Compromised notch-1 cleavage and premature cell differentiation in neural progenitor cells expressing reduced levels of PS1 can be partially rescued by exogenous NICD.

<p><b>A</b>. Reduced expression of PS1-NTF, NICD (80 & 120 kDa fragments) and PDGFRα in neural progenitor cells that were infected with lentiviral vectors expressing PS1 shRNA compared to cells infected with control RL shRNA. <b>B<i>i</i>-B<i>ii</i></b>. Densitometry analysis of Western blots. <b>C</b>. Scheme of infection of lentiviral vectors followed by NICD transfection. <b>D, F</b>. Phase contrast images of neural progenitor cells infected with lentiviral vectors expressing either control RL shRNA (D) or PS1 shRNA (F) followed by transfection with either control- or NICD-expressing vector. <b>E, G</b>. Quantification of round cells following NICD transfection show no change in the control condition (<b>E)</b> and a significant increase in the PS1 condition <b>(G)</b> indicating a reversal in morphological change following transfection with NICD-expressing vector with cells exhibiting round morphology (unpaired t-test; *P<0.05). Error bars indicate ±SEM.</p

PS1 downregulation in new neurons decreases dendritic arborization and spine density.

<p><b>A</b>. Compressed Z-stack confocal images (Zeiss LSM 510) of newly mature GFP⁺ neurons (GFP⁺BrdU+NeuN⁺) in the granular cell layer of the dentate gyrus of RL shRNA- and PS1 shRNA- injected mice 6 months after injection. Images show a dramatic decrease in the number of dendritic arborization in mature GFP⁺ neurons in the PS1 shRNA- injected mice. <b>B</b>. Sholl analysis of the number of intersections measured from n = 16 neurons per group. PS1 animals show decreased number of intersections (two-way ANOVA (treatment and distance from soma) F_{(treatment)1,120} = 14.75, P<0.001, F_{(distance)19,120} = 4.10, P<0.0001). <b>C, D</b>. PS1 downregulation results in decreased number of spines <b>(C)</b> as a function of increasing distance from the soma (two-way ANOVA (treatment and distance from soma) F_{(treatment)1,60} = 13.10, P<0.001, F_{(distance)19,60} = 3.18, P<0.001) and mean spine density <b>(D)</b> (unpaired t-test, *P<0.05). <b>F, G</b>. Dendritic surface area (<b>F)</b> and volume <b>(G)</b> were unaffected by PS1 downregulation. Error bars indicate ±SEM.</p

Presenilin-1 Dependent Neurogenesis Regulates Hippocampal Learning and Memory

<div><p>Presenilin-1 (PS1), the catalytic core of the aspartyl protease γ-secretase, regulates adult neurogenesis. However, it is not clear whether the role of neurogenesis in hippocampal learning and memory is PS1-dependent, or whether PS1 loss of function in adult hippocampal neurogenesis can cause learning and memory deficits. Here we show that downregulation of PS1 in hippocampal neural progenitor cells causes progressive deficits in pattern separation and novelty exploration. New granule neurons expressing reduced PS1 levels exhibit decreased dendritic branching and dendritic spines. Further, they exhibit reduced survival. Lastly, we show that PS1 effect on neurogenesis is mediated via β-catenin phosphorylation and notch signaling. Together, these observations suggest that impairments in adult neurogenesis induce learning and memory deficits and may play a role in the cognitive deficits observed in Alzheimer’s disease.</p></div

Compromised survival of new neurons expressing reduced levels of PS1 in the granular cell layer of the dentate gyrus of adult mice.

<p>Unbiased stereological analysis of green fluorescent protein positive (GFP⁺) cell populations in the SGL and GCL of the DG at 3 and 6 months post-injection. <b>A</b>. Less mature neurons within the GCL (GFP⁺BrdU^-NeuN⁺; *P<0.05) of mice injected with PS1 shRNA. <b>B</b>. Separate Comparisons within RL and PS1 groups at 3 and 6 months post injection reveals reduced survival of new neurons in the GCL of PS1 shRNA (GFP⁺BrdU⁺NeuN⁺; *P<0.05). Error bars indicate ±SEM. <b>C</b>. Confocal image (Zeiss LSM 510) representing colocalization of GFP with NeuN and BrdU (63x). <b>(D,E)</b>. Comparisons within RL and PS1 groups at 3 and 6 months post injection shows reduced rate of survival of PDGFRα⁺ NPCs in the SGL (GFP⁺PDGFRα⁺Nestin^-; **P<0.01) <b>(D)</b> and of total neurons and oligodendrocytes within the SGL (GFP⁺PDGFRα⁺; **P<0.01), <b>(E)</b>. Error bars indicate ±SEM. <b>F</b>. Confocal image (Zeiss LSM 510) representing colocalization of GFP, nestin and PDGFRα (63x).</p

Downregulation of PS1 expression alters β-catenin metabolism and disrupts CREB expression.

<p><b>A</b>. Schematics of neurogenic signals that interact with PS1. <b>B</b>. Confocal image (Zeiss LSM 510) representing colocalization of pCREB and Nestin in the dentate gyrus of the hippocampus (40x). <b>C, D</b>. (<i>i</i>) Expression of total β-Catenin were similar in both groups. However, levels of phospho-β-Catenin (<i>ii</i>) were significantly decreased in neural progenitor cells with reduced PS1 expression. Levels of total GSK3β (<i>iii</i>), phospho-GSK3β <i>(iv)</i> and CREB (<i>v</i>) were similar in both groups. Phospho-CREB (<i>vi</i>) shows decreasing trend, albeit statistically not significant. Western blot analysis significance was determined by unpaired t-test with Welch’s correction, *P<0.05, **P<0.01. Error bars indicate ±SEM.</p

Downregulation of PS1 expression in adult neural progenitor cells induces the expression of differentiation markers.

