An Alzheimer's disease-linked PS1 variant rescues the developmental abnormalities of PS1-deficient embryos

Mutations in presenilin 1 (PS1) cosegregate with approximately 25% of early onset familial Alzheimer's disease (FAD) pedigrees. A variety of in vitro and in vivo paradigms have established that one mechanism by which PS1 variants cause AD is by elevating the production of highly amyloidogenic Abeta1-42/43 peptides. PS1 is homologous to sel-12, a C. elegans protein that facilitates signaling mediated by the Notch/lin-12 family of receptors. Wild-type human PS1 complements an egg-laying defect in C. elegans lacking sel-12, while FAD-linked PS1 variants exhibit reduced rescue activity. These data suggested that mutant PS1 may cause disease as a result of reduction in PS1 function. To test the function of FAD-linked PS1 in mammals, we examined the ability of the A246E PS1 variant to complement the embryonic lethality and axial skeletal defects in mice lacking PS1. Finally, to examine the influence of reduced PS1 levels on Abeta production, we quantified Abeta1-42/43 peptide levels in PS1 heterozygous null mice (PS1[+/-] mice). We now report that both human wild-type and A246E PS1 efficiently rescue the phenotypes observed in PS1(-/-) embryos, findings consistent with the view that FAD-linked PS1 mutants retain sufficient normal function during mammalian embryonic development. Moreover, the levels of Abeta1-42/43 and Abeta1-40 peptides between PS1(+/-) and control mice are indistinguishable. Collectively, these data lead us to conclude that mutant PS1 causes AD not by loss of normal PS1 function but by influencing amyloid precursor protein (APP) processing in a manner that elevates Abeta1-42/43 production.     

Expression and localization in the fish retina of a homologue of the Alzheimer's related PS1 gene

Early-onset familial Alzheimer's disease (early-onset FAD) has been linked with mutations in the presenilin gene, PS1. Mutations in PS1 may affect the processing/trafficking of b-amyloid precursor-protein (bAPP) and favour the production of toxic amyloid-b fragments that are associated with neural degeneration. This study reports the expression of a PS1-like cDNA in the carp (Cyprinus carpio) retina (the encoded protein shows 76% identity to the human PS1 protein). Carp PS1 mRNA was localized by in situ hybridization to the photoreceptor cell, inner nuclear and ganglion cell layers. Expression of the PS1 gene in the rat retina was also confirmed. The retinal expression of PS1 raises the possibility that PS1 mutations also lead to neural degeneration in the retina.     

Dual roles of proteasome in the metabolism of presenilin 1

Presenilin 1 (PS1) has been identified as a causative gene for most early-onset familial Alzheimer's disease. Biochemical studies revealed that PS1 exists predominantly as two processed fragments in cells and brain tissues. We prepared stably transfected cells expressing the wild-type and familial Alzheimer's disease-associated mutants of PS1 and investigated the enzyme that participates in the metabolism of PS1. After treatment of the cells with proteasome inhibitors, the full-length PS1 was significantly accumulated. The levels of N- and C-terminal fragments were also increased. The accumulation of PS1 with a deletion of exon 10, which is unable to be processed, on treatment of the transfected cells with lactacystin indicated that proteasome can degrade full-length PS1. A synthetic peptide that includes the processing region of PS1 was cleaved by 20S proteasome at the putative processing sites after Met288 and Glu299. Metabolic labeling experiments showed that the appearance of the N-terminal fragment was attenuated by the inhibitor. Finally, 28-kDa N- and 20-kDa C-terminal fragments were generated by purified PS1 in vitro. These data indicated that the proteasome pathway is involved in PS1 processing. These results demonstrate that the proteasome pathway plays dual roles in processing and degradation of PS1.     

