Expression Of Presenilin Research Articles

Immunohistochemical characterization of γ‐secretase activating protein expression in Alzheimer's disease brains

A recent study showed that γ-secretase activating protein (GSAP), derived from a C-terminal fragment of pigeon homolog (PION), increases amyloid-β (Aβ) production by interacting with presenilin-1 (PS1) and the β-secretase-cleaved C-terminal fragment of amyloid precursor protein (APP-CTF). In the study, knockdown of GSAP reduces production of Aβ and plaque formation in the brain of APPswe and PS1ΔE9 double transgenic mice without affecting the Notch-dependent pathway. Therefore, GSAP is an ideal target for designing γ-secretase modulators with least side effects in Alzheimer's disease (AD). However, at present, the precise distribution of GSAP in AD brains remains to be characterized. By immunohistochemistry, we studied GSAP expression in the frontal cortex and the hippocampus of 11 aged AD and 17 age-matched control cases. GSAP immunoreactivity exhibited distinct morphological features, such as fine granular cytoplasmic deposits, dense nodular and patchy deposits, beads and string-like deposits, and diffuse dot-like deposits. In both AD and control brains, a fairly small subset of cerebral cortical and hippocampal neurones expressed fine granular cytoplasmic deposits, while diffuse dot-like deposits were more frequently found in the neuropil and neuronal processes, particularly enriched in the hippocampal CA2 and CA3 regions. Among GSAP-immunoreactive deposits, dense nodular and patchy deposits, located in the neuropil and closely associated with PS1 expression and Aβ deposition, indicated the most distinguishing features of AD pathology. Aberrant regulation of GSAP expression plays a key role in acceleration of γ-cleavage of APP-CTF and accumulation of Aβ in AD brains.

Lack of Evidence for Presenilins as Endoplasmic Reticulum Ca2+ Leak Channels

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.

Presenilin-2 regulates the degradation of RBP-Jk protein through p38 mitogen-activated protein kinase

Transcriptional regulation performs a central role in Notch1 signaling by recombining binding protein Suppressor of Hairless (RBP-Jk)--a signaling pathway that is widely involved in determination of cell fate. Our earlier work demonstrated the possible regulation of the Notch1-RBP-Jk pathway through protein degradation of RBP-Jk; however, the potential regulator for the degradation of RBP-Jk remains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cells treated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found that PS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase ∂ (MAPK) had significantly increased levels of RBP-Jk. p38 MAPK phosphorylates RBP-Jk at Thr339 by physical binding, which subsequently induces the degradation and ubiquitylation of the RBP-Jk protein. Collectively, our results indicate that PS2 modulates the degradation of RBP-Jk through phosphorylation by p38 MAPK.

Intraperitoneal injection of JNK-specific inhibitor SP600125 inhibits the expression of presenilin-1 and Notch signaling in mouse brain without induction of apoptosis

Presenilin-1 (PS1) is a multifunctional protein involved in many cellular functions including the processing of type 1 membrane proteins such as β-amyloid precursor protein (APP) and Notch 1 receptor. PS1 acts as the catalytic subunit of the γ-secretase complex, and participates in Notch 1 processing to release Notch intracellular domain (NICD) in the cytoplasm. NICD subsequently migrates to the nucleus and causes Notch signaling by increasing the expression of the Hes1 gene. We have previously shown that inhibition of basal activity of c-jun-NH2-terminal kinase (JNK) with JNK-specific inhibitor SP600125 represses the expression of PS1 and γ-secretase activity by increasing p53 level in SK-N-SH cell line in vitro (Lee and Das, 2008, 2010). However, it is largely unknown whether PS1 can be effectively suppressed in vivo in adult mouse brains. In this report we showed that intraperitoneal (i.p) injection of JNK-specific inhibitor SP600125 decreased p-JNK level, and reduced PS1 expression by increasing p53 level in adult mouse brains. We also showed that suppression of PS1 expression by SP600125 reduced γ-secretase activity which decreased Notch 1 processing to reduce NICD in mouse brains. Furthermore, inhibition of Notch 1 processing by SP600125 decreased Notch 1 signaling by reducing the expression of the NICD target Hes1 gene in mouse brains without induction of apoptosis. These results provide insights for further study on PS1-mediated reduction of Notch 1 and APP processing for the treatment of Alzheimer's disease.