<p>Quantitation of Western blot analysis reported as percentage of control values all normalized to actin. Western blot analysis (<b>A</b>) and quantification (<b>B</b>) of neurogenic signals in neural progenitor cell culture infected with lentiviral vectors expressing either control RL shRNA or PS1 shRNA. <b>(B<i>i-iii</i>)</b>. Expression of PS1-NTF (<i>i</i>), and Nestin (<i>ii</i>) are significantly reduced, while expression of Cyclin D1 (<i>iii</i>) and epidermal growth factor receptor (EGFR, <i>iv</i>) are slightly reduced following PS1 downregulation in protein extract of neurosphere cultures infected with lentivirus expressing PS1 shRNA. This suggests that reduced PS1 expression decreases proliferation and progenitor phase. Expression of Neurofilament-L (<i>v</i>) increased in neural progenitors infected with PS1 shRNA, supporting enhanced premature neuronal differentiation following PS1 downregulation in these cells. Levels of platelet derived growth factor receptor α (PDGFRα, <i>vi</i>) were not significantly different. Unpaired t-test with Welch’s correction, *P<0.05, **P<0.005. Error bars indicate ±SEM.</p

Impairments in novel object recognition at 3 and 6 months after PS1 knockdown in neural progenitor cells.

<p>Percentage of time spent exploring the novel or familiar object by mice injected with either the control RL shRNA or PS1 shRNA at 3 <b>(A)</b> and 6 months <b>(B)</b>. In contrast to RL-injected mice, PS1 shRNA-injected mice show no preference for the novel object at both time-points (unpaired t-test, *p<0.05). Error bars indicate ±SEM.</p

Impairments in pattern separation 3 months after PS1 knockdown in neural progenitor cells.

<p><b>A</b>. Experimental timeline. GFP expression in the dentate gyrus of the hippocampus at 2 weeks, 6 weeks, 3 and 6 months after lentivirus injection. Two weeks following injection, GFP-infected cells are located in the subgranular layer of the dentate gyrus. Migration of GFP+ cells into the granular cell layer is apparent at 6 weeks post injection, while at 3 and 6 months post-injection many GFP⁺ cells are incorporated in the granular cell layer. <b>B-C</b> Percentage of freezing in context A (shock) vs. context B (similar context) of mice 3 months following injection with either control (15) shRNA (<b>B</b>) or with PS1 shRNA (<b>C</b>) (two-way repeated measures (context and days) ANOVA, F_{(context)1,16} = 5.02, P<0.05; and F_{(context)1,18} = 6.18, P<0.05 respectively). Paired t-test, *p<0.05, **p<0.005. Error bars indicate ± SEM. <b>D</b>. Discrimination ratio [(A-B)/max(A,B)] between the control and the PS1 injected animals (two-way ANOVA [treatment and days] with repeated measures, F_{(treatment)1,153} = 1.38, P>0.05) Error bars indicate ±SEM. <b>E</b>. Post-hoc comparisons between contexts and treatments on single days during the recognition memory paradigm (paired t-test, *p<0.05).</p

Progressive impairments in pattern separation 6 months after PS1 knockdown in neural progenitor cells.

<p>Percentage of freezing in context A (shock) vs. context B (similar context) of 14 month old mice, 6 months following injection with either control shRNA (<b>A</b>) or with PS1 shRNA (<b>B</b>) (two-way repeated measures (context and days) ANOVA, F_{(context)1,14} = 6.92, P<0.05 and F_{(context)1,14} = 1.88, P>0.05, respectively). Paired t-test, *P<0.05, **P<0.005. Error bars indicate ± SEM. <b>C</b>. Discrimination ratio [(A-B)/max(A,B)] analysis reveals a significant difference between the RL shRNA and PS1 shRNA injected animals (two-way ANOVA (treatment and days) with repeated measures, F_{(treatment)1,126} = 3.95, *P<0.05). Error bars indicate ±SEM. <b>D</b>. Post-hoc comparisons between contexts and treatments on individual days in the recognition memory paradigm (paired t-test, *p<0.05).</p

Ischemic preconditioning (IPC) does not occur in Cav-1 KO mice.

<p>(<b>A</b>) Hippocampal synaptosomes from Cav-1 KO (Yg) showed a similar pattern to Ag, with a decrease in PSD-95, NR2A, NR2B, and AMPAR. PSD-95 IPs of Cav-1 KO synaptosomes revealed minimal detection in PSD-95, NR2A, NR2B, and AMPAR. (<b>B</b>) WT or Cav-1 KO mice were subjected to 3 min (ischemic preconditioning, IPC) and/or 12 min (lethal ischemia, LI) induced by bilateral carotid artery occlusion (BCAO). Intact neurons in CA1 hippocampal (HP) region were counted from Cresyl Violet stained paraffin fixed sections. IPC (3 min, BCAO) significantly protected CA1 neurons against LI (12 min, BCAO) in WT mice (<b>iv</b>). There was a significant increase in CA1 neuronal death in Cav-1 KO animals subject to IPC (<b>viii</b>) versus WT IPC + LI. Representative Cresyl Violet stained CA1 hippocampal images from (<b>i</b>) WT sham, (<b>ii</b>) WT IPC, (<b>iii</b>) WT LI, and (<b>iv</b>) WT IPC and (<b>v</b>) Cav-1 KO sham, (<b>vi</b>) Cav-1 KO IPC, (<b>vii</b>) Cav-1 KO LI, and (<b>viii</b>) Cav-1 KO IPC. Quantitation of images is presented by the graph.</p