Determination of a cleavage site of presenilin 2 protein in stably transfected SH-SY5Y human neuroblastoma cell lines

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes are associated with early-onset autosomal dominant familial Alzheimer's disease, and the gene products are endoproteolytically processed to yield N-terminal fragments (NTF) and C-terminal fragments (CTF). We have studied the cleavage site of the PS2 protein in stably transfected human neuroblastoma cells. The 23 kD PS2-CTF was isolated by a combination of anion exchange chromatography and affinity chromatography and directly sequenced. The N-terminus of the PS2-CTF started at residue 307, which indicated that the cleavage occurs between Lys306 and Leu307 in the proximal portion of the large hydrophilic loop. This site is close to the cleavage positions observed in the PS1 protein.     

Presenilin 1 regulates the processing of beta-amyloid precursor protein C-terminal fragments and the generation of amyloid beta-protein in endoplasmic reticulum and Golgi

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. To establish the subcellular localization of the PS1 regulation of APP processing to Abeta, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) were markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases were documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP was detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, formed complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. When the same fractions were analyzed by enzyme-linked immunosorbent assays for Abetatotal and Abeta42, Abeta42 was the major Abeta species in the ER fraction (Abeta42:Abetatotal ratio 0.5-1.0), whereas absolute levels of both Abeta42 and Abeta40 were higher in the Golgi fraction and the Abeta42:Abetatoal ratio was 0.05-0.16 there. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles.     

Stable association of presenilin derivatives and absence of presenilin interactions with APP

Mutations in two related genes, presenilin 1 and 2 presenilin 2 (PS1 and PS2), cosegregate with Alzheimer's disease. PS1 and PS2 are highly homologous polytopic membrane proteins that are subject to endoproteolytic cleavage in vivo. The resulting N- and C-terminal derivatives are the preponderant PS-related species that accumulate in cultured cells and tissue. In earlier studies, we demonstrated that PS1 N- and C-terminal derivatives accumulate to 1:1 stoichiometry and that the absolute levels of fragments are established by a tightly regulated and saturable mechanism. These findings led to the suggestion that the levels of PS1 derivatives might be determined by their association with limiting cellular components. In this study, we use in situ chemical cross-linking and coimmunoprecipitation analyses to document that the N- and C-terminal derivatives of either PS1 or PS2 can be coisolated. Moreover, and in contrast to published reports which documented that PS1 and PS2 form stable heteromeric assemblies with the beta-amyloid precursor protein (APP), we have failed to provide evidence for physiological complexes between PS1 and PS2 holoproteins or their derivatives with APP.     

Presenilin proteins undergo heterogeneous endoproteolysis between Thr291 and Ala299 and occur as stable N- and C-terminal fragments in normal and Alzheimer brain tissue

Humans inheriting missense mutations in the presenilin (PS)1 and -2 genes undergo progressive cerebral deposition of the amyloid beta-protein at an early age and develop a clinically and pathologically severe form of familial Alzheimer's disease (FAD). Because PS1 mutations cause the most aggressive known form of AD, it is important to elucidate the structure and function of this multitransmembrane protein in the brain. Using a panel of region-specific PS antibodies, we characterized the presenilin polypeptides in mammalian tissues, including brains of normal, AD, and PS1-linked FAD subjects, and in transfected and nontransfected cell lines. Very little full-length PS1 or -2 was detected in brain and untransfected cells; instead the protein occurred as a heterogeneous array of stable N- and C-terminal proteolytic fragments that differed subtly among cell types and mammalian tissues. Sequencing of the major C-terminal fragment from PS1-transfected human 293 cells showed that the principal endoproteolytic cleavage occurs at and near Met298 in the proximal portion of the large hydrophilic loop. Full-length PS1 in these cells is quickly turned over (T1/2 approximately 60 min), in part to the two major fragments. The sizes and amounts of the PS fragments were not significantly altered in four FAD brains with the Cys410Tyr PS1 missense mutation. Our results indicate that presenilins are rapidly processed to N- and C-terminal fragments in both neural and nonneural cells and that interference with this processing is not an obligatory feature of FAD-causing mutations.     

