All in the family: how the APPs regulate neurogenesis

Recent intriguing evidence suggests that metabolites of amyloid precursor protein(APP), mutated in familial forms of Alzheimer’s disease(AD), play critical roles indevelopmental and post natal neurogenesis. Of note is soluble APPα (sAPPα) that regulates neural progenitor cell proliferation. The APP family encompasses a group of ubiquitously expressed and evolutionarily conserved, type I transmembrane glycoproteins, whose functions have yet to be fully elucidated. APP can undergo proteolytic cleavage by mutually exclusive pathways. The subtle structural differences between metabolites generated in the different pathways, as well as thei requilibrium, maybe crucial for neuronal function. The implications of this newbody of evidence are significant. Miscleavage of APP would readily impact developmental and postnatal neurogenesis, which might contribute to cognitive deficits characterizing Alzheimer’s disease. This review will discuss the implications of the role of the APP family in neurogenes is for neuronal development, cognitive function, and brain disorders that compromise learning and memory, such as AD

Presenilin-1 Regulates Neural Progenitor Cell Differentiation in the Adult Brain

Presenilin-1 (PS1) is the catalytic core of the aspartyl protease γ-secretase. Previous genetic studies using germ-line deletion of PS1 and conditional knock-out mice demonstrated that PS1 plays an essential role in neuronal differentiation during neural development, but it remained unclear whether PS1 plays a similar role in neurogenesis in the adult brain. Here we show that neural progenitor cells infected with lentiviral vectors-expressing short interfering RNA (siRNA) for the exclusive knockdown of PS1 in the neurogenic microenvironments, exhibit a dramatic enhancement of cell differentiation. Infected cells differentiated into neurons, astrocytes and oligodendrocytes, suggesting that multipotentiality of neural progenitor cells is not affected by reduced levels of PS1. Neurosphere cultures treated with γ-secretase inhibitors exhibit a similar phenotype of enhanced cell differentiation, suggesting that PS1 function in neural progenitor cells is γ-secretase-dependent. Neurospheres infected with lentiviral vectors expressing siRNA for the targeting of PS1 differentiated even in the presence of the proliferation factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), suggesting that PS1 dominates EFG and bFGF signaling pathways. Reduction of PS1 expression in neural progenitor cells was accompanied by a decrease in EGF receptor and β-catenin expression level, suggesting that they are downstream essential transducers of PS1 signaling in adult neural progenitor cells. These findings suggest a physiological role for PS1 in adult neurogenesis, and a potential target for the manipulation of neural progenitor cell differentiation

Molecular Mechanisms of Environmental Enrichment: Impairments in Akt/GSK3β, Neurotrophin-3 and CREB Signaling

<div><p>Experience of mice in a complex environment enhances neurogenesis and synaptic plasticity in the hippocampus of wild type and transgenic mice harboring familial Alzheimer's disease (FAD)-linked APPswe/PS1ΔE9. In FAD mice, this experience also reduces levels of tau hyperphosphorylation and oligomeric β-amyloid. Although environmental enrichment has significant effects on brain plasticity and neuropathology, the molecular mechanisms underlying these effects are unknown. Here we show that environmental enrichment upregulates the Akt pathway, leading to the downregulation of glycogen synthase kinase 3β (GSK3β), in wild type but not FAD mice. Several neurotrophic signaling pathways are activated in the hippocampus of both wild type and FAD mice, including brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and this increase is accompanied by the upregulation of the BDNF receptor, tyrosine kinase B (TrkB). Interestingly, neurotrophin-3 (NT-3) is upregulated in the brains of wild type mice but not FAD mice, while insulin growth factor-1 (IGF-1) is upregulated exclusively in the brains of FAD mice. Upregulation of neurotrophins is accompanied by the increase of N-Methyl-D-aspartic acid (NMDA) receptors in the hippocampus following environmental enrichment. Most importantly, we observed a significant increase in levels of cAMP response element- binding (CREB) transcripts in the hippocampus of wild type and FAD mice following environmental enrichment. However, CREB phosphorylation, a critical step for the initiation of learning and memory-required gene transcription, takes place in the hippocampus of wild type but not of FAD mice. These results suggest that experience of wild type mice in a complex environmental upregulates critical signaling that play a major role in learning and memory in the hippocampus. However, in FAD mice, some of these pathways are impaired and cannot be rescued by environmental enrichment.</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

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

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

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

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

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