein has reduced interactions with both GSK3α and β ( Lipina et al., 2010 ). However, there is also evidence that other pathways involving DISC1 and GSK3 can affect dendritic growth, including NDEL1 and GSK3-protease activated receptor 3, respectively (Ozeki et al, 2003, Kamiya et al, 2005, and Hur and Zhou, 2010). More research is required to precisely characterize the relationship of DISC1 and GSK3α and their precise roles in dendritic development.
Dendritic spines are critical in synaptic connections and provide the basis for functional neural circuit connectivity within the brain ( Calabrese et al., 2006 ). The Wnt signaling pathway has been implicated in the regulation of synapse formation (Ahmad-Annuar et al, 2006 and Lovestone et al, 2007). Surprisingly, spine density in L100P/GSK3α−/− mice was significantly rescued and comparable to WT andGSK3α−/− mice, which may represent a possible mechanism underlying the normalized behavioral phenotypes observed in these double mutants. Since DISC1 regulates GSK3 and subsequent downstream Wnt proteins, a plausible speculation is that our L100P mutation disrupts Disc1 interactions with GSK3, leading to degradation of specific proteins. However, in contrast to the effects on dendritic development, the deletion ofGSK3αmay potentially stabilize Wnt proteins, resulting in normal spine development and behaviors. However, the specific Wnt proteins responsible for spine development that are directly regulated by DISC1 remain to be determined.
Behavioral deficits often involve dysfunction of multiple brain regions and can be regulated by many molecular networks. In particular, GSK3 signaling affects neural development through a wide range of different pathways ( Hur and Zhou, 2010 ) and DISC1 regulation of GSK3 is only part of this complex network. Our study is novel in demonstrating that GSK3α affects neurite outgrowth and that abnormal dendritic spines inDisc1-L100P mutant mice may be rescued byGSK3αgene inactivation. This enhances our understanding of the mechanisms by which GSK3 inhibitors may be effective as treatments for psychiatric disorders. Further experiments are required to determine whether the restoration of dendritic spines inDisc1-L100P mice byGSK3αinactivation is necessary or sufficient for improved behavioral function.
Role of funding source
Funding of this study was provided by Canadian Institute of Health Research; the CIHR had no further role in study designs; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Oksana Kaidanovich-Beilin, James Woodgett and Albert Wong designed the study. Oksana Kaidanovich-Beilin prepared all the animals and performed Western blot experiments. Frankie Lee performed all other experiments and statistical analysis. Frankie Lee and Albert Wong prepared the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
Conflict of interest
All authors declare that they have no competing financial interests.
We would like to thank the Canadian Institutes of Health Research for supporting AHCW with a Clinician Scientist Phase II Fellowship. We would also like to thank Mawahib Semeralul for blinding of data during analysis and Dr. Jose Nobrega for his advice with statistical analyses.
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a Neuroscience Division, Centre for Addiction and Mental Health, 250 College St, Toronto, Ontario, Canada M5T 1R8
b Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
c Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5
d Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8
e Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada M5T 1R8
f Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada M5S 1A8
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