More dramatic increases in apoptosis in the lateral cortex of <i>Psen</i> cDKO mice.

<p>Black vertical lines superimposed on an image of a Nissl-stained coronal brain section depict the relative positions of sagittal brain sections spaced 300 µm apart that were analyzed for either Fluoro-Jade B+ or TUNEL+ cells. The average (± s.e.m.) of the three medial-most sections (M) was compared to the average of the three lateral-most sections (L). Scale bar: 1 mm. For each timepoint, 4 mice per genotype (10 sections per mouse) were analyzed. (<i>A, B</i>) Quantification of numbers of Fluoro-Jade B+ cells in sagittal brain sections from mice at 2 months (<i>A</i>) or 4 months (<i>B</i>). At 2 months of age, increases in degenerating neurons are more pronounced in the lateral sections (control: 1.7±0.7; cDKO: 34.1±3.1; p<0.01) than in the medial sections (control: 2.5±0.3; cDKO: 3.4±0.3; p>0.05). By 4 months of age, the number of degenerating neurons increases significantly in medial sections of cDKO mice, compared to controls (control: 2.6±0.3; cDKO: 14.3±1.1; p<0.01). Furthermore, the number of degenerating neurons in lateral sections of cDKO mice is much greater than in medial sections (control: 1.4±0.1 vs. <i>Psen</i> cDKO: 32.1±3.0; p<0.02). *, p<0.05; **, p<0.01; ***, p<0.001. (<i>C, D</i>) Quantification of numbers of TUNEL+ cells in sagittal brain sections from mice age 2 months (<i>C</i>) or 4 months (<i>D</i>). (<i>C</i>) No significant increase was observed in TUNEL+ cells in the <i>Psen</i> cDKO medial cortex at 2 months (control: 3.0±0.3 vs. <i>Psen</i> cDKO: 4.9±0.5; p = 0.05), in contrast to a large increase in lateral <i>Psen</i> cDKO cortex (control: 3.4±0.4 vs. <i>Psen</i> cDKO: 54.8±8.0; p<0.05). (<i>E</i>) At 4 months, lateral <i>Psen</i> cDKO cortex showed a significant increases in TUNEL+ cells (control: 2.3±0.5 vs. <i>Psen</i> cDKO: 32.9±4.1; p<0.05), while medial <i>Psen</i> cDKO cortex showed only a trend toward increased apoptosis (control: 1.3±0.3 vs. <i>Psen</i> cDKO: 11.9±2.3; p = 0.05).</p

Mitochondrial defects in the cerebral cortex of <i>Psen</i> cDKO mice.

<p>(<i>A</i>) (<i>Top</i>) Electron micrographs of mitochondria from the neocortex at 2 months and 6 months of age. Red arrows point to mitochondria typical of those quantified in (<i>A–C</i>). Scale bar, 1 µm. (<i>Bottom</i>) Quantification of mitochondria per 100 µm² in neocortex. The density of <i>Psen</i> cDKO mitochondria was comparable to controls at 2 months (control: 62.5±4.1 vs. <i>Psen</i> cDKO: 68.4±4.1; p>0.05) but reduced by 6 months (control: 61.7±4.8 vs. <i>Psen</i> cDKO: 44.2±4.6; p<0.001), revealing age-dependent loss of mitochondria in <i>Psen</i> cDKO (n = 4 mice per genotype (2 mo) and 3 mice per genotype (6 mo)). (<i>B,C</i>) Analysis of size distribution of cortical mitochondria determined that <i>Psen</i> cDKO has no change in mitochondrial morphology at 2 months (<i>B</i>), but a sizeable reduction in small mitochondria by 6 months of age (<i>C</i>) (0.01–0.1 µm² bin: control, 40.0±4.4 vs. <i>Psen</i> cDKO, 28.6±4.3 (p<0.001); 0.11–0.2 µm² bin: control, 15.5±2.3 vs. <i>Psen</i> cDKO, 7.3±1.6 (p<0.00001)). (<i>D</i>) (<i>Top</i>) High-magnification electron micrographs of cortical mitochondria from mice at 6 months of age. Both control (<i>left</i>) and <i>Psen</i> cDKO samples (<i>middle</i>) have normal mitochondria with clearly visible cristae and outer membrane structures; however, <i>Psen</i> cDKO mice also have a slight increase in the number of swollen mitochondria (<i>right</i>). Scalebar, 0.4 µm (all 3 images). (<i>Bottom</i>) Quantification of large (area >0.3 µm²) mitochondria from mice age 2 months and 6 months. The percentage of large <i>Psen</i> cDKO cortical mitochondria relative to the total number was normal at 2 months of age (control: 0.028±0.017 vs. <i>Psen</i> cDKO: 0.024±0.009; p>0.05), but at 6 months of age, the percentage of large <i>Psen</i> cDKO mitochondria is twice the number observed in controls (control: 0.048±0.018 vs. <i>Psen</i> cDKO: 0.098±0.035; p<0.05), implicating a dysregulation of mitochondrial morphology in the absence of PS function. (n = 4 at 2 months and 3 at 6 months). All data are presented as the mean ± s.e.m.</p

Quantitative analysis of neurodegeneration and cell death in <i>Psen</i> cDKO Cx^†^‡.

