Association of orexin receptor polymorphisms with antipsychotic-induced weight gain

<p><i>Objectives</i>: Antipsychotic-induced weight gain (AIWG) is a common side effect of treatment with antipsychotics such as clozapine and olanzapine. The orexin gene and its receptors are expressed in the hypothalamus and have been associated with maintenance of energy homeostasis. In this study, we have analysed tagging single nucleotide polymorphisms (SNPs) in orexin receptors 1 and 2 (HCRTR1 and HCRTR2) for association with AIWG. <i>Methods</i>: Schizophrenia or schizoaffective disorder subjects (<i>n</i> = 218), treated mostly with clozapine and olanzapine for up to 14 weeks, were included. Replication was conducted in a subset of CATIE samples (<i>n</i> = 122) treated with either olanzapine or risperidone for up to 190 days. Association between SNPs and AIWG was assessed using analysis of covariance (ANCOVA) with baseline weight and duration of treatment as covariates. <i>Results</i>: Several SNPs in HCRTR2 were nominally associated with AIWG in patients of European ancestry treated with either clozapine or olanzapine (<i>P</i><0.05). In the replication analysis two SNPs rs3134701 (<i>P</i> = 0.043) and rs12662510 (<i>P</i> = 0.012) were nominally associated with AIWG. None of the SNPs in HCRTR1 were associated with AIWG. <i>Conclusion</i>: This study provides preliminary evidence supporting the role of HCRTR2 in AIWG. However, these results need to be confirmed in large study samples.</p

Impact of histamine receptors H1 and H3 polymorphisms on antipsychotic-induced weight gain

<p><b>Objectives:</b> A positive correlation between antipsychotic-induced weight gain (AIWG) and the antagonist effect of antipsychotic drugs at the histamine H1 receptor (HRH1) as well as the agonist effect at the histamine H3 receptor (HRH3) in the brain has been consistently demonstrated. We investigated the potential impact of single-nucleotide polymorphisms (SNPs) in HRH1 and HRH3 genes on AIWG.</p> <p><b>Methods:</b> We analysed 40 tagSNPs in HRH1 (<i>n</i> = 34) and HRH3 (<i>n</i> = 6) in schizophrenia/schizoaffective disorder patients (<i>n</i> = 193) primarily treated with clozapine or olanzapine for up to 14 weeks. Linear regression was used to evaluate the association between SNPs and AIWG, with baseline weight and treatment duration as covariates.</p> <p><b>Results:</b> In HRH1, a nominal association of rs7639145 with AIWG was observed in patients of European ancestry treated with either clozapine or olanzapine (<i>P</i>= 0.043; β = 1.658; <i>n</i> = 77). We observed nominal association for two HRH1 SNPs rs346074 (<i>P = </i>0.002; β = –5.024) and rs13064530 (<i>P</i>= 0.004; β = –5.158) in patients of African ancestry treated with either clozapine or olanzapine (<i>n</i> = 37). However, the above associations are not significant after correcting for multiple testing. In HRH3, we did not observe association in either ancestry.</p> <p><b>Conclusions:</b> The current study suggests that SNPs in HRH1 and HRH3 may not have a major role in AIWG.</p

GFP and Ki-67 expression do not overlap in the majority of the cells in cell lines or in the tumors derived from them.

<p>A- LC25-R-p9, GFP (1:750), 40X; B- LC25-R-p9, Ki-67(1:200), 40X; C- LCAS-R, GFP (1:750), 5X (insert 40x digital); D- LCAS-R, Ki-67 (1:200), 5X (insert 40x digital).</p

3D imaging of the mice brain using a confocal microscope at one month A- or two months B- post—injection (LCAS-R).

<p>Top left panels reconstitute images of the brains placed horizontally, top right panels reconstitute coronal sections at the indicated lines 1–3. Bottom left panels reconstitute 3D image of the brains oriented as shown by the red plot lines. The section images at the white dotted lines shown at the bottom right panels. A—anterior; P—posterior; R-right; L—left.</p

Illustration of the orthotopic transplantation sites (red arrows).

<p>A- Cerebellum. B- Motor cortex. C- SVZ of 3^rd ventricle. (The images were taken from Harvard Medical School High Resolution Mouse brain Atlas).</p

Ossification within tumor mass (LCAS-R—12 weeks post-injection).

