Wild-type Presenilin 1 Research Articles

Genetically Augmenting Aβ42 Levels in Skeletal Muscle Exacerbates Inclusion Body Myositis-Like Pathology and Motor Deficits in Transgenic Mice

The pathogenic basis of inclusion body myositis (IBM), the leading muscle degenerative disease afflicting the elderly, is unknown, although the histopathological features are remarkably similar to those observed in Alzheimer's disease. One leading hypothesis is that the buildup of amyloid-beta (Abeta) peptide within selective skeletal muscle fibers contributes to the degenerative phenotype. Abeta is a small peptide derived via endoproteolysis of the amyloid precursor protein (APP). To determine the pathogenic effect of augmenting Abeta42 levels in skeletal muscle, we used a genetic approach to replace the endogenous wild-type presenilin-1 (PS1) allele with the PS1(M146V) allele in MCK-APP mice. Although APP transgene expression was unaltered, Abeta levels, particularly Abeta42, were elevated in skeletal muscle of the double transgenic (MCK-APP/PS1) mice compared to the parental MCK-APP line. Elevated phospho-tau accumulation was found in the MCK-APP/PS1 mice, and the greater activation of GSK-3beta and cdk5 were observed. Other IBM-like pathological features, such as inclusion bodies and inflammatory infiltrates, were more severe and prominent in the MCK-APP/PS1 mice. Motor coordination and balance were more adversely affected and manifested at an earlier age in the MCK-APP/PS1 mice. The data presented here provide experimental evidence that Abeta42 plays a proximal and critical role in the muscle degenerative process.

Equimolar Production of Amyloid β-Protein and Amyloid Precursor Protein Intracellular Domain from β-Carboxyl-terminal Fragment by γ-Secretase

We showed previously that cells expressing wild-type (WT) beta-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.

Modeling Clinically Heterogeneous Presenilin Mutations with Transgenic Drosophila

To assess the potential of Drosophila to analyze clinically graded aspects of human disease, we developed a transgenic fly model to characterize Presenilin (PS) gene mutations that cause early-onset familial Alzheimer's disease (FAD). FAD exhibits a wide range in severity defined by ages of onset from 24 to 65 years . PS FAD mutants have been analyzed in mammalian cell culture, but conflicting data emerged concerning correlations between age of onset and PS biochemical activity . Choosing from over 130 FAD mutations in Presenilin-1, we introduced 14 corresponding mutations at conserved residues in Drosophila Presenilin (Psn) and assessed their biological activity in transgenic flies by using genetic, molecular, and statistical methods. Psn FAD mutant activities were tightly linked to their age-of-onset values, providing evidence that disease severity in humans primarily reflects differences in PS mutant lesions rather than contributions from unlinked genetic or environmental modifiers. Our study establishes a precedent for using transgenic Drosophila to study clinical heterogeneity in human disease.

Presenilin‐1 controls the growth and differentiation of endothelial progenitor cells through its β‐catenin‐binding region

Presenilin-1 (PS1) is a gene responsible for the development of early-onset familial Alzheimer's disease. Targeted disruption of the PS1 gene in mice suggested that PS1 might be involved in angiogenesis. We have used an in vitro embryonic stem (ES) cell culture system to prepare endothelial progenitor cells (EPC) lacking PS1 and investigated the roles of PS1 in endothelial cell lineage. With this system, Flk-1+ E-cadherin- EPC were generated from PS1-deficient ES cells, and the EPC lacking PS1 as well as wild-type EPC grew to form VE-cadherin+ endothelial colonies supported by a layer of OP9 stromal cells. Although the endothelial colonies from PS1-deficient EPC showed morphology similar to those from wild-type EPC, the PS1-deficient EPC formed a large number of the colonies compared to wild-type EPC. The enhanced colony-forming ability of PS1-deficient EPC was attenuated by the inductions of wild-type human PS1. To differentiate multiple activities of PS1 for colony-forming ability, we used two types of human PS1 mutants: one (hPS1D257A) with the aspartate to alanine mutation at residue 257 that impairs the proteolytic activity of PS1, and the other (hPS1Deltacat) deleting amino acids 340-371 of the cytosolic loop sequence essential for beta-catenin binding. hPS1D257A showed activity to regulate the colony-forming ability of PS1-deficient EPC, while hPS1Deltacat failed to exhibit this activity. These results suggest that PS1 regulates the growth and differentiation of endothelial progenitor cells through its beta-catenin-binding region and that the defect of PS1 function in endothelial cell lineage could contribute to the induction of vascular pathology.