Cellular consequences of the expression of Alzheimer's disease-causing presenilin 1 mutations in human neuroblastoma (SH-SY5Y) cells

Mutations in the presenilin 1 (PS1) gene lead to early-onset Alzheimer's disease with the S170F mutation causing the earliest reported age of onset. Expression of this, and other PS1 mutations, in SH-SY5Y cells resulted in significant loss of cellular viability compared to control cells. Basal Ca2+ concentrations in PS1 mutants were never lower than controls and prolonged incubation in Ca2+-free solutions did not deplete Ca2+ stores, demonstrating there was no difference in Ca2+ leak from endoplasmic reticulum (ER) stores in PS1 mutants. Peak muscarine-evoked rises of [Ca2+]i were variable, but the integrals were not significantly different, suggesting, while kinetics of Ca2+ store release might be affected in PS1 mutants, store size was similar. However, when Ca2+-ATPase activity was irreversibly inhibited with thapsigargin, the S170F and ΔE9 cells showed larger capacitative calcium entry indicating a direct effect on Ca2+ influx pathways. There was no significant effect of any of the mutations on mitochondrial respiration. Amyloid β(Aβ(1–40)) secretion was reduced, and Aβ(1–42) secretion increased in the S170F cells resulting in a very large increase in the Aβ42/40 ratio. This, rather than any potential disruption of ER Ca2+ stores, is likely to explain the extreme pathology of this mutant.

Endoplasmic reticulum stress enhances γ-secretase activity

The endoplasmic reticulum (ER) copes with unfolded proteins in the lumen (ER stress) by activating three distinct intracellular signaling pathways of unfolded protein response (UPR). ER stress contributes to the pathogenesis of obesity and diabetes, which are risk factors for Alzheimer’s disease (AD) that accelerate the pathogenesis of AD. However, whether ER stress is involved in the development of AD remains unclear. In this study, we demonstrate that ER stress induces presenilin-1 expression through activating transcription factor 4 (ATF4), resulting in increased amyloid-β (Aβ) secretion by γ-secretase activity, which is suppressed by quercetin by modifying UPR signaling. This result suggests that ER stress may be stimulated in obesity and type 2 diabetes, thereby enhancing γ-secretase activity that is the underlying molecular mechanism affecting the pathogenesis of AD.

Genistein downregulates presenilin 1 and ubiquilin 1 expression

The aim of this study was to determine the effects of several food ingredients and chemical inhibitors on the expression of presenilin, a molecule involved in γ-secretase activity and the generation of amyloid-β peptide in Alzheimer's disease. Western blotting revealed the downregulation of presenilin 1 protein expression by stimulation with genistein in vitro, while the effects on presenilin 1 gene expression examined by reverse transcriptase-polymerase chain reaction (RT-PCR) were unaltered in Daudi cells. Genistein likely downregulates presenilin via the inhibition of ubiquilin 1 expression in lymphoid cells. Our findings provide new insights that may help to establish preventive strategies against Alzheimer's disease.

Presenilin genes are downregulated during somitogenesis in the cadmium-induced omphalocele chick model

