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Accumulation of proteolytic fragments of mutant presenilin 1 and accelerated amyloid deposition are co-regulated in transgenic mice
Abstract
The activities of presenilin 1 (PS1) and 2 modulate the proteolytic processing of amyloid precursor proteins to produce Abeta1-42, and mutations in these proteins are associated with an accelerated rate of Abeta deposition. PS1 and PS2 themselves are subject to a highly-regulated endoproteolytic cleavage to generate stable 27 kDa N-terminal and 17 kDa C-terminal fragments. Here, we examined the relationship between the regulated cleavage of PS1 and the acceleration of Abeta deposition in transgenic mice that co-express Mo/Hu APPswe and varied levels mutant PS1 (A246E variant). The steady-state levels of the N- and C-terminal fragments of mutant PS1 in mice expressing low levels of mRNA were similar to that of mice expressing high levels of mRNA. Only mice expressing high levels of transgene mRNA accumulated uncleaved full-length protein. In mice co-expressing low levels of PS1A246E mRNA with Mo/Hu APPswe the age of appearance of Abeta deposits was similar to that of mice co-expressing expressing Mo/Hu APPswe with very high levels of mutant PS1. Our findings demonstrate that the levels of accumulated human PS1 N- and C-terminal fragments do not increase in proportion to the level of transgene mRNA and that similarly, the magnitude by which mutant PS1 accelerates the deposition of beta-amyloid is not proportional to the level of transgene expression.
... Subsequent work in cell models demonstrated that co-expression of FAD-mutant PS1 with APP reproduced the rise in Ab42 relative to Ab40 that had been seen in patients with PS1-associated AD (39)(40)(41). This shift in Ab40 : Ab42 ratio was also found in the brains of transgenic mice co-expressing one of several FAD PS1 mutations (M146V, M146L, A246E or M286L) with the Swedish mutation of APP (39,(42)(43)(44). Similar shifts in the steady-state ratios of mouse Ab40 : Ab42 were also noted in mice expressing only FAD-variant PS1 (45) or PS2 (46). ...
... Transgenic mice co-expressing mutated PS1 and APP developed amyloid pathology much earlier than mice expressing only APP; the acceleration in amyloid formation directly correlated with the change in the Ab40 : Ab42 ratios (42,47,48). In contrast, co-expression of wild-type human PS1 with mutant human APP had no impact on the rate of amyloid formation (44). ...
... In previous studies we (44,47) and others (42,43,48) have shown that the co-expression mutant PS1 with APPswe can accelerate the rate of amyloid deposition. By contrast, co-expression of wild-type human PS1, even at very high levels, has no effect on the rate of amyloid deposition (44). ...
Amyloid precursor protein (APP) is endoproteolytically processed by BACE1 and gamma-secretase to release amyloid peptides (Abeta40 and 42) that aggregate to form senile plaques in the brains of patients with Alzheimer's disease (AD). The C-terminus of Abeta40/42 is generated by gamma-secretase, whose activity is dependent upon presenilin (PS 1 or 2). Missense mutations in PS1 (and PS2) occur in patients with early-onset familial AD (FAD), and previous studies in transgenic mice and cultured cell models demonstrated that FAD-PS1 variants shift the ratio of Abeta40 : 42 to favor Abeta42. One hypothesis to explain this outcome is that mutant PS alters the specificity of gamma-secretase to favor production of Abeta42 at the expense of Abeta40. To test this hypothesis in vivo, we studied Abeta40 and 42 levels in a series of transgenic mice that co-express the Swedish mutation of APP (APPswe) with two FAD-PS1 variants that differentially accelerate amyloid pathology in the brain. We demonstrate a direct correlation between the concentration of Abeta42 and the rate of amyloid deposition. We further show that the shift in Abeta42 : 40 ratios associated with the expression of FAD-PS1 variants is due to a specific elevation in the steady-state levels of Abeta42, while maintaining a constant level of Abeta40. These data suggest that PS1 variants do not simply alter the preferred cleavage site for gamma-secretase, but rather that they have more complex effects on the regulation of gamma-secretase and its access to substrates.
