Developmental Expression of Orphan G Protein-Coupled Receptor 50 in the Mouse Brain

Mental disorders have a complex etiology resulting from interactions between multiple genetic risk factors and stressful life events. Orphan G protein-coupled receptor 50 (GPR50) has been identified as a genetic risk factor for bipolar disorder and major depression in women, and there is additional genetic and functional evidence linking GPR50 to neurite outgrowth, lipid metabolism, and adaptive thermogenesis and torpor. However, in the absence of a ligand, a specific function has not been identified. Adult GPR50 expression has previously been reported in brain regions controlling the HPA axis, but its developmental expression is unknown. In this study, we performed extensive expression analysis of GPR50 and three protein interactors using rt-PCR and immunohistochemistry in the developing and adult mouse brain. Gpr50 is expressed at embryonic day 13 (E13), peaks at E18, and is predominantly expressed by neurons. Additionally we identified novel regions of Gpr50 expression, including brain stem nuclei involved in neurotransmitter signaling: the locus coeruleus, substantia nigra, and raphe nuclei, as well as nuclei involved in metabolic homeostasis. Gpr50 colocalizes with yeast-two-hybrid interactors Nogo-A, Abca2, and Cdh8 in the hypothalamus, amygdala, cortex, and selected brain stem nuclei at E18 and in the adult. With this study, we identify a link between GPR50 and neurotransmitter signaling and strengthen a likely role in stress response and energy homeostasis

Developmental Expression of Orphan G Protein-Coupled Receptor 50 in the Mouse Brain

Mental disorders have a complex etiology resulting from interactions between multiple genetic risk factors and stressful life events. Orphan G protein-coupled receptor 50 (GPR50) has been identified as a genetic risk factor for bipolar disorder and major depression in women, and there is additional genetic and functional evidence linking GPR50 to neurite outgrowth, lipid metabolism, and adaptive thermogenesis and torpor. However, in the absence of a ligand, a specific function has not been identified. Adult GPR50 expression has previously been reported in brain regions controlling the HPA axis, but its developmental expression is unknown. In this study, we performed extensive expression analysis of GPR50 and three protein interactors using rt-PCR and immunohistochemistry in the developing and adult mouse brain. Gpr50 is expressed at embryonic day 13 (E13), peaks at E18, and is predominantly expressed by neurons. Additionally we identified novel regions of Gpr50 expression, including brain stem nuclei involved in neurotransmitter signaling: the locus coeruleus, substantia nigra, and raphe nuclei, as well as nuclei involved in metabolic homeostasis. Gpr50 colocalizes with yeast-two-hybrid interactors Nogo-A, Abca2, and Cdh8 in the hypothalamus, amygdala, cortex, and selected brain stem nuclei at E18 and in the adult. With this study, we identify a link between GPR50 and neurotransmitter signaling and strengthen a likely role in stress response and energy homeostasis

Developmental Expression of Orphan G Protein-Coupled Receptor 50 in the Mouse Brain

Mental disorders have a complex etiology resulting from interactions between multiple genetic risk factors and stressful life events. Orphan G protein-coupled receptor 50 (GPR50) has been identified as a genetic risk factor for bipolar disorder and major depression in women, and there is additional genetic and functional evidence linking GPR50 to neurite outgrowth, lipid metabolism, and adaptive thermogenesis and torpor. However, in the absence of a ligand, a specific function has not been identified. Adult GPR50 expression has previously been reported in brain regions controlling the HPA axis, but its developmental expression is unknown. In this study, we performed extensive expression analysis of GPR50 and three protein interactors using rt-PCR and immunohistochemistry in the developing and adult mouse brain. Gpr50 is expressed at embryonic day 13 (E13), peaks at E18, and is predominantly expressed by neurons. Additionally we identified novel regions of Gpr50 expression, including brain stem nuclei involved in neurotransmitter signaling: the locus coeruleus, substantia nigra, and raphe nuclei, as well as nuclei involved in metabolic homeostasis. Gpr50 colocalizes with yeast-two-hybrid interactors Nogo-A, Abca2, and Cdh8 in the hypothalamus, amygdala, cortex, and selected brain stem nuclei at E18 and in the adult. With this study, we identify a link between GPR50 and neurotransmitter signaling and strengthen a likely role in stress response and energy homeostasis