Effect of γ-secretase inhibitors on muscarinic receptor-mediated calcium signaling in human salivary epithelial cells

Altered intracellular Ca(2+) signaling has been observed in cells derived from Alzheimer's disease patients, and a possible link between gamma-secretase activity and the content of intracellular Ca(2+) stores has been suggested. To test this hypothesis we studied the effects of several gamma-secretase inhibitors on muscarinic receptor-mediated intracellular calcium release in the human salivary gland cell line HSG. Although several inhibitors in the peptide aldehyde class partially blocked carbachol-induced Ca(2+) transients, these effects did not appear to be due to gamma-secretase inhibition, and overall we found no evidence that inhibition of gamma-secretase activity had any significant effect on agonist-induced intracellular calcium release in HSG cells. In complementary experiments with presenilin-null cells we found that the reconstitution of gamma-secretase activity by transfection with wild-type presenilin 1 likewise had no significant effect on thapsigargin-induced Ca(2+) release. In a test of the specific hypothesis that the level of APP intracellular domain (AICD), the intracellular fragment of the beta-amyloid precursor protein (APP) resulting from gamma-secretase cleavage, can modulate the Ca(2+) content of the endoplasmic reticulum, we were unable to demonstrate any effect of APP small interfering RNA on the magnitude of carbachol-induced intracellular calcium release in HSG cells. Together our data cast considerable doubt on the hypothesis that there is a direct link between gamma-secretase activity and the content of intracellular Ca(2+) stores.

Formation of Tau Inclusions in Knock-in Mice with Familial Alzheimer Disease (FAD) Mutation of Presenilin 1 (PS1)

Mutations in the presenilin 1 (PS1) gene are responsible for the early onset of familial Alzheimer disease (FAD). Accumulating evidence shows that PS1 is involved in gamma-secretase activity and that FAD-associated mutations of PS1 commonly accelerate Abeta(1-42) production, which causes Alzheimer disease (AD). Recent studies suggest, however, that PS1 is involved not only in Abeta production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant (I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles (NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of Abeta deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing Abeta(1-42) production but by also accelerating the formation and accumulation of filamentous tau.

Expression of a Familial Alzheimer's Disease-Linked Presenilin-1 Variant Enhances Perforant Pathway Lesion-Induced Neuronal Loss in the Entorhinal Cortex

Alzheimer's disease (AD) is characterized by neuronal loss in the hippocampus and entorhinal cortex that is manifested by progressive memory impairment and cognitive decline. Autosomal-dominant, familial forms of AD (FAD) are caused by mutations in genes encoding amyloid precursor protein, presenilin-1 (PS1), and presenilin 2. Although it is established that expression of mutant PS1 variants leads to increased production of highly fibrillogenic amyloidbeta42 (Abeta42) peptides that deposit in the brains of patients with AD, the mechanism(s) by which Abeta deposition and expression of mutant genes induce lamina- and region-specific vulnerability of neuronal populations is not known. We have examined the hypothesis that expression of transgene-encoded FAD-linked mutant PS1 variants in entorhinal cortex neurons exacerbates the vulnerability of these cells to lesion-induced neuronal loss. To test this notion, we transected the perforant pathway (PP) of transgenic mice harboring either wild-type human PS1 (PS1HWT) or the FAD-linked mutant PS1DeltaE9 variant and examined neuronal survival in layer II of the entorhinal cortex (ECL2). Remarkably, PP transections lead to marked reductions in the numbers of ECL2 neurons in the ECL2 of mice expressing mutant PS1, compared with ECL2 neurons in PP-lesioned PS1HWT mice. Finally, and in contrast to studies in nontransgenic mice and in mice expressing PS1HWT, ECL2 neurons that express mutant PS1 and the calcium binding protein calbindin-D28k in ECL2 are also susceptible to lesion-induced neuronal loss. We conclude that expression of FAD-linked mutant PS1 variants enhances the vulnerability of neurons in the entorhinal cortex to PP lesion-induced cytotoxicity.