†<p>Values for Fluoro-Jade B and TUNEL represent percentage of positive cells based on stereological estimates of total cell number. Note that although <i>Psen1</i> gene inactivation, measured by <i>Psen1</i> mRNA levels, occurs by 3–4 weeks, death is not increased at this timepoint. Since Psen1 protein levels persist and gradually diminish between P22 and 6 weeks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010195#pone.0010195-Yu2" target="_blank">[14]</a>, the delay in onset of death is likely due to the continued presence of Psen1 protein subsequent to gene inactivation.</p>‡<p>All calculations of total % death are based on stereological estimates of cortical cell number at 2 months of age (4.8×10⁶ cells/hemisphere; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010195#pone.0010195-Saura1" target="_blank">[10]</a> and at 4 months of age (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010195#pone-0010195-g001" target="_blank">Fig. 1</a>) combined with an estimate of 5 mm width of one hemisphere. Raw data (# dead cells/20 µm-thick sagittal section) was converted to a % by the following calculations: 1) the fraction of a hemisphere represented by one 20-µm thick section (1 section =  20 µm/5000 µm, or 1/250^th of a hemisphere), 2) the average number of cortical cells per 20 µm -thick section (4,800,000 cells/hemisphere (2 mo) or 1,500,000 cells/hemisphere (4 mo) ×1/250^th of a hemisphere  = 19,200 cells/20 µm section), and 3) the percentage of dying cells per section (average number of dead cells per 20 µm section/19,200 total cells).</p><p>#actual value = −9.6% (p = 0.09; n = 4 pairs); <i>ND</i>, not determined. *<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010195#pone.0010195-Saura1" target="_blank">[10]</a></p

Apoptotic neuronal death in the cerebral cortex of <i>Psen</i> cDKO mice.

<p>(<i>A</i>) Left: Confocal images of TUNEL-stained cells in the neocortex of control and cDKO mice at low magnification (20×). Right: High magnification (100×) images of individual TUNEL+ cells. Scale bar: 10 µm. (<i>B</i>) Quantification of TUNEL+ cells at the ages of 6 weeks, 2 and 4 months. At 6 weeks, similar low numbers of TUNEL+ cells are present in the neocortex between control and <i>Psen</i> cDKO mice. In contrast, dramatic increases in TUNEL+ cells are observed in the cDKO neocortex at 2 months (7.4-fold increase) and 4 months (15.2-fold increase)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). (<i>C</i>) Quantification of cells positive for activated caspase-9 or caspase-3. The number of active caspase-9+ cells is significantly increased in the neocortex of cDKO mice at 2 and 4 months of age (2m: p<0.005; 4m: p<0.001). The number of active caspase-3+ cells is not significantly different in the cDKO neocortex at 2 months (p>0.05), but is significantly increased in the cDKO neocortex at 4 months (1.7-fold increase; p<0.01)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). Data are presented as the mean ± s.e.m.</p

Increases in degenerating cortical neurons at 2 months followed by significant loss of cortical neurons and volume at 4 months in <i>Psen</i> cDKO mice.

<p>(<i>A</i>) Nissl-stained images of coronal sections from age-matched control (<i>left</i>) and <i>Psen</i> cDKO mutant (<i>right</i>) brains from 2 to 22 months of age are shown. Black horizontal bars delineate neocortical layers. At 2 months, no detectable difference is found in size or shape of the <i>Psen</i> cDKO brain relative to control. However, subsequent ages reveal a gradual decrease in cortical thickness in <i>Psen</i> cDKO mice. Scale bar: 1 mm. (<i>B</i>) Stereological measurement of cortical volume from control (open bars) and <i>Psen</i> cDKO mutant (black bars) brains at 2 and 4 months. Values are presented per hemisphere. At 2 months of age, the cortical volume is similar between control and cDKO mice (p>0.05). At 4 months, the cortical volume in cDKO mice is significantly smaller (21.7% reduction; p<0.001). NS, not significant; ***, p<0.001; n = 4 mice per genotype. (<i>C</i>) Stereological quantification of neuron number in the neocortex. At 4 months, an 8.7% decrease in neuron number (1.15×10⁶) is present in <i>Psen</i> cDKO mice, relative to controls (1.05×10⁶; **p<0.01)(n = 3 control mice +4 cDKO mice). (<i>D</i>) Quantification of degenerative neurons. <i>Top</i>: Fluoro-Jade B labels degenerating neurons in brain sections, and high magnification (100×) confocal images of single cells stained with Fluoro-Jade B. Scale bar: 10 µm. <i>Bottom</i>: Increased numbers of Fluoro-Jade-positive neurons are detected at 2 months in the <i>Psen</i> cDKO cortex (7.6-fold increase; p<0.0001) relative to control, with even greater numbers of dying neurons present by 4 months of age (9.0-fold increase; p<0.0001)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). Data are presented as the mean ± s.e.m.</p

Neurodegeneration and increased adult neurogenesis in the hippocampus of <i>Psen</i> cDKO mice.

