Neuropeptide precursor VGF is genetically associated with social anhedonia and underrepresented in the brain of major mental illness: its downregulation by DISC1

In a large Scottish pedigree, disruption of the gene coding for DISC1 clearly segregates with major depression, schizophrenia and related mental conditions. Thus, study of DISC1 may provide a clue to understand the biology of major mental illness. A neuropeptide precursor VGF has potent antidepressant effects and has been reportedly associated with bipolar disorder. Here we show that DISC1 knockdown leads to a reduction of VGF, in neurons. VGF is also downregulated in the cortices from sporadic cases with major mental disease. A positive correlation of VGF single-nucleotide polymorphisms (SNPs) with social anhedonia was also observed. We now propose that VGF participates in a common pathophysiology of major mental disease

Oligomer assembly of the C-terminal DISC1 domain (640-854) is controlled by self-association motifs and disease-associated polymorphism S704C

Genetic studies have established a role of disrupted-in-schizophrenia-1 (DISC1) in chronic mental diseases (CMD). Limited experimental data are available on the domain structure of the DISC1 protein although multiple interaction partners are known including a self-association domain within the middle part of DISC1 (residues 403-504). The DISC1 C-terminal domain is deleted in the original Scottish pedigree where DISC1 harbors two coiled-coil domains and disease-associated polymorphisms at 607 and 704, as well as the important nuclear distribution element-like 1 (NDEL1) binding site at residues 802-839. Here, we performed mutagenesis studies of the C-terminal domain of the DISC1 protein (residues 640-854) and analyzed the expressed constructs by biochemical and biophysical methods. We identified novel DISC1 self-association motifs and the necessity of their concerted action for orderly assembly: the region 765-854 comprising a coiled-coil domain is a dimerization domain and the region 668-747 an oligomerization domain; dimerization was found to be a prerequisite for orderly assembly of oligomers. Consistent with this, disease-associated polymorphism C704 displayed a slightly higher oligomerization propensity. The heterogeneity of DISC1 multimers in vitro was confirmed with a monoclonal antibody binding exclusively to HMW multimers. We also identified C-terminal DISC1 fragments in human brains, suggesting that C-terminal fragments could carry out DISC1-dependent functions. When the DISC1 C-terminal domain was transiently expressed in cells, it assembled into a range of soluble and insoluble multimers with distinct fractions selectively binding NDEL1, indicating functionality. Our results suggest that assembly of the C-terminal domain is controlled by distinct domains including the disease-associated polymorphism 704 and is functional in vivo

Biophysical insights from a single chain camelid antibody directed against the Disrupted-in-Schizophrenia 1 protein

<div><p>Accumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis. V_HH domains derived from camelid heavy chain only antibodies are minimal antigen binding modules with appreciable solubility and stability, which makes them well suited for the stabilizing proteins prior to structural investigation. Here, we report on the generation of a V_HH domain derived from an immunized <i>Lama glama</i>, displaying high affinity for the human DISC1 C region (aa 691–836), and its characterization by surface plasmon resonance, size exclusion chromatography and immunological techniques. The V_HH-DISC1 (C region) complex was also used for structural investigation by small angle X-ray scattering analysis. In combination with molecular modeling, these data support predictions regarding the three-dimensional fold of this DISC1 segment as well as its steric arrangement in complex with our V_HH antibody.</p></div

<i>Ab initio</i> SAXS reconstructions and molecular models for the DISC1^691-836 protein, the V_HH domain, and their complex.

<p>The hypothetical DISC1 model (upper left) features a single domain containing three longer and three shorter α-helices. Similar to the terminal segments, the two extended loops (tilde symbols) are suggested to be poorly ordered. The positions of functionally important residues S704 and S713 are indicated in a close-up view. Also note the proline-rich motif (ball-and-stick representation, marked by a hash), which is predicted to be readily accessible for protein-protein interactions. The 691–715 region, suggested to participate in the epitope for the nanobody, is highlighted in gold while cloning artifacts at the termini are colored dark gray. The model of the V_HH antibody (lower left) displays a canonical immunoglobulin fold, including a conserved disulfide bridge (ball-and-stick model, marked by an asterisk). The CDR segments, as defined by the Chothia criteria [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191162#pone.0191162.ref060" target="_blank">60</a>], are highlighted in cyan. Finally, the DISC1^691-836 fragment and the nanobody are predicted to interact in a complex (right) featuring a 1:1 stoichiometry.</p

Parameters derived from the SAXS data (MM, molecular mass).

<p>Parameters derived from the SAXS data (MM, molecular mass).</p

Sequence and purification of the generated anti-DISC1 V_HH B5 antibody.

<p>(A) Sequence of the anti-DISC1 V_HH B5 protein showing the three complementarity determining regions (CDRs). (B) SEC profile of the purified anti-DISC1 V_HH B5 antibody eluting with an apparent molecular mass of 13 kDa. (C) Coomassie-stained SDS gel loaded with different SEC elution fractions (samples numbered in accordance with the chromatogram). Fraction 1 corresponds to the void volume, fractions 3 and 4 contain the V_HH B5 antibody.</p

Mapping the binding site of the anti-DISC1 V_HH B5 antibody on the DISC1 protein.

<p>(A) The binding epitope of the anti-DISC1 V_HH B5 antibody was mapped by western blot using truncated DISC1 protein constructs. (+) and (-) indicate positive and negative control, respectively. All constructs were detected using an anti-His tag antibody, whereas only the constructs covering the 691–715 segment were detected using the anti-DISC1 V_HH B5 antibody. The multiple bands with higher apparent mass observed in each lane correspond to SDS-resistant oligomers, which are a typical feature of DISC1 protein fragments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191162#pone.0191162.ref019" target="_blank">19</a>]. (B) Detection of human and mouse DISC1 protein in the lysates of transfected NLF cells by western blot, using either anti-rodent Disc1 C-term antibody, control MEM media, anti-DISC1 V_HH B5 antibody or anti-human DISC1 14F2 antibody. The four samples tested are untransfected NLF cell lysate, mouse Disc1 transfected NLF cell lysate, human DISC1 transfected NLF cell lysate, recombinant human DISC1 (50 ng). (C) Alignment of the human DISC1 epitope (residues 691–715) with the corresponding region of mouse Disc1.</p

Investigation of DISC1-V_HH complex formation by SAXS.

<p>(A) Primary scattering data (Intensity <i>I</i> as a function of momentum transfer <i>q</i>, the latter being defined as 4π sinθ / λ) for the V_HH domain (blue), the DISC1^691-836 protein (red), and their complex (yellow). The inset shows the linear regions of the respective Guinier plots at low <i>q</i> values, as suggested by <i>AUTORG</i>; the slope of the linear fit is related to the radius of gyration of the respective sample. (B) Normalized Kratky plot of the data shown in (A). A peak at low <i>qR</i>_<i>g</i> values is considered typical of folded globular proteins. (C) Distance distribution functions derived from the data shown in (A), using the indirect Fourier transformation implemented in <i>DATGNOM</i>. For each species, the intersection with the positive abscissa corresponds to the maximum particle diameter. A.u., arbitrary units.</p