Presenilin clinical mutations can affect γ‐secretase activity by different mechanisms

Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimer's disease. Presenilins are polytopic proteins that harbour the catalytic site of the gamma-secretase complex and cleave many type I transmembrane proteins including beta-amyloid precursor protein (APP), Notch and syndecan 3. Contradictory results have been published concerning whether PS mutations cause 'abnormal' gain or (partial) loss of function of gamma-secretase. To avoid the possibility that wild-type PS confounds the interpretation of the results, we used presenilin-deficient cells to analyse the effects of different clinical mutations on APP, Notch, syndecan 3 and N-cadherin substrate processing, and on gamma-secretase complex formation. A loss in APP and Notch substrate processing at epsilon and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the gamma site was affected in variable ways. PS1-Delta9 and PS1-L166P mutations caused a reduction in beta-amyloid peptide Abeta40 production whereas PS1-G384A mutant significantly increased Abeta42. Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Abeta than PS1. Finally, subtle differences in gamma-secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect gamma-secretase structure or function in multiple ways.

Morphological Change by Overexpression of D385A Dominant Negative Presenilin 1 in Human Neuroblastoma SH-SY5Y Cells

Presenilin 1 (PS1) is a multifunctional protein, and its mutations are highly related to familial Alzheimer’s disease (AD). In this study, we examined the effects of PS1 overexpression on neuronal morphology using SH-SY5Y cells. Overexpression of dominant-negative D385A PS1 induced morphological change and impairment of neurite formation, while those of wild-type and pathogenic P117L mutant PS1 did not change cellular morphology compared with native cells. Moreover, filopodium-formation-related proteins were decreased only in cells overexpressing D385A PS1. Therefore, PS1 may be involved in neuritogenesis and morphological change in SH-SY5Y cells, and P117L mutation may linked to AD by different mechanisms.

Detection of presenilin-1 homodimer formation in intact cells using fluorescent lifetime imaging microscopy

Presenilin-1 (PS1) is a multipass transmembrane domain protein, which is believed to be the catalytic component of the γ-secretase complex. The complex is comprised of four major components: PS1, nicastrin, Aph-1, and Pen-2. The exact stoichiometric relationship between the four components remains unclear. It has been shown that γ-secretase exists as high molecular weight complexes, suggesting the possibility of dimer/multimer formation. We combined a biochemical approach with a novel morphological microscopy assay to analyze PS1 dimer formation and subcellular distribution in situ, in intact mammalian cells. Both coimmunoprecipitation and fluorescent lifetime imaging microscopy approaches showed that wildtype PS1 molecules form dimers. Moreover, PS1 holoproteins containing the D257A mutation also come into close enough proximity to form a dimer, suggesting that cleavage within the loop is not necessary for dimer formation. Taken together these data suggest that PS1 dimerization occurs during normal PS1 function.

