Brain size and brain/intracranial volume ratio in major mental illness

<p>Abstract</p> <p>Background</p> <p>This paper summarizes the findings of a long term study addressing the question of how several brain volume measure are related to three major mental illnesses in a Colorado subject group. It reports results obtained from a large N, collected and analyzed by the same laboratory over a multiyear period, with visually guided MRI segmentation being the primary initial analytic tool.</p> <p>Methods</p> <p>Intracerebral volume (ICV), total brain volume (TBV), ventricular volume (VV), ventricular/brain ratio (VBR), and TBV/ICV ratios were calculated from a total of 224 subject MRIs collected over a period of 13 years. Subject groups included controls (C, N = 89), and patients with schizophrenia (SZ, N = 58), bipolar disorder (BD, N = 51), and schizoaffective disorder (SAD, N = 26).</p> <p>Results</p> <p>ICV, TBV, and VV measures compared favorably with values obtained by other research groups, but in this study did not differ significantly between groups. TBV/ICV ratios were significantly decreased, and VBR increased, in the SZ and BD groups compared to the C group. The SAD group did not differ from C on any measure.</p> <p>Conclusions</p> <p>In this study TBV/ICV and VBR ratios separated SZ and BD patients from controls. Of interest however, SAD patients did not differ from controls on these measures. The findings suggest that the gross measure of TBV may not reliably differ in the major mental illnesses to a degree useful in diagnosis, likely due to the intrinsic variability of the measures in question; the differences in VBR appear more robust across studies. Differences in some of these findings compared to earlier reports from several laboratories finding significant differences between groups in VV and TBV may relate to phenomenological drift, differences in analytic techniques, and possibly the "file drawer problem".</p

Hecate/Grip2a acts to reorganize the cytoskeleton in the symmetry-breaking event of embryonic axis induction.

Maternal homozygosity for three independent mutant hecate alleles results in embryos with reduced expression of dorsal organizer genes and defects in the formation of dorsoanterior structures. A positional cloning approach identified all hecate mutations as stop codons affecting the same gene, revealing that hecate encodes the Glutamate receptor interacting protein 2a (Grip2a), a protein containing multiple PDZ domains known to interact with membrane-associated factors including components of the Wnt signaling pathway. We find that grip2a mRNA is localized to the vegetal pole of the oocyte and early embryo, and that during egg activation this mRNA shifts to an off-center vegetal position corresponding to the previously proposed teleost cortical rotation. hecate mutants show defects in the alignment and bundling of microtubules at the vegetal cortex, which result in defects in the asymmetric movement of wnt8a mRNA as well as anchoring of the kinesin-associated cargo adaptor Syntabulin. We also find that, although short-range shifts in vegetal signals are affected in hecate mutant embryos, these mutants exhibit normal long-range, animally directed translocation of cortically injected dorsal beads that occurs in lateral regions of the yolk cortex. Furthermore, we show that such animally-directed movement along the lateral cortex is not restricted to a single arc corresponding to the prospective dorsal region, but occur in multiple meridional arcs even in opposite regions of the embryo. Together, our results reveal a role for Grip2a function in the reorganization and bundling of microtubules at the vegetal cortex to mediate a symmetry-breaking short-range shift corresponding to the teleost cortical rotation. The slight asymmetry achieved by this directed process is subsequently amplified by a general cortical animally-directed transport mechanism that is neither dependent on hecate function nor restricted to the prospective dorsal axis

Molecular identification of the <i>hecate</i> locus.