Although the precise pathogenesis of ventral body wall (VBW) defects is not clearly understood, it has recently postulated that disruption of somite development during early embryogenesis may cause failure of proper VBW formation. The administration of cadmium (Cd) after 60 h of incubation induces omphalocele spectrum in the chick embryo. Previous studies have shown that one of the earliest histological changes seen in this model is abnormal cell death in the somite, occurring at 4 h post treatment (4H). However, the molecular mechanism by which Cd acts in this critical period of embryogenesis still remains unclear. Presenilins are expressed in somites and play an important role in vertebrate development, including somitogenesis and thus VBW formation. We designed this study to test the hypothesis that gene expression levels of presenilin 1 (PSEN1) and presenilin 2 (PSEN2) are downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele in the chick model. After 60 h of incubation, chick embryos were exposed to either saline or 50 μM cadmium and divided into two groups: control and Cd (n = 8 at each time point for each group). Real-time RT-PCR was performed to evaluate the relative mRNA expression levels of PSEN1 and PSEN2 in the Cd-induced omphalocele chick model. Differences between two groups at each time point were analysed statistically and the significance was accepted at p < 0.05. Immunofluorescence confocal microscopy was performed to evaluate the protein expression/distribution of presenilins in the somite of chick embryo. The relative mRNA expression levels of PSEN1 and PSEN2 were significantly downregulated in the Cd group at 4H compared with controls (p < 0.005) (Table). However, there were no significant differences at the other time points. At 4H, immunofluorescence of presenilins (green) was markedly diminished in the Cd-treated embryos, whereas strong immunofluorescence of them was seen in the somite (dermomyotome) in controls (Fig. 1). 1 Immunofluorescence Confocal Microscopy for PSEN1 and PSEN2 in the dermomyotome of the somite in the trunk level of chick embryo 4H post treatment. Intensity of PSEN1 immunofluorescence (green) was markedly diminished in Cd-treated embryos, whereas strong PSEN1 expression was seen in the dermomyotome in controls. PSEN2 immunofluorescence was also decreased in the Cd-treated embryos, whereas strong PSEN2 immunofluorescence (green) was observed predominantly in the dermomyotome in controls. Immunofluorescence in orange is DNA counter staining by DAPI CONCLUSION: We provide evidence, for the first time, that gene expression of presenilins is downregulated during the narrow window of very early embryogenesis in the Cd chick model. Decreased expression of presenilins may contribute to omphalocele phenotype in Cd chick model, by disrupting somite development.

Curcumin mediates presenilin-1 activity to reduce β-amyloid production in a model of Alzheimer’s disease

AbstractCurcumin has been reported to inhibit the generation of Aβ, but the underlying mechanisms by which this occurs remain unknown. Aβ is thought to play an important role in the pathogenesis of Alzheimer’s disease (AD). The amyloid hypothesis argues that aggregates of Aβ trigger a complex pathological cascade that leads to neurodegeneration. Aβ is generated by the processing of APP (amyloid precursor protein) by β- and γ-secretases. Presenilin 1 (PS1) is central to γ-secretase activity and is a substrate for GSK-3β, both of which are implicated in the pathogenesis of AD. The present study aimed to investigate the effects of curcumin on the generation of Aβ in cultured neuroblastoma cells and on the in vitro expression of PS1 and GSK-3β. To stimulate Aβ production, a plasmid expressing APP was transfected into human SH-SY5Y neuroblastoma cells. The transfected cells were then treated with curcumin at 0–20µM for 24h or with 5µM curcumin for 0–48h, and the extracellular levels of Aβ40/42 were determined by ELISA. The levels of PS1 and GSK-3β mRNA were measured by RT-PCR, and the expression of the PS1 and GSK-3β proteins (including the phosphorylated form of GSK-3β, p-GSK-3β-Ser9) were evaluated by western blotting. Curcumin treatment was found to markedly reduce the production of Aβ40/42. Treatment with curcumin also decreased both PS1 and GSK-3β mRNA and protein levels in a dose- and time-dependent manner. Furthermore, curcumin increased the inhibitory phosphorylation of GSK-3β protein at Ser9. Therefore, we propose that curcumin decreases Aβ production by inhibiting GSK-3β-mediated PS1 activation.

Docosahexaenoic Acid Reduces Amyloid β Production via Multiple Pleiotropic Mechanisms