The presenilin 1 mutation (M146V) linked to familial Alzheimer's disease attenuates the neuronal differentiation of NTera 2 cells

Mutations in presenilin 1 (PS1) gene are the major cause of early-onset familial Alzheimer's disease. The biological functions of PS1 remain elusive, although accumulating evidence suggests that PS1 may play an important role in development and differentiation. To learn about the significance of PS1 in the differentiation of neuronal cells, we established NTera 2 (NT2) cell lines stably expressing wild-type (wt) or M146V mutant human PS1, and compared the differentiation of both types of cell lines into postmitotic neurons upon retinoic acid (RA) treatment. After 25 days of RA treatment, a significant proportion of cells differentiated into neurons in NT2 cells expressing wt PS1 (27.7% of total cells), which was comparable to that in untransfected cells, whereas very few cells differentiated into neurons in NT2 cells expressing M146V mutant PS1 (2.6% of total cells). These results suggest that mutant PS1 attenuates the potentials of NT2 cells to differentiate into neurons.     

Short-term β-amyloid vaccinations do not improve cognitive performance in cognitively impaired APP+PS1 mice.

Prior work demonstrated that β-amyloid (Aβ) immunotherapy for 8 months prevented cognitive impairment in 16-month-old APP+PS1 transgenic mice. In the present study, 4 immunizations administered biweekly to cognitively impaired 16-month-old transgenic mice could not reverse deficits in working memory or reference memory in the radial arm water maze or in visual platform recognition, possibly because of inadequate antibody exposure. Nontransgenic mice showed cognitive savings between the 16- and 18-month test periods, but the transgenic groups did not. These results suggest that a longer period of active immunotherapy, or passive immunization, may be required to provide sufficient antibody titers to improve cognition in older transgenic mice. Aβ-based immunotherapy for Alzheimer's disease will likely be more successful prophylactically than therapeutically. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Presenilin 1 mutations linked to familial Alzheimer's disease increase the intracellular levels of amyloid beta-protein 1-42 and its N-terminally truncated variant(s) which are generated at distinct sites

Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid beta-protein (A beta) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuroblastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or delta exon 10). The induction of mutated PS1 increased the intracellular levels of two distinct A beta species ending at residue 42 that were likely to be A beta1-42 and its N-terminally truncated variant(s) A beta x-42. The induction of mutated PS1 resulted in a higher level of intracellular A beta1-42 than of intracellular A beta x-42, whereas extracellular levels of A beta1-42 and A beta x-42 were increased proportionally. In addition, the intracellular generation of these A beta42 species in wt and mutated PS1-induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular A beta x-42 versus inhibition of intracellular A beta1-42 generation. These data strongly suggest that A beta x-42 is generated in a proximal Golgi, whereas A beta1-42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific gamma-secretase cleavage that occurs in the normal beta-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to beta-secretase cleavage or (b) in the distinct sites where A beta x-42 and A beta1-42 are generated.     

Amyloid-beta deposition in skeletal muscle of transgenic mice: possible model of inclusion body myopathy

Inclusion body myopathy is a progressive muscle disorder characterized by nuclear and cytoplasmic inclusions and vacuolation of muscle fibers. Affected muscle fibers contain deposits of congophilic amyloid, amyloid-beta immunoreactive filaments, and paired helical filaments, all of which are pathological hallmarks of Alzheimer's disease in brain. Accumulations of amyloid-beta and its precursor are thought to play important roles in the pathogenesis of both inclusion body myopathy and Alzheimer's disease. Overexpression of mutant forms of beta protein precursor in transgenic mice by neuron-specific promoters has been reported to cause amyloid deposits in the brain. Here we report that overexpression in transgenic mice of the signal plus 99-amino acid carboxyl-terminal sequences of beta protein precursor, under the control of a cytomegalovirus enhancer/beta-actin promoter, resulted in vacuolation and increasing accumulation of the 4-kd amyloid-beta and the carboxyl-terminus in skeletal muscle fibers during aging. These deposits in transgenic muscle only rarely showed Congo red birefringence. Thus, overexpression of part of beta protein precursor in transgenic mice led to development of some of the characteristic features of inclusion body myopathy. These mice may be a useful model of inclusion body myopathy, which shares a number of pathological markers with Alzheimer's disease.     