<p>A, B, C. Mature and newly formed bone is observed in intimate correlation with the tumor mass (HE- 10X, 20X and 40X respectively). D, E and F. Bone marrow is observed within mature bone. It shows hematopoietic cells including erythrocytes, leucocytes and megakaryocytes in different stages of maturation suggesting active hematopoietic activity (HE- 10X, 20X and 40X respectively). BM- bone marrow, TU-tumor.</p

Western blot.

<p>TJP1 protein quantitative difference between the RG cell lines grown for 72 hours in normoxic versus hypoxic conditions. The quantitative differences of TJP1 protein calculated after the correction for actual protein loaded per lane using the β-Actin protein control.</p

EMT markers show loss of expression by immunohistochemistry in tumors when compared to their corresponding wild type RG cell lines.

<p>TJP1 A- Diffuse membrane expression in wild type RG cells, B- Multifocal loss of expression in tumors, C- Loss of expression around necrotic areas (LCAS-R—12 weeks post-injection) (40X, inserts digital magnification 4X); TJP3 D- 2/3 of cells present cytoplasmic expression in wild type RG cells; E- Extensive areas of loss of expression in tumors (LCAS-R—12 weeks post-injection), F- Loss of expression in areas surrounding necrosis (LC26-R—12 weeks post-injection) (40X, inserts digital magnification 4X); β-catenin G- Diffuse membrane expression in wild type RG cells, H- Almost complete loss of expression in corresponding tumors (LCAS-R—12 weeks post-injection), I- Loss of expression in areas surrounding necrosis (LC26-R—12 weeks post-injection) (40X, inserts digital magnification 4X); N-cadherin, J- Low membrane expression in wild type RG cells, K- Extensive membrane expression in tumor cells (LCAS-R—12 weeks post-injection) (40X, inserts digital magnification 4X), L- Extensive membrane expression in the areas surrounding necrosis (LC26-R—12 weeks post-injection) (40X, inserts digital magnification 4X). N—Necrosis.</p

Orthotopic transplantation of the wild type RG cells to different brain areas of the mice.

<p>A- Cerebellum: Ki-67 (red) overlay with GFP (green) and DAPI (blue), white rectangle identifies area magnified in Fig 5C (LCAS-R—6 weeks post-injection) (tissue slides, 2.5X); B- Motor Cortex: Ki-67 (red) overlay with GFP (green) and DAPI (blue), white rectangle identifies area magnified in Fig 5D (LCAS-R—6 weeks post-injection) (tissue slides, 2.5X), C, D- Cerebellum an Motor Cortex: Ki-67 (red) overlay with GFP (green) (LCAS-R—6 weeks post-injection) (tissue slides, 10X).</p

The tumor cells invade the ventricular system.

<p>A- Septo-Striatal section, phase contrast (tissue slides, 1X); B- The same Septo-Striatal section: Ki-67 (red) overlay with GFP (green) and DAPI (blue) (LC25-R—8 weeks post-injection) (tissue slides, 1X); C- (LC26-R—12 weeks post-injection), D- (LC35TR-R—12 weeks post-injection), and E- (LCAS-R—12 weeks post-injection)—tumor growing within the parenchyma of the subventricular zone protruding to the ventricle (HE- 5X, 20X and 10X respectively); F- tumor growing within the parenchyma and invading lateral ventricle (LC26-R—12 weeks post-injection) (HE- 5X); G- tumor growing within the parenchyma, Ki-67 staining (LC26-R—12 weeks post-injection) (HE- 20X); H- tumor growing within the ventricular system including forth ventricle and subarachnoid space permeating the cerebellum (LC26-R—12 weeks post-injection) (HE- 5X); I- Tumor growing inside the parenchyma and protruding into the ventricular space. (CM14R - 8 weeks post-injection) (HE- 10X, inserts- 4X digital); J- and K- poorly differentiated neuroblastic tumor with rosette formation (LC26-R—12 weeks post-injection) (HE- 40X), rosettes—orange arrows; L- and N- frequent atypical mitoses—yellow arrows and inserts (LC26-R—12 weeks post-injection) (HE- 40X, inserts- 6X digital); M- tumor perivascular invasion (LCAS-R—12 weeks post-injection) (HE— 40X); N- and O—prominent angiogenesis—red arrows and insert (LC26-R—12 weeks post-injection) (HE- 40X, insert- 5X digital); P- geographic necrosis with pseudo-palisades (LCAS-R—12 weeks post-injection) (HE— 10X).TU—tumor; P—parenchyma; V—ventricle; CP—choroid plexus; CBL—cerebellum; N—necrosis.</p