... Transgenic mice expressing a chimeric mouse/human APP695 cDNA harboring the Swedish K670M/N671L mutations (Mo/HuAPPswe; line C3-3) and human PS1with the exon-9 deletion mutation (PS1dE9; line S-9) have been described in previous publications234519]. Both of these transgenes were expressed in a vector (MoPrP.XhoI) [2], which drives high protein expression in neurons and astrocytes of the CNS [21]. ...
... Total RNA was extracted using Trizol Reagent (Gibco BRL, Rockville, MD, USA) according to the manufacturer's recommendations. Eight to ten micrograms of total RNA from each sample was then electrophoresed in 1% MOPS–formaldehyde agarose gels, transferred to 0.45 m Optitran nitrocellulose (Schleicher and Schuell) in 10× SSC, UV cross-linked, and then probed with radiolabeled cDNA fragments as previously described [3]. ...
... Mice transgenic for APPswe and hemizygous for PS1 (n = 7) were examined for amyloid pathology at 12 months of age. This time point falls between the very early onset seen in Mo/HuAPPswe mice co-expressing PS1dE9 (4.5–6 months of age), and the very late onset seen in Mo/HuAPPswe single transgenic animals (>18 months; line C3-3; [3,4]). The rationale being the following: If a two-fold reduction in endogenous PS1 levels caused a proportional two-fold acceleration in A deposition, then initial deposits should be visible at 9 months of age in C3-3 mice hemizygous for endogenous PS1. ...
More than 70 different mutations in presenilin 1 (PS1) have been associated with inherited early onset Alzheimer's disease (AD). How all these different mutations cause disease has not been clearly delineated. Our laboratory has previously shown that co-expression of mutant PS1 in mice transgenic for amyloid precursor protein (APPswe) dramatically accelerates the rate of amyloid deposition in the brain. In our original animals mutant PS1 was substantially over-expressed, and the stabilized pool of mouse PS1 fragments was largely replaced by the human protein. In this setting the accelerated amyloid pathology in the double transgenic mice could have been due, in part, to decreased endogenous PS1 activity. To investigate this possibility, we generated APP transgenic mice with reduced levels of endogenous PS1. We find that mice harboring only one functional PS1 allele and co-expressing Mo/HuAPPswe do not develop amyloid deposits at ages comparable to mice expressing mutant PS1. We next tested whether hypo-expression of mutant PS1 could accelerate the rate of amyloid deposition using an unusual line of transgenic mice expressing PS1dE9 at low levels, finding no significant acceleration. Our findings demonstrate that the accelerated amyloid pathology, caused by so many different mutations in PS1, is clearly not a result of haplo-insufficiency that might result from inactivating mutations. Instead, our data are consistent with a gain of property mechanism.
... In fact, there was a significant linear correlation between the Ab content and the relative hPS1-CTF expression in both hippocampus (Fig. 3B ) and cortex (not shown) of 6- month-old PS1xAPP tg mice. The total amount of PS1- CTF (hCTF + mCTF) increased only slightly in the PS1xAPP tg mice (as compared with WT) and displayed a clear saturation plateau (Lee et al., 1997; Borchelt et al., 2002) (Fig. 3 ). The total PS1-CTF presented in the double tg mice represented 165 6 49% (n ¼ 11), as compared with WT mice. ...
... Therefore, these data indicated that variations in the expression of tg form of the hPS1 strongly influenced the incorporation of the mutated hPS1 into functional g-secretase complexes, the production of Ab peptides and, in consequence, the detrimental effects of the production of Ab peptides. These results are apparently in conflict with those reported by others (Borchelt et al., 2002). This apparent discrepancy could be due to differences in hPS1 expression between our tg model and those reported previously in other tg models. ...
... In our case, the detected levels of hPS1-CTF were in the same range of the WT counterpart (Fig. 3A). Even in the low hPS1 expressing tg mice from Borchelt et al. (2002) the hPS1-CTF was considerable higher than that present in WT mice. The high expression of hPS1 protein observed in other tg models could saturate the assembling process of the g-secretase complex and, in consequence, the hPS1 incorporated into the complexes should be independent of the mRNA expression. ...