<p>A) Linkage map of the <i>hec</i> locus. The number of recombinants over the total number of analyzed meiosis is indicated. <i>hec</i> linkage was initially identified between SSLP markers z59658 and z24511 on chromosome 8. Fine mapping analysis with newly identified RFLP markers further narrowed the region between the gene <i>gpd1a-1</i> and the RFLP zC150E8y. B) Contig of five BAC clones covering the <i>hec</i> critical region. CH73-233M11, CH73-272M14, CH73-250D21, DKEY-43H14 and CH211-150E8 are five sequenced and overlapping BAC clones in this interval. C) Exon-intron structure of the <i>hec/grip2a</i> gene, which contains 16 exons. The <i>hec^p06ucal</i>, <i>hec^t2800</i> and <i>hec^p08ajug</i> alleles each cause a premature stop-codon in exon 4, exon 10 and exon 12, respectively. D) Sequence traces of the cDNA products from wild-type and the three mutant <i>hec</i> alleles. Nucleotide substitutions are indicated by the red box. Mutant cDNAs show a C-A transversion in codon 118 (<i>hec^p06ucal</i>), a C-T transversion in codon 414 (<i>hec^t2800</i>), or a C-T transversion in codon 499 (<i>hec^p08ajug</i>), all creating premature STOP codons. E) Schematic diagram showing the protein domain structures of Grip2a in the wild-type and mutant alleles. Red boxes represent conserved PDZ domains.</p

Amplification of <i>hecate/grip2a</i>-dependent symmetry breaking event by a general animal-directed long-range transport system.

<p>A) Cortical shifts of various vegetally localized components, including <i>wnt8a</i> mRNA, Sybu protein and <i>grip2a</i> mRNA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Nojima2" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Lu1" target="_blank">[7]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Tran1" target="_blank">[8]</a>; this report) are short-range and dependent on microtubule bundling and alignment, itself dependent on <i>hec</i> function. In wild-type embryos, such a short-range shift generates a symmetry breaking event that is subsequently amplified by long-range, animally-directed transport mechanism independent of <i>hec</i> function and not restricted to the prospective dorsal axis. B) In <i>hec</i> mutant embryos, neither reorganization of vegetal microtubules into aligned bundles nor a short-range shift occur, so that, even though long-range transport remains intact, vegetal determinant transport to the animal pole is affected. The mechanistic basis for the long-range transport, occurring in the region of a loosely organized mediolateral microtubule cortical network remains to be determined (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#s3" target="_blank">Discussion</a>).</p

Microtubule reorganization at the vegetal cortex is affected in <i>hecate</i> mutants.

<p>A–F) Cortical microtubule network at the vegetal pole in wild-type (A) and <i>hec</i> mutant (B–F) embryos at 20 mpf. Microtubules appear oriented in the same direction and bundled in wild-type embryos (A). The extent of co-orientation and bundling is greatly reduced in <i>hec</i> mutant embryos (B), where microtubules form multiple aster-like structures which can have a well focused-center (C,D) or can exhibit a central microtubule-free zone (E,F) and often overlap (F) or interdigitate (D). The relatively unbundled microtubule arrangement shown in (B) also corresponds to a sector of a large aster-like structure emanating from a not shown central core. Up to 6 aster-like structures were observed in the vegetal cortex of a single embryo. G,H) Cortex in mediolateral regions shows a loose and apparently random network of microtubules which appears similar in both wild-type (G) and <i>hec</i> mutant (H) embryos (n = 8 for wild-type and mutants). All images are z-axis projections of confocal image stages. The phenotype was fully (100%) penetrant according to the two main categories (wild-type, aligned and bundled microtubules; mutant, radialy oriented and unbundled microtubules, with 10 wt and 25 <i>hec</i> mutant embryos imaged. Magnification bar in (H) corresponds to 40 µm for all panels.</p

Localization of <i>grip2a</i> mRNA in wild-type and mutant oocytes.

