Biochemical and functional interaction of disrupted-in-schizophrenia 1 and amyloid precursor protein regulates neuronal migration during mammalian cortical development - PubMed (original) (raw)

Biochemical and functional interaction of disrupted-in-schizophrenia 1 and amyloid precursor protein regulates neuronal migration during mammalian cortical development

Tracy L Young-Pearse et al. J Neurosci. 2010.

Abstract

Although clinically distinct, schizophrenia and Alzheimer's disease are common and devastating disorders that profoundly impair cognitive function. For Alzheimer's disease, key mechanistic insights have emerged from genetic studies that identified causative mutations in amyloid precursor protein (APP) and presenilin. Several genes have been associated with schizophrenia and other major psychoses, and understanding their normal functions will help elucidate the underlying causes of these disorders. One such gene is disrupted-in-schizophrenia 1 (DISC1). DISC1 and APP have been implicated separately in cortical development, with each having roles in both neuronal migration and neurite outgrowth. Here, we report a previously unrecognized biochemical and functional interaction between DISC1 and APP. Using in utero electroporation in the living rat brain, we show that DISC1 acts downstream of APP and Disabled-1 to regulate cortical precursor cell migration. Specifically, overexpression of DISC1 rescues the migration defect caused by a loss of APP expression. Moreover, knockdown of APP in cultured embryonic neurons results in altered subcellular localization of DISC1. Using transfected cells and normal brain tissue, we show that APP and DISC1 coimmunoprecipitate and that the intracellular domain of APP interacts with the N-terminal domain of DISC1. Based on these findings, we hypothesize that the APP cytoplasmic region transiently interacts with DISC1 to help regulate the translocation of DISC1 to the centrosome, where it plays a key role in controlling neuronal migration during cortical development.

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Figures

Figure 1.

Figure 1.

Functional domains of DISC1 involved in cortical migration. A, Schematic representation of the DISC1 gene outlining constructs used in this study. B–D, E15.5 rat cortices were coelectroporated with GFP and constructs listed. Three days later, brains were fixed, sectioned coronally, and immunostained for MAP2 (red), and the percentage of electroporated cells (green) that migrated into the cortical plate was quantified (C, D). Electroporations tested the ability of various DISC1 constructs to rescue the migration defect observed with DISC1 shRNA (C) and also the migration effects of overexpression of each DISC1 construct in the absence of DISC1 shRNA (D). Representative images for some conditions are shown (B). Scale bars, 100 μm. Data for each construct represent the average from at least three independent brains. Error bars represent SD. *p < 0.05; ***p < 0.001.

Figure 2.

Figure 2.

DISC1 acts downstream of APP and DAB1 in cortical migration. E15.5 rat cortices were coelectroporated with GFP and the constructs listed. The APP shRNA used here is the “active” shRNA shown in supplemental Figure 1 (available at

www.jneurosci.org

as supplemental material). Three days later, brains were fixed, sectioned coronally, and immunostained for MAP2 (red). Representative images for some conditions are shown (A–C). The percentage of electroporated cells (green) that migrated into the cortical plate was quantified (D). Scale bars, 100 μm. Data for each construct represents the average from at least three independent brains. Error bars represent SDs. ***p < 0.001.

Figure 3.

Figure 3.

APP and DISC1 interact biochemically. COS cells were transiently transfected with constructs as listed and harvested 48 h later in 1% NP-40 lysis buffer. IPs were performed for HA using 3F10-affinity resin (A–C, E, F), or with 22C11 (anti-APP; Millipore) (D). Western blots were run of lysates and IPs as shown. In A, the asterisk (*) denotes relative sizes of each FLAG-tagged protein. In E, the asterisk (*) denotes the IgG heavy chain band resulting from the immunoprecipitation. In F, the asterisk (*) marks co-IP of endogenous APP with overexpressed DISC1-HA. G, Membrane and cytosolic preps were made from adult rat brains as described in Materials and Methods. Antibodies used for IP were anti-DISC1 (Invitrogen) and anti-APP (C9).

Figure 4.

Figure 4.

APP knockdown at E15.5 does not affect proliferation. E15.5 rat cortices were coelectroporated with GFP and the constructs listed. Twenty-four hours later, BrdU was injected intraperitoneally, and brains were harvested 24 h after BrdU injection. Brains were fixed, sectioned coronally, and immunostained for BrdU (red) and Ki67 (blue). Representative images are shown (A–C). The percentage of electroporated cells (green) that migrated into the cortical plate (D) was quantified. The percentage of cells in the VZ/SVZ/IZ that incorporated BrdU (E) also was quantified (D). Scale bars, 50 μm. Data for each construct represent the average from at least three independent brains. Error bars represent SDs. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5.

Figure 5.

APP knockdown results in altered DISC1 localization in cortical neurons. Primary rat cortical neurons were cotransfected with GFP alone or with GFP plus control shRNA or APP shRNA constructs. Three days later, neurons were fixed and immunostained for DISC1 (Abcam) (shown in red in A, C, D, F, G, I; or white in B, E, H) and pericentrin (blue). Confocal images were acquired, and representative images are shown (A–I). The insets show magnified views of the cells indicated by red arrows. Identities of the images were deidentified, and DISC1 localization was examined in GFP-positive cotransfected cells (indicated by white arrows). Cells were classified as having focal (B, E, arrows) or diffuse DISC1 (H, arrow) immunostaining. Data from three independent experiments are shown in J. At least 50 cells were quantified for each condition. Error bars represent SDs between the three experiments. **p < 0.01; ***p < 0.001.

Figure 6.

Figure 6.

Summary of data showing APP and DISC1 interactions in cortical cell migration. In wild-type cells, APP exists in a complex with DAB1 and DISC1 through distinct binding domains in its cytoplasmic region. In addition to interacting with APP at the cell surface and/or on intracellular membranes, DISC1 localizes to the centrosome with its binding partners NDEL1 and LIS1, where it carries out its role in nucleokinesis (Morris et al., 2003; Kamiya et al., 2005; Bradshaw et al., 2008). When APP is knocked down, DISC1 is localized more diffusely throughout the cytoplasm and cannot perform its function in migration. However, when cells lacking APP are flooded with excess DISC1, the need for the APP–DISC1 biochemical interaction is bypassed and the migration defect is rescued. The small red ellipse represents the centrosome, and the blue oval represents the nucleus.

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