The detection of the early phenotypic modifications of Alzheimer's disease (AD) models is fundamental to understand the progression and identify pharmacologic targets of this pathology. However, a large variability within different models and between age-matched mice from the same model has been observed. This variability could be due to heterogeneity in the Abeta production. Present results showed the existence of a large variability in the Abeta deposition in both hippocampus and cortex in 6-month-old PS1xAPP mice. This variability was not due to the expression of hAPP751SL, however, linear relationship between hPS1M146L mRNA and Abeta production was identified. The Abeta content was related to the incorporation of the hPS1M146L into functional gamma-secretase complexes, detected by the presence of the corresponding human or endogenous PS1-CTFs. Animals expressing low amount of hPS1M146L mRNA, displayed low hPS1-CTF incorporation and produced a low amount of Abeta peptides. Conversely, mice with relatively high hPS1 mRNA expression displayed high hPS1-CTF and high Abeta deposition. Furthermore, the Abeta total and Abeta1-42 content was increased dramatically by the expression of hPS1M146L (as compared with transgenic APPsl littermates). Therefore, variations in the expression of transgenic form of hPS1M146L in this model, or even between different models, influenced strongly the incorporation of the mutated PS1 into functional gamma-secretase complexes, the production of Abeta peptides and, in consequence, the detrimental effects of Abeta peptides. These data might implicate an "apparent gain-of-function" of the gamma-secretase complex by the expression of the mutated PS1M146L.
... MoPrP.hSOD1-G37R was injected into mouse embryos, by mixing with MoPrP.hPS1-WT (1:1 molar ratio) as a potential selection marker (see Materials and methods and Supplemental information); previous work demonstrated that mice expressing human wild-type presenilin, via this vector, are phenotypically normal ( Borchelt et al., 2001;Thinakaran et al., 1996); and that the embryonic lethality of mice with targeted deletion of endogenous PS1 genes is rescued by MoPrP.hPS1-WT transgenes ( Davis et al., 1998). ...
... However, we note that the level of mutant SOD1 expression required to induce disease in the PrP-SOD1 mice was very similar to that of mice harboring genomic constructs. We also note that we have previously established that very high levels of hPS1-WT expression cause no obvious phenotypes in mice ( Borchelt et al., 2001;Thinakaran et al., 1996). To test whether expression of hPS1-WT influences the toxicity of hSOD1-G37R, we generated a small cohort of mice that were doubly transgenic for PrP.hPS1-WT and Gn.SOD1-G37Rline 29, noting no obvious change in the age of disease onset by the added expression of the wild-type human PS1 protein. ...
Familial amyotrophic lateral sclerosis (FALS) has been modeled in transgenic mice by introducing mutated versions of human genomic DNA encompassing the entire gene for Cu,Zn superoxide dismutase (SOD1). In this setting, the transgene is expressed throughout the body and results in mice that faithfully recapitulate many pathological and behavioral aspects of FALS. By contrast, transgenic mice made by introducing recombinant vectors, encoding cDNA genes, that target mutant SOD1 expression to motor neurons, only, or astrocytes, only, do not develop disease. Here, we report that mice transgenic for human SOD1 cDNA with the G37R mutation, driven by the mouse prion promoter, develop motor neuron disease. In this model, expression of the transgene is highest in CNS (both neurons and astrocytes) and muscle. The gene was not expressed in cells of the macrophage lineage. Although the highest expressing hemizygous transgenic mice fail to develop disease by 20 months of age, mice homozygous for the transgene show typical ALS-like phenotypes as early as 7 months of age. Spinal cords and brain stems from homozygous animals with motor neuron disease were found to contain aggregated species of mutant SOD1. The establishment of this SOD1-G37R cDNA transgenic model indicates that expression of mutant SOD1 proteins in the neuromuscular unit is sufficient to cause motor neuron disease. The expression levels required to induce disease coincide with the levels required to induce the formation of SOD1 aggregates.
... The main studies with transgenic animals involving curcumin are focused on the expression of the APPswe/PS1dE9 genes and crossing between 5 x FAD transgenic male animals and C57BL/6J females. The animal model used in these studies has a higher Ab42/40 ratio possibly due to the PS1 mutation [92,93], which is a gene established to induce AD and accelerate the deposition of Aβ [76,77]. In the 5x FAD transgenic model, Zheng et al. (2017) coexpress five familial AD mutations (APPK670N / M671L + V717I + I716V and PS1 L286V + M146L) [61]. ...