Alzheimer disease is characterized by accumulation of the β-amyloid peptide (Aβ) generated by β- and γ-secretase processing of the amyloid precursor protein (APP). The intake of the polyunsaturated fatty acid docosahexaenoic acid (DHA) has been associated with decreased amyloid deposition and a reduced risk in Alzheimer disease in several epidemiological trials; however, the exact underlying molecular mechanism remains to be elucidated. Here, we systematically investigate the effect of DHA on amyloidogenic and nonamyloidogenic APP processing and the potential cross-links to cholesterol metabolism in vivo and in vitro. DHA reduces amyloidogenic processing by decreasing β- and γ-secretase activity, whereas the expression and protein levels of BACE1 and presenilin1 remain unchanged. In addition, DHA increases protein stability of α-secretase resulting in increased nonamyloidogenic processing. Besides the known effect of DHA to decrease cholesterol de novo synthesis, we found cholesterol distribution in plasma membrane to be altered. In the presence of DHA, cholesterol shifts from raft to non-raft domains, and this is accompanied by a shift in γ-secretase activity and presenilin1 protein levels. Taken together, DHA directs amyloidogenic processing of APP toward nonamyloidogenic processing, effectively reducing Aβ release. DHA has a typical pleiotropic effect; DHA-mediated Aβ reduction is not the consequence of a single major mechanism but is the result of combined multiple effects.

Insulin Suppresses the Expression of Amyloid Precursor Protein, Presenilins, and Glycogen Synthase Kinase-3β in Peripheral Blood Mononuclear Cells

Our objective was to determine whether peripheral blood mononuclear cells express amyloid precursor protein (APP) and other mediators involved in the pathogenesis of Alzheimer's disease and whether their expression is suppressed by insulin. Ten obese type 2 diabetic patients were infused with insulin (2 U/h with 100 ml 5% dextrose/h) for 4 h. Patients were also infused with 5% dextrose/h or normal physiological saline for 4 h, respectively, on two other days as controls. Blood samples were obtained at 0, 2, 4, and 6 h. Insulin infusion significantly suppressed the expression of APP, presenilin-1, presenilin-2, and glycogen synthase kinase-3β in peripheral blood mononuclear cells. Dextrose and saline infusions did not alter these indices. Insulin infusion also caused significant parallel reductions in nuclear factor-κB binding activity and plasma concentrations of serum amyloid A and intercellular adhesion molecule-1. A low dose infusion of insulin suppresses APP, presenilin-1, presenilin-2, and glycogen synthase kinase-3β, key proteins involved in the pathogenesis of Alzheimer's disease, in parallel with exerting its other antiinflammatory effects.

Presenilins Promote the Cellular Uptake of Copper and Zinc and Maintain Copper Chaperone of SOD1-dependent Copper/Zinc Superoxide Dismutase Activity

Dyshomeostasis of extracellular zinc and copper has been implicated in β-amyloid aggregation, the major pathology associated with Alzheimer disease. Presenilin mediates the proteolytic cleavage of the β-amyloid precursor protein to release β-amyloid, and mutations in presenilin can cause familial Alzheimer disease. We tested whether presenilin expression affects copper and zinc transport. Studying murine embryonic fibroblasts (MEFs) from presenilin knock-out mice or RNA interference of presenilin expression in HEK293T cells, we observed a marked decrease in saturable uptake of radiolabeled copper and zinc. Measurement of basal metal levels in 6-month-old presenilin 1 heterozygous knock-out (PS1(+/-)) mice revealed significant deficiencies of copper and zinc in several tissues, including brain. Copper/zinc superoxide dismutase (SOD1) activity was significantly decreased in both presenilin knock-out MEFs and brain tissue of presenilin 1 heterozygous knock-out mice. In the MEFs and PS1(+/-) brains, copper chaperone of SOD1 (CCS) levels were decreased. Zinc-dependent alkaline phosphatase activity was not decreased in the PS null MEFs. These data indicate that presenilins are important for cellular copper and zinc turnover, influencing SOD1 activity, and having the potential to indirectly impact β-amyloid aggregation through metal ion clearance.

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.

HPRT Deficiency Coordinately Dysregulates Canonical Wnt and Presenilin-1 Signaling: A Neuro-Developmental Regulatory Role for a Housekeeping Gene?