<p>(<i>A</i>) Images of Nissl-stained brain sections at 2 and 22 months of age. At 2 months, the neocortex (NCx), hippocampal areas CA1 and CA3, and dentate gyrus (DG) are indistinguishable between <i>Psen</i> cDKO and age-matched controls. By 22 months, extensive loss of white and grey matter is evident in the neocortex of <i>Psen</i> cDKO mice. In the hippocampus, loss of white matter in cDKO mice coincides with the increase in neurons in the dentate gyrus. Scale bar: 1 mm. (<i>B</i>) Stereological quantification of hippocampal volumes. The volume of the <i>Psen</i> cDKO hippocampus is normal at 2 months (control: 4.85×10⁵; cDKO: 4.54×10⁵; p>0.05) and 4 months (control: 4.78×10⁵; cDKO: 4.61×10⁵; p>0.05). By 16–23 months, a 15.5% reduction in hippocampal volume is observed (control: 4.76×10⁵; cDKO: 4.02×10⁵; *p<0.05). NS: not significant; n = 4 mice per genotype (2 mo, 4 mo); n = 6 mice per genotype (16–23 mo). (<i>C</i>) Stereological quantification of the number of cells within the hippocampal dentate gyrus (DG). Cell number in the DG is comparable between cDKO and control mice at 2 months (control: 2.94×10⁵; cDKO: 3.21×10⁵; p>0.05) and 6 months (control: 2.80×10⁵; cDKO: 3.15×10⁵; p>0.3). In contrast, by 19 months, <i>Psen</i> cDKO mice have significantly greater numbers of cells in the DG (control: 3.18×10⁵; cDKO: 4.51×10⁵; **p<0.01)(n = 4 mice per genotype per age). (<i>D</i>) Quantification of Fluoro-Jade B+ degenerating neurons. At 2 months, similar numbers of Fluoro-Jade B+ cells are present in control and cDKO mice (p>0.05), in contrast to a 2.3-fold increase in <i>Psen</i> cDKO mice by 4 months of age (*p<0.05)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). (<i>E</i>) Quantification of apoptotic neurons. Increases in TUNEL+ cells are present in the <i>Psen</i> cDKO hippocampus at 2 months (*p<0.05) and 4 months (***p<0.001), whereas the number of TUNEL+ cells is similar in control and cDKO mice at 6 weeks (p> 0.05)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). (<i>F</i>) More active caspase-9+ cells are present in the <i>Psen</i> cDKO hippocampus at 2 months (p<0.05) and 4 months (p<0.05)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). (<i>G</i>) The number of cells positive for active caspase-3+ in the hippocampus is increased in cDKO mice at 2 months (p<0.05), but not significantly increased at 4 months (p>0.05)(n = 4 mice per genotype per age; 10 sections analyzed per mouse). Data are presented as the mean ± s.e.m.</p

Selected box plots for metabolites of interest for PD (left) and RLS (right).

<p>Selected box plots for metabolites of interest for PD (left) and RLS (right).</p

Ten Rare, Non-synonymous Variants Shared by Individuals IV:11 and IV:18 of Family PARK_0005.

<p>The rare variants common to the two affected individuals were genotyped in 975 cases and 1014 controls. Penetrance with regard to the PD phenotype was assessed in 6 family members belonging to the same generation as the affected individuals. EA = European American.</p

Qualitative multifactorial interaction network of <i>PLXNA4</i> and genetic factors with known and hypothetical relevance to PD.

<p>Edges obtained from CIDeR are highlighted in blue, PD-specific pathways from KEGG are given in green, red edges denote annotations from OMIM and edges extracted from literature, protein-protein interaction databases or high-confidence predictions are colored black. Undirected protein-protein interactions hold circular ends, directed molecular relations are marked by arcs, whereas general regulations have arrows with no filling, activations have filled arrows and inhibitions have blunted end. Dashed lines indicate indirect effects.</p

Assessment of cell viability and subcellular protein localization in fibroblasts.

<p>(A) The presence of <i>PLXNA4</i> p.Ser657Asn do not affect cell viability as assay by live-dead staining and FACS. (B) Immunohistochemistry shows similar subcellular localization of <i>PLXNA4</i> (anti-PLXNA4, Sigma, 1∶500) in fibroblasts with and without the p.Ser657Asn amino acid substitution (scale bar = 50 µm).</p