Background:
The formation of senile plaques and neurofibrillary tangles of the tau protein are the main pathological mechanism of Alzheimer's disease (AD). Current therapies for AD offer discrete benefits to the clinical symptoms and do not prevent the continuing degeneration of neuronal cells. Therefore, novel therapeutic strategies have long been investigated, where curcumin (Curcuma longa) has shown some properties that can prevent the deleterious processes involved in neurodegenerative diseases.
Objective:
The aim of the present work is to review studies that addressed the effects of curcumin in experimental models (in vivo and in vitro) for AD.
Method:
This study is a systematic review conducted between January and June 2017, in which a consultation of scientific articles from indexed periodicals was carried out in Science Direct, United States National Library of Medicine (PubMed), Cochrane Library and Scielo databases, using the following descriptors: "Curcuma longa", "Curcumin" and "Alzheimer's disease".
Results:
A total of 32 studies were analyzed, which indicated that curcumin supplementation reverses neurotoxic and behavioral damages in both in vivo and in vitro models of AD.
Conclusion:
The administration of curcumin in experimental models seems to be a promising approach in AD, even though it is suggested that additional studies must be conducted using distinct doses and through other routes of administration.
Keywords: Alzheimer’s disease, curcumin, Aβ aggregation, oxidative stress, therapeutics, brain.Alzheimer’s disease, curcumin, Aβ aggregation, oxidative stress, therapeutics, brain.Alzheimer’s disease, curcumin, Aβ aggregation, oxidative stress, therapeutics, brain.
... Since Aβ1–42, the first Aβ specie deposited in the brain of AD patients [16], [39], is more prone to aggregation when compared to shorter Aβ species [40], [41], [42], it has been implicated in the seeding of amyloid plaques in AD patients with PSEN1 mutations [43]. This was further validated in vivo as the overexpression of PS1 mutants in APP transgenic mice accelerated the rate of Aβ accumulation and deposition in the brain [44], [45]. However, Bentahir and colleagues challenged this view by showing that several PSEN1 mutations were also capable to decrease total Aβ production in PS1/PS2 knockout cells [23]. ...
Mutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1-42/Aβ1-40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1-42 production, decreasing Aβ1-40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q.
We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1-40, Aβ1-42 and APP intracellular domain productions in vitro.
Our data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1-42/Aβ1-40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer's disease.
... This was further nurtured by the finding of a genetic component associated to the hypothesis of Aβ deposition. Mutations in the amyloid precursor protein (APP) that facilitate its cleavage to generate amyloid peptide and/or mutations in presenilin-1 (PS-1) or presenilin-2 (PS-2), that promote amyloid peptide formation and consequently Aβ deposition have also been reported [10] [11] [12]. Although the genetic component is documented, we have to mention that far less than 1% of the worldwide AD cases are based on APP or PS ...
Alzheimer’s disease (AD) is defined by the concurrence of accumulation of abnormal aggregates composed of two proteins: Amyloid beta (A
β
) and tau, and of cellular changes including neurite degeneration and loss of neurons and cognitive functions. Based on their strong association with disease, genetically and pathologically, it is not surprising that there has been a focus towards developing therapies against the aggregated structures. Unfortunately, current therapies have but mild benefit. With this in mind we will focus on the relationship of synaptic plasticity with A
β
and tau protein and their role as potential targets for the development of therapeutic drugs. Finally, we will provide perspectives in developing a multifactorial strategy for AD treatment.
... Mice expressing Mo/Hu APPswe (line C3-3), expressing wild-type human PS1 (line S8-4), the A246E variant of PS1 (lines N-5, F3.2 and I2-4), and the exon 9 deleted (dE9) of PS1 (line S-9) have been previously described (Borchelt et al., 1996a;Borchelt et al., 1996b;Thinakaran et al., 1996;Lee et al., 1997;Borchelt et al., 1997;Borchelt et al., 2001). ...