We have used microarray-based methods of global gene expression together with quantitative PCR and Western blot analysis to identify dysregulation of genes and aberrant cellular processes in human fibroblasts and in SH-SY5Y neuroblastoma cells made HPRT-deficient by transduction with a retrovirus stably expressing an shRNA targeted against HPRT. Analysis of the microarray expression data by Gene ontology (GO) and Gene Set Enrichment Analysis (GSEA) as well as significant pathway analysis by GeneSpring GX10 and Panther Classification System reveal that HPRT deficiency is accompanied by aberrations in a variety of pathways known to regulate neurogenesis or to be implicated in neurodegenerative disease, including the canonical Wnt/β-catenin and the Alzheimer's disease/presenilin signaling pathways. Dysregulation of the Wnt/β-catenin pathway is confirmed by Western blot demonstration of cytosolic sequestration of β-catenin during in vitro differentiation of the SH-SY5Y cells toward the neuronal phenotype. We also demonstrate that two key transcription factor genes known to be regulated by Wnt signaling and to be vital for the generation and function of dopaminergic neurons; i.e., Lmx1a and Engrailed 1, are down-regulated in the HPRT knockdown SH-SY5Y cells. In addition to the Wnt signaling aberration, we found that expression of presenilin-1 shows severely aberrant expression in HPRT-deficient SH-SY5Y cells, reflected by marked deficiency of the 23 kDa C-terminal fragment of presenilin-1 in knockdown cells. Western blot analysis of primary fibroblast cultures from two LND patients also shows dysregulated presenilin-1 expression, including aberrant proteolytic processing of presenilin-1. These demonstrations of dysregulated Wnt signaling and presenilin-1 expression together with impaired expression of dopaminergic transcription factors reveal broad pleitropic neuro-regulatory defects played by HPRT expression and suggest new directions for investigating mechanisms of aberrant neurogenesis and neuropathology in LND and potential new targets for restoration of effective signaling in this neuro-developmental defect.

A Novel Role for γ-Secretase: Selective Regulation of Spontaneous Neurotransmitter Release from Hippocampal Neurons

With a multitude of substrates, γ-secretase is poised to control neuronal function through a variety of signaling pathways. Presenilin 1 (PS1) is an integral component of γ-secretase and is also a protein closely linked to the etiology of Alzheimer's disease (AD). To better understand the roles of γ-secretase and PS1 in normal and pathological synaptic transmission, we examined evoked and spontaneous neurotransmitter release in cultured hippocampal neurons derived from PS1 knock-out (KO) mice. We found no changes in the size of evoked synaptic currents, short-term plasticity, or apparent calcium dependence of evoked release. The rate of spontaneous release from PS1 KO neurons was, however, approximately double that observed in wild-type (WT) neurons. This increase in spontaneous neurotransmission depended on calcium influx but did not require activation of voltage-gated calcium channels or presynaptic NMDA receptors or release of calcium from internal stores. The rate of spontaneous release from PS1 KO neurons was significantly reduced by lentivirus-mediated expression of WT PS1 or familial AD-linked M146V PS1, but not the D257A PS1 mutant that does not support γ-secretase activity. Treatment of WT neuronal cultures with γ-secretase inhibitor mimicked the loss of PS1, leading to a selective increase in spontaneous release without any change in the size of evoked synaptic currents. Together, these results identify a novel role for γ-secretase in the control of spontaneous neurotransmission through modulation of low-level tonic calcium influx into presynaptic axon terminals.

Turmeric and curcumin suppress presenilin 1 protein expression in Jurkat cells

In the present study, we aimed to determine the effects of herbs or spices on the expression of presenilin 1, a molecule involved in γ-secretase activity and the generation of amyloid-β peptide in Alzheimer's disease. Western blot analysis revealed that presenilin 1 protein expression was down-regulated by stimulation with turmeric or cinnamon extracts in vitro, while the effects on presenilin 1 gene expression examined by reverse transcriptase-polymerase chain reaction were unaltered. Our results showed that curcumin, a component of turmeric, induced the down-regulation of presenilin 1 protein in Jurkat and K562 cell lines.

Presenilin-Dependent Receptor Processing Is Required for Axon Guidance

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.

Mutation Analysis of the Presenilin 1 N-terminal Domain Reveals a Broad Spectrum of γ-Secretase Activity toward Amyloid Precursor Protein and Other Substrates

The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease β-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.