Calcium oscillations in type-1 astrocytes, the effect of a presenilin 1 (PS1) mutation

Stimulation of type-1 astrocytes, by a number of agonists, has been shown to increase cytosolic Ca 2+ concentrations in an oscillatory manner. However, it is unknown how these are driven or altered by aging, injury or disease. Therefore, we characterized the signaling properties of rat type-1 astrocytes in monolayer cultures. Ca 2+ responses were recorded in astrocytes stimulated with ATP or glutamate. Oscillations were evident in cultures at 3 days in vitro (DIV 3) with a peak percentage (26%) of cells responding by DIV 7. The presence or absence of serum in the culture medium did not influence this percentage. Likewise, long-term culture (DIV 30+) did not increase the oscillating cell numbers. ATP was found to have a more potent effective dose (50 μM) than glutamate (10 mM). Membrane potential was recorded with fluorescent voltage-sensitive dye (membrane potential dye for FLIPR™) and was similar regardless of the culture age and intracellular Ca 2+ response. This suggests that mechanisms associated with the intracellular release of Ca 2+ from endogenous Ca 2+ stores, rather than ion fluxes across the plasma membrane, may contribute to the oscillations in the astrocytes. In order to identify a possible pathological significance of this response, we transfected astrocytes with wild-type presenilin (PS1) as well as PS1 harboring a mutation linked to familial Alzheimer's disease (FAD). PS mutation expressing astrocytes oscillated at lower ATP and glutamate concentrations when compared to wild-type PS1 expressing astrocytes. This indicates that PS1 mutation may introduce aberrations in the intracellular Ca 2+ mobilization in astrocytes contributing to the accelerated pathogenesis in FAD.

γ-Cleavage Is Dependent on ζ-Cleavage during the Proteolytic Processing of Amyloid Precursor Protein within Its Transmembrane Domain

beta-Amyloid precursor protein apparently undergoes at least three major cleavages, gamma-, epsilon-, and the newly identified zeta-cleavage, within its transmembrane domain to produce secreted beta-amyloid protein (Abeta). However, the roles of epsilon- and zeta-cleavages in the formation of secreted Abeta and the relationship among these three cleavages, namely epsilon-, zeta-, and gamma-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known gamma-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that Abeta(46) is an intermediate precursor of secreted Abeta. Our co-immunoprecipitation data also reveal that, as an intermediate, Abeta(46) is tightly associated with presenilin in intact cells. Furthermore, we identified a long Abeta species that is most likely the long sought after intermediate product, Abeta(49), generated by epsilon-cleavage, and this Abeta(49) is further processed by zeta- and gamma-cleavages to generate Abeta(46) and ultimately the secreted Abeta(40/42). More interestingly, our data demonstrate that gamma-cleavage not only occurs last but also depends on zeta-cleavage occurring prior to it, indicating that zeta-cleavage is crucial for the formation of secreted Abeta. Thus, we conclude that the C terminus of secreted Abeta is most likely generated by a series of sequential cleavages, namely first epsilon-cleavage which is then followed by zeta- and gamma-cleavages, and that Abeta(46) produced by zeta-cleavage is the precursor of secreted Abeta(40/42).

Mutant presenilin 2 increases acetylcholinesterase activity in neuronal cells

A presenilin 2 mutation is believed to be involved in the development of Alzheimer's disease. In addition, transgenic mice with a presenilin 2 mutation have been reported to have learning and memory impairments. In this study, exposing PC12 cells expressing mutant presenilin 2 to 50 microM AP25-35, 30 mM L-glutamate and 50 microM H2O2 caused a significant increase in acetylcholine esterase activity. An in vivo study revealed high levels of this enzyme activity in the mutant presenilin 2 transgenic brains compared with the wild type presenilin 2 transgenic and nontransgenic samples. These results suggest that a mutant presenilin 2-induced neurodegeneration in Alzheimer's disease might be involved in the increase in acetylcholinesterase activity. These findings might help in the development of an appropriate therapeutic intervention targeting mutant presenilin 2-induced Alzheimer's disease.