In Alzheimer disease, the extracellular deposition of beta-amyloid (Abeta) in the brain is accompanied by the intracellular accumulation of aggregated forms of hyperphosphorylated tau. In developing animal models of AD, the authors and others have been able to reproduce extracellular amyloid pathology in the brains of mice by expressing mutant amyloid precursor proteins (APP). The co-expression of APP with mutant presenilin leads to a dramatic acceleration in Abeta deposition, leading to very high amyloid burdens in mice. In the current study, the authors have examined whether the brains of mice with high burdens of amyloid deposition also contain aggregated forms of tau, using a cellulose acetate filter trap assay. Although discrete accumulations of phosphorylated tau immunoreactivity were apparent in neurites proximal to cored deposits of Abeta, little if any of this tau was in a SDS-resistant state of aggregation. By contrast, the brains of AD patients contained large amounts of aggregated tau. Overall, this study demonstrates that, in mice, deposition of Abeta does not cause endogenous tau to aggregate.
... We tentatively attribute this outcome to structural masking of the epitope. It is common practice to denature tissue sections in formic acid when these antibodies are used in immunocytochemistry Borchelt et al., 2001). Hence, the lack of reactivity of A trapped in the filters may be due to structural features of the aggregate. ...
Extracellular and/or intracellular aggregates are pathological features of many, if not all, neurodegenerative diseases. In Alzheimer disease (AD), extracellular aggregates of beta-amyloid (Abeta) and intracellular aggregates of tau or a-synuclein are key diagnostic markers of the disease. We report here a method to rapidly detect these protein aggregates that relies on size exclusion filtration and immunostaining of trapped material, a method termed filter trapping. We demonstrate that aggregated forms of Abeta and tau are readily trapped in 0.22 microm cellulose acetate filter membranes, which can then be immunostained with specific antibodies in a manner similar to the standard immunoblot. Coupling this method with serial dilution permits a rapid assessment of relative aggregate burden.
... It is intriguing that in our study we observed clearance of amyloid and reduced toxicity as measured by examining the structural alterations of neurites near plaques, despite the increase in soluble Ab42/40 ratio which has been implicated as causative in AD (Walker et al. 2005). The animal model used in this work, APPswe/PS1dE9, has a higher Ab42/40 ratio probably due to the PS1 mutation (Borchelt et al. 2002;Garcia-Alloza et al. 2006b), and the immediate effect of curcumin tends to exacerbate this tendency. Further studies with mouse models that favor the Ab40/42 ratio could also help to elucidate this question. ...
Alzheimer's disease (AD) is characterized by senile plaques and neurodegeneration although the neurotoxic mechanisms have not been completely elucidated. It is clear that both oxidative stress and inflammation play an important role in the illness. The compound curcumin, with a broad spectrum of anti-oxidant, anti-inflammatory, and anti-fibrilogenic activities may represent a promising approach for preventing or treating AD. Curcumin is a small fluorescent compound that binds to amyloid deposits. In the present work we used in vivo multiphoton microscopy (MPM) to demonstrate that curcumin crosses the blood-brain barrier and labels senile plaques and cerebrovascular amyloid angiopathy (CAA) in APPswe/PS1dE9 mice. Moreover, systemic treatment of mice with curcumin for 7 days clears and reduces existing plaques, as monitored with longitudinal imaging, suggesting a potent disaggregation effect. Curcumin also led to a limited, but significant reversal of structural changes in dystrophic dendrites, including abnormal curvature and dystrophy size. Together, these data suggest that curcumin reverses existing amyloid pathology and associated neurotoxicity in a mouse model of AD. This approach could lead to more effective clinical therapies for the prevention of oxidative stress, inflammation and neurotoxicity associated with AD.
... These mouse models emulate with great success the deposition of beta-amyloid in the neocortex and the hippocampus, but not necessarily neurodegenerative changes, such as neurofibrillary tangles. Transgenic mice typically overexpress the amyloid precursor protein (APP) with familial AD mutations under the control of various promoters, and although there are more than a dozen strains available, most studies are done on the PDAPP, Tg2576, APP23 and most recently, the mo/hu APPswe/PS1dE9 mice (140)(141)(142)(143). The mutations carried in the hAPP