DISC1: Structure, Function, and Therapeutic Potential for Major Mental Illness

<i>Disrupted in schizophrenia 1 (DISC1)</i> is well established as a genetic risk factor across a spectrum of psychiatric disorders, a role supported by a growing body of biological studies, making the DISC1 protein interaction network an attractive therapeutic target. By contrast, there is a relative deficit of structural information to relate to the myriad biological functions of DISC1. Here, we critically appraise the available bioinformatics and biochemical analyses on DISC1 and key interacting proteins, and integrate this with the genetic and biological data. We review, analyze, and make predictions regarding the secondary structure and propensity for disordered regions within DISC1, its protein-interaction domains, subcellular localization motifs, and the structural and functional implications of common and ultrarare <i>DISC1</i> variants associated with major mental illness. We discuss signaling pathways of high pharmacological potential wherein DISC1 participates, including those involving phosphodiesterase 4 (PDE4) and glycogen synthase kinase 3 (GSK3). These predictions and priority areas can inform future research in the translational and potentially guide the therapeutic processes

Hippocampal Neuroanatomical Measurements between <i>Disc1</i>^31L/31L, <i>Disc1</i>^100P/100P and wild-type mice.

<p>A significant difference only in dentate granule cell layer thickness between wild-type and <i>Disc1</i>^100P/100P mice was noted (ANOVA F(2,20) = 4.07, P = 0.032; <i>post-hoc</i> Bonferroni p<0.05), though Student's <i>t</i> test indicated a strong correlation (p = 0.06) between wild-type and <i>Disc1</i>^31L/31L mice. Within the hippocampus, the thinning of the granule cell layer in the mutants is specific to the dentate gyrus, as no significant differences are observed between genotypes in the thickness of the pyramidal cell layer in the CA1 and CA3 regions, and the overall height and width of the hippocampus. Data presented as mean ± SEM.</p><p>Hippocampal Neuroanatomical Measurements between <i>Disc1</i>^31L/31L, <i>Disc1</i>^100P/100P and wild-type mice.</p

Hypothetical model for how DISC1 mutation may affect interacting proteins within the cell populations residing in the dentate gyrus.

<p>Neural stem cells (NSCs) generate neurons which extend processes across the granule cell layer through the generation of Tbr2⁺ intermediate progenitors (IPCs) as shown in the ‘wild type’ illustration. Our data suggests that the homozygous Q31L mutation in DISC1 leads to a loss of IPCs which may arise from a reduction in the formation of DISC1-GSK3β complexes in the NSCs leading to increased quiescence. With the homozygous L100P mutation, however, we note a normal number of IPCs, but instead observe alterations in the morphology and migration of the DCX⁺ postmitotic neurons, which we hypothesize may be due to changes in the NDEL1-LIS1 complex mediated by DISC1 interactions with PDE4.</p

Deficits in cell proliferation are restricted to <i>Disc1</i>^31L/31L mice.

<p>(<b>A</b>) Missense mutations do not affect expression of full length DISC1 in tissue lysates taken from the cortex and hippocampus, as shown using a C-terminal DISC1 antibody that recognizes a specific 100 kDa band in the adult mouse tissue brain homogenates that are absent in <i>Disc1</i>^Δ2Δ3 mice. (<b>B</b>) Significantly fewer primary neurospheres (P = 0.007; <i>post-hoc</i> Bonferroni p<0.05) derived from dissociated adult hippocampal cells in <i>Disc1</i>^31L/31L mice (n = 6) compared with either wild-type (n = 6) or <i>Disc</i>1^100P/100P (n = 4) mutants. (<b>C</b>) Confocal z-stacks of mouse sections labeled with an antibody raised against the neural progenitor marker Tbr2 (red) and nuclei label Hoechst 33242 (blue) indicate that <i>Disc1</i>^31L/31L mutants (n = 8) have significantly (<b>D</b>) fewer Tbr2 labelled cells (ANOVA, P = 0.014) than either wild-type (n = 9, <i>post-hoc</i> Bonferroni p<0.05) or <i>Disc1</i>^100P/100P (n = 9, <i>post-hoc</i> Bonferroni p<0.05) mice. (<b>E</b>) Cell death as measured by activated caspase-3 immunoreactivity was not significantly different between genotypes (ANOVA, p = 0.74, n = 4 wild type, <i>Disc1</i>^100P/100P; n = 5 <i>Disc1</i>^31L/31L; Scale bar 100 µm. Data presented as mean ± SEM.</p