Presenilin/γ-Secretase-mediated Cleavage of the Voltage-gated Sodium Channel β2-Subunit Regulates Cell Adhesion and Migration

The voltage-gated sodium channel beta2-subunit (beta2) is a member of the IgCAM superfamily and serves as both an adhesion molecule and an auxiliary subunit of the voltage-gated sodium channel. Here we found that beta2 undergoes ectodomain shedding followed by presenilin (PS)-dependent gamma-secretase-mediated cleavage. 12-O-Tetradecanoylphorbol-13-acetate treatment or expression of an alpha-secretase enzyme, ADAM10, resulted in ectodomain cleavage of beta2 in Chinese hamster ovary cells. Subsequent cleavage of the remaining 15-kDa C-terminal fragment (beta2-CTF) was independently inhibited by three specific gamma-secretase inhibitors, expression of the dominant negative form of PS1, and in PS1/PS2 knock-out cells. gamma-Secretase inhibitor treatment also increased endogenous beta2-CTF levels in neuroblastoma cells and mouse primary neuronal cultures. In a cell-free gamma-secretase assay, we detected gamma-secretase activity-dependent generation of a 12 kDa beta2 intracellular domain (ICD), which was loosely associated with the membrane fraction. To assess the functional role of beta2 processing by gamma-secretase, we tested whether N-[N-(3,5-difluorophenylacetyl-l-alanyl)]-S-phenylglycine t-butylester (DAPT), a specific gamma-secretase inhibitor, would alter beta2-mediated cell adhesion and migration. We found that DAPT inhibited cell-cell aggregation and migration in a wound healing assay carried out with Chinese hamster ovary cells expressing beta2. DAPT also reduced migration of neuroblastoma cells in a modified Boyden chamber assay. Since DAPT treatment resulted in increased beta2-CTF levels, we also tested whether beta2-CTFs or beta2-ICDs would directly affect cell migration by overexpressing recombinant proteins. Interestingly, elevated levels of beta2-CTFs, but not ICDs, also blocked cell migration by 81 to 93%. Together, our findings show for the first time that beta2 is a PS/gamma-secretase substrate and gamma-secretase mediated cleavage of beta2-CTF is required for cell-cell adhesion and migration of beta2-expressing cells.

Presenilin-1 D257A and D385A Mutants Fail to Cleave Notch in Their Endoproteolyzed Forms, but Only Presenilin-1 D385A Mutant Can Restore Its γ-Secretase Activity with the Compensatory Overexpression of Normal C-terminal Fragment

The enzyme gamma-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the gamma-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other gamma-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of gamma-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their gamma-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349-467). However, the defective PS-1 D257A mutant could not restore their gamma-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the gamma-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.

Study on expression of PS1 in APP-PS1 double gene stably transfected cell lines and its relation to gamma-secretase

To study the role of presenilin1 (PS1) in the processing of beta-amyloid precursor protein (APP) to amyloid beta-peptide (Abeta) and its relation to gamma-secretase in the pathogenesis of Alzheimer's disease (AD). Several CHO cell lines stably transfected with either wide-type or mutant PS1 (M(146)L) along with APP(751) genes were established. The expression of PS1 and its half-life were determined by immunoprecipitation, Western blotting and pulse-chase experiment. Abeta released into the conditional media was quantitated by ELISA. PS1 transfected CHO cells expressed an expected 45,000 full length protein. This over-expressed full length PS1 was subject to fast degradation with a half-life of less than 1 hour. In contrast to full length PS1, the truncated N-terminal and C-terminal proteins of PS1 were significantly more stable with a longer half-life of nearly 16 hours. Although the total amount of Abeta released into the conditional media did not show a significant difference between wild-type and mutant PS1 (M(146)L) transfected APP cells, mutant PS1 (M(146)L) transfected APP cells increase Abeta(1 - 42) (a subspecies of total Abeta) production with nearly a 2 fold increase, comparing to untransfected or wild-type PS1 transfected APP cells. PS1 is involved in the processing of APP to Abeta, a nearly 2 fold increase of Abeta production in mutant PS1 (M(146)L) transfected APP cells indicates that PS1 may be the expected gamma-secretase itself.