Site-Dependent E-Cadherin Cleavage and Nuclear Translocation in a Metastatic Colorectal Cancer Model

Metastases are frequently found during colorectal cancer diagnoses and are the main determinants of clinical outcome. The lack of reliable models of metastases has precluded their mechanistic understanding and our capacity to improve outcome. We studied the effect of E-cadherin and Snail1 expression on metastagenesis in a colorectal cancer model. We microinjected SW480-ADH human colorectal cancer cells, transfected with an empty vector (Mock) or overexpressing Snail1 (Snail1(OE)) or E-cadherin (E-cadherin(OE)), in the ceca of nude mice (eight per group) and analyzed tumor growth, dissemination, and Snail1, E-cadherin, β-catenin, and Presenilin1 (PS1) expression in local tumors and/or metastatic foci. Snail1(OE) cells disseminated only to lymph nodes, whereas Mock or E-cadherin(OE) cells spread to lymph nodes and peritoneums. Peritoneal tumor foci developed by E-cadherin(OE) cells presented an increase in E-cadherin proteolysis and nuclear translocation, and enhanced expression of proteolytically active PS1, which was linked to increased tumor growth and shortened mouse survival. Interestingly, local and lymph node tumors in mice bearing E-cadherin(OE) cells overexpressed E-cadherin, but they did not show E-cadherin proteolysis or nuclear translocation. Remarkably, E-cadherin nuclear translocation and enhanced expression of active PS1 were found in a patient with colorectal signet-ring cell carcinoma. In conclusion, we have established a colorectal cancer metastasis model in which E-cadherin proteolyis and nuclear translocation associates with aggressive foci growth only in the peritoneal microenvironment.

TRANSLATIONAL IMPACT

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. Some cases of the disease (FAD) are inherited through causative defects in the genes for amyloid precursor protein (APP), presenilin 1 (PS1) or presenilin 2 (PS2). APP is a type 1 (single-pass) transmembrane protein that is cleaved by γ-secretase; presenilin contains two essential aspartyl residues that constitute the active site of the protease. γ-Secretase functions as a multiprotein complex with the accessory proteins Nicastrin, Pen2 and Aph1. Pathogenesis of FAD results from the misregulation of γ-secretase cleavage of APP, rather than by the absence of cleavage.γ-secretase cleaves other type 1 transmembrane proteins and plays a role in diverse clinically relevant pathways, including cell adhesion and cell differentiation. γ-Secretase cleavage of Notch is essential for normal embryogenesis, and loss of any of the γ-secretase components in mammals has an embryonic lethal, Notch-like phenotype.The authors show that the ancient organism Dictyostelium has highly diverged γ-secretase subunits that can precisely process ectoptic human APP. Thus, this key regulatory pathway is evolutionarily conserved through hundreds of millions of years. In Dictyostelium, γ-secretase activity is necessary for phagocytosis, a process also required for innate immune response in mammalian cells. Similar to the complex metazoa, γ-secretase components in Dictyostelium are required for cell-fate determination. Dictyostelium is a unique system that is experimentally amenable to study γ-secretase function.Understanding the function and regulation of presenilin-dependent γ-secretase cleavage events is essential for identifying new therapeutic approaches for treating AD. By expressing FAD mutations in Dictyostelium, we might be able to recapitulate aberrant cleavage of APP. Such cell lines might present a relatively simple and inexpensive system for the high-throughput screening of small-molecule libraries to identify novel pharmaceutical agents. Expression of mutant presenilin can also be used to study causative effects of γ-secretase misregulation on phagocytosis and developmental pathways.This model also has potential for understanding complex pathways involved in immunity. γ-Secretase in Dictyostelium functions in a primary role to regulate nutrient-particle capture via phagocytosis during cellular growth. In mammals, macrophages engulf invading pathogens and apoptotic cells by a mechanistically identical pathway. γ-secretase signaling is central both to phagocytosis in Dictystelium and to the immune response in mammalian cells. Studies in Dictyostelium that identify essential transmembrane protein targets that are cleaved by γ-secretase might uncover novel and conserved mechanistic pathways that regulate phagocytosis. The conservation of γ-secretase in Dictyostelium suggests it as a tractable model to understand AD and macrophage phagocytosis.