Loss of immature neurons in <i>Disc1</i>^100P/100P mice.

<p>(<b>A</b>) Immunolabelling of immature neurons residing in the subgranular zone with an antibody raised against doublecortin (DCX) identify frequent gaps (arrows) in DCX staining solely in <i>Disc1</i>^100P/100P mice that reflect a significant loss (<b>B</b>) of DCX⁺ cell bodies compared to wild-type controls (<i>post-hoc</i> Bonferroni p<0.05; n = 8 wild type, n = 5 <i>Disc1</i>^31L/31L. n = 6 <i>Disc1</i>^100P/100P). Data presented as mean ± SEM. Scale Bar (A) 100 µm (B) 25 µm</p

Ectopic migration of select populations of immature neurons in <i>Disc1</i>^100P/100P mice.

<p>(<b>A–C</b>) An analysis of doublecortin (DCX) positive cells prematurely migrating away from the subgranular zone (SGZ) (<b>A</b>, upper boundary of GCL demarcated with dashed line) reveal no significant (<b>B</b>) differences (ANOVA, p = 0.35, n = 7 wild type, n = 5 <i>Disc1</i>^31L/31L, n = 5 <i>Disc1</i>^100P/100P) between genotypes. Arrowheads point to DCX⁺ cell somas positioned in the GCL within 70 µm of the SGZ, while the arrow in <i>Disc1</i>^100P/100P section shows DCX⁺ cell positioned greater than 70 µm away from SGZ. (<b>C</b>) Binning of DCX⁺ cells in the GCL into 10 µm distance segments from 20 µm away from the SGZ reveal a small but significant (ANOVA F(2,14) = 5.180, p = 0.021; post-hoc Tukey p<0.05; n = 7 wild type, n = 5 <i>Disc1</i>^31L/31L, n = 5 <i>Disc1</i>^100P/100P) percentage of cells that migrate a distance greater than 70 µm in the <i>Disc1</i>^100P/100P mice compared to wild-type and <i>Disc1</i>^31L/31L mice. Data presented as mean ± SEM. Scale Bar 50 µm</p

NDE1 and GSK3β Associate with TRAK1 and Regulate Axonal Mitochondrial Motility: Identification of Cyclic AMP as a Novel Modulator of Axonal Mitochondrial Trafficking

Mitochondria are essential for neuronal function, providing the energy required to power neurotransmission, and fulfilling many important additional roles. In neurons, mitochondria must be efficiently transported to sites, including synapses, where their functions are required. Neurons, with their highly elongated morphology, are consequently extremely sensitive to defective mitochondrial trafficking which can lead to neuronal ill-health/death. We recently demonstrated that DISC1 associates with mitochondrial trafficking complexes where it associates with the core kinesin and dynein adaptor molecule TRAK1. We now show that the DISC1 interactors NDE1 and GSK3β also associate robustly with TRAK1 and demonstrate that NDE1 promotes retrograde axonal mitochondrial movement. GSK3β is known to modulate axonal mitochondrial motility, although reports of its actual effect are conflicting. We show that, in our system, GSK3β promotes anterograde mitochondrial transport. Finally, we investigated the influence of cAMP elevation upon mitochondrial motility, and found a striking increase in mitochondrial motility and retrograde movement. DISC1, NDE1, and GSK3β are implicated as risk factors for major mental illness. Our demonstration that they function together within mitochondrial trafficking complexes suggests that defective mitochondrial transport may be a contributory disease mechanism in some cases of psychiatric disorder