Shedding and γ-secretase-mediated intramembrane proteolysis of the mucin-type molecule CD43

CD43 is a transmembrane molecule that contains a 123-aminoacids-long cytoplasmic tail and a highly O-glycosylated extracellular domain of mucin type. Endogenous CD43 expressed in COLO 205, K562 and Jurkat cells revealed a membrane-associated, 20 kDa CD43-specific cytoplasmic tail fragment (CD43-CTF) upon inhibition of gamma-secretase. This fragment was formed by an extracellular cleavage, as it was not accumulated after treating cells with 1,10-phenanthroline, a metalloprotease inhibitor. When CD43 was transfected into HEK-293 cells expressing dominant-negative PS1 (presenilin-1), the CD43-CTF was accumulated, but not in cells with wild-type PS1. Owing to its accumulation in the presence of a non-functional PS variant, it may thus be a novel gamma-secretase substrate. This CTF is formed by an extracellular cleavage close to the membrane, is a fragment that can be concluded to be a substrate for gamma-secretase. However, the intracellular gamma-secretase product has not been possible to detect, suggesting a quick processing of this product. During normal growth the CTF was not found without gamma-secretase inhibition, but when the cells (COLO 205) were very confluent the fragment could be detected. The intracellular domain of CD43 has previously been shown to contain a functional nuclear localization signal, and has been suggested to be involved in gene activation. From this and the present results, a novel way to explain how mucin-type molecules may transduce intracellular signals can be proposed.

Ryanodine receptor-mediated interference of neuronal cell differentiation by presenilin 2 mutation

Neuronal cell differentiation alterations induced by mutant presenilin 2 (PS2) were investigated in transgenic mice expressing wild-type or mutant-type PS2. Progressive increases in differentiation and marker protein expression were found in neuronal cells expressing wild-type PS2, whereas these processes were much perturbed in mutant-type PS2 with elevated ryanodine-receptor (RyR) expression and intracellular calcium levels. Moreover, dantrolene, a blocker of RyR reduced the PS2 mutation-induced interference of cell differentiation and calcium release, but caffeine, an activator of RyR, exacerbated PS2 mutation-induced interference with cell differentiation. Our results indicate that mutant PS2 inhibits normal neuronal cell differentiation and that RyR-mediated calcium overrelease may be a significant factor.

Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway.

Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579–589). Here, we demonstrate that TLN is not a substrate for γ-secretase cleavage, but displays a prolonged half-life in PS1−/− hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from γ-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a γ-secretase–independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.

The presenilin-1 familial Alzheimer disease mutant P117L impairs neurogenesis in the hippocampus of adult mice

The functions of presenilin 1 (PS1) and how PS1 mutations cause familial Alzheimer's disease (FAD) are incompletely understood. PS1 expression is essential for neurogenesis during embryonic development and may also influence neurogenesis in adult brain. We examined how increasing PS1 expression or expressing an FAD mutant would affect neurogenesis in the adult hippocampus. A neuron-specific enolase (NSE) promoter was used to drive neuronal overexpression of either wild-type human PS1 or the FAD mutant P117L in transgenic mice, and the animals were studied under standard-housing conditions or after environmental enrichment. As judged by bromodeoxyuridine (BrdU) labeling, neural progenitor proliferation rate was mostly unaffected by increasing expression of either wild-type or FAD mutant PS1. However, in both housing conditions, the FAD mutant impaired the survival of BrdU-labeled neural progenitor cells leading to fewer new β-III-tubulin-immunoreactive neurons being generated in FAD mutant animals during the 4-week postlabeling period. The effect was FAD mutant specific in that neural progenitor survival and differentiation in mice overexpressing wild-type human PS1 were similar to nontransgenic controls. Two additional lines of PS1 wild-type and FAD mutant transgenic mice showed similar changes indicating that the effects were not integration site-dependent. These studies demonstrate that a PS1 FAD mutant impairs new neuron production in adult hippocampus by decreasing neural progenitor survival. They also identify a new mechanism whereby PS1 FAD mutants may impair normal neuronal function and may have implications for the physiological functioning of the hippocampus in FAD.