<p>A–C) Whole mount in situ hybridization of dissected ovaries from wild-type (A), <i>bucky ball (buc</i>, B) and <i>magellan (mgn</i>, C) mutant females. In (A–C) oocytes at stage III of development are indicated. Smaller oocytes are at stages I and II, which are difficult to differentiate in whole mounts at this magnification. The <i>grip2a</i> mRNA localization domain is observed in an asymmetric cortical position in wild-type oocytes (A) but is unlocalized and diffuse in <i>buc</i> oocytes (B) and internally-located in <i>mgn</i> mutants (C). D–J) Sections of wild-type and mutant oocytes at the indicated stages after labeling to detect <i>grip2a</i> mRNA. Stages are as indicated in the panel and were determined by size and oocyte morphology according to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Selman1" target="_blank">[101]</a>. D–F) Wild-type oocytes showing localization to the presumptive Balbiani Body (D) and subsequent localization to a cortical domain of the oocyte corresponding to the presumptive vegetal pole (E–F). G,H) <i>buc</i> mutant oocytes lack the Balbiani body <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Bontems1" target="_blank">[39]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Marlow1" target="_blank">[43]</a> and the <i>grip2a</i> mRNA subcellular localization domain in stage I and II oocytes. I) <i>mgn</i> mutant stage I oocytes exhibit an enlarged Balbiani body <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Gupta1" target="_blank">[40]</a> and displayed an enlarged <i>grip2a</i> mRNA localization domain. J) Stage II mgn mutant oocytes fail to localize transcripts to the vegetal pole which instead persist in an internal domain <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Gupta1" target="_blank">[40]</a>, as observed also for <i>grip2a</i> mRNA. Number of oocytes examined were as follows: wild-type: early stage I: 13, stage II: 12; <i>buc</i>: early stage I: 35, stage II: 26; <i>mgn</i>: early stage I: 23, stage II: 18. Magnification bar in (C) corresponds to 250 µm for panels (A–C), and in (J) to 50 µm for panels (D–J).</p

Defects in the vegetal localization of <i>wnt8a</i> mRNA and Sybu protein.

<p>A–D) Off-center shift of <i>wnt8a</i> mRNA is affected in <i>hec</i> mutants. Whole mount in situ hybridization of wild-type embryos (A,C) and <i>hec</i> mutant embryos (B,D) at the 1- (A,B, 30 mpf) and 4- (C,D, 60 mpf) cell stages. Images show representative embryos. A majority of wild-type embryos showed a clear off-center shift (85%, n = 27 at 30 mpf and 74%, n = 47 at 60 mpf). A majority of <i>hec</i> mutant embryos showed vegetal localization without a shift at 30 mpf (79%, n = 33, remaining embryos show no localization) and absence of localization at 60 mpf (89%, n = 38, remaining embryos show reduced vegetal localization without a shift). The apparent label at the base of the blastodisc is observed in a majority of mutant embryos (71%, n = 38) but not in wild-type (C) or control embryos labeled with other probes (not shown) and may reflect remaining <i>wnt8a</i> mRNA that has lost anchoring at the vegetal pole and has moved animally through the action of axial streamers <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Fuentes1" target="_blank">[83]</a>. E–I) Localization of Sybu protein is affected in <i>hec</i> mutants. Whole mount immunofluorescence to detect Sybu protein of untreated wild-type (E,G) and <i>hec</i> mutant (F,H,) embryos and nocodazole-treated wild-type embryos (I) at the indicated stages. In wild-type embryos, an off-center shift in Sybu protein can be observed starting at 30 mpf (G). In <i>hec</i> mutants, Sybu protein becomes undetectable levels by this same time point (H). Patterns of localization of Sybu protein at 10 mpf and 20 mpf time points (combined n: 32 WT, 19 mutant for 10–20 mpf), and 30 mpf and 40 mpf time points were similar and have been combined. 59% (n = 32) of wild-type and 63% (n = 19) of <i>hec</i> mutant embryos showed centered vegetal localization during 10–20 mpf. At 30–40 mpf, the percent of embryos that showed vegetal localization, now with an off-center shift, was reduced to 25% (n = 28) in wild-type, and 0% (n = 25) of <i>hec</i> mutants showed any localization at these time points. Treatment of wild-type embryos with nocodazole inhibits the shift but does not result in delocalization from the vegetal cortex (I, embryo at 40 mpf), as previously shown <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004422#pgen.1004422-Nojima2" target="_blank">[6]</a>. Magnification bars in (D) and (I) correspond to 100 µm for panels sets (A–D) and (E–I), respectively.</p