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

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

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

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