Atomic force microscopy examination of tobacco mosaic virus and virion RNA

The majority of early-onset familial Alzheimer's disease (FAD) is associated with mutations in the presenilin-1 (PS1) gene. We describe a novel Polish PS1 mutation of Pro117Leu, associated with the earliest average age of onset and death so far reported in a PS-linked, FAD kindred. Human kidney 293 and mouse neuroblastoma N2a cells were stably transfected with wild-type and PS1 P117L. There was a significant increase in the amyloid beta42/40 ratio in the N2a P117L PS1 transfected cells compared with N2a transfected with wild-type PS1. What role PS has in the pathogenesis of AD remains to be determined, however, the severity of the clinical picture associated with this PS1 mutation stresses the importance of presenilin.     

Amyloidogenic role of cytokine TGF-beta1 in transgenic mice and in Alzheimer's disease

Deposition of amyoid-beta peptide in the central nervous system is a hallmark of Alzheimer's disease and a possible cause of neurodegeneration. The factors that initiate or promote deposition of amyloid-beta peptide are not known. The transforming growth factor TGF-beta1 plays a central role in the response of the brain to injury, and increased TGF-beta1 has been found in the central nervous system of patients with Alzheimer's disease. Here we report that TGF-beta1 induces amyloid-beta deposition in cerebral blood vessels and meninges of aged transgenic mice overexpressing this cytokine from astrocytes. Co-expression of TGF-beta1 in transgenic mice overexpressing amyloid-precursor protein, which develop Alzheimer's like pathology, accelerated the deposition of amyloid-beta peptide. More TGF-beta1 messenger RNA was present in post-mortem brain tissue of Alzheimer's patients than in controls, the levels correlating strongly with amyloid-beta deposition in the damaged cerebral blood vessels of patients with cerebral amyloid angiopathy. These results indicate that overexpression of TGF-beta1 may initiate or promote amyloidogenesis in Alzheimer's disease and in experimental models and so may be a risk factor for developing Alzheimer's disease.     

Transgenic mouse models of Alzheimer's disease and amyotrophic lateral sclerosis

Over the past several years, there has been enormous progress in generating transgenic mice that model aspects of human neurodegenerative diseases. These studies build upon the efforts of molecular geneticists who have identified a number of genes that, when mutated, cause familial forms of these diseases. In this review, we focus on the mutations that cause familial forms of Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), and transgenic mouse models that develop clinical and pathological abnormalities resembling those occurring in the human diseases.     

Suppression of somatotroph function induced by growth hormone treatment in neonatal pigs

The Her-2/neu protooncogene is associated with malignant transformation and aggressive disease. Because of its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. By identifying potential HLA-A2.1-binding peptides from the Her-2/neu sequence, peptides were selected as candidate T-cell epitopes. The immunogenicity of each peptide was evaluated by priming double transgenic mice expressing both the human (hu) CD8 and HLA-A2.1 molecules with synthetic peptides corresponding to these sequences. Because of the lack of interaction between murine CD8 and HLA-A2.1, expression of huCD8 on murine cells facilitates recognition of HLA molecules on human tumor cell lines. This led to the identification of two peptides that elicit an A2-restricted CTL response, one of which has not been previously identified. Both peptide-specific CTL populations were able to specifically lyse A2.1 and Her-2/neu expressing human tumor cells originating from a variety of tissues, demonstrating the utility of this murine model in identifying peptides presented by human cells. However, several Her-2/neu peptides previously reported to be immunogenic for human CTL were found not to be immunogenic in transgenic mice. The basis for these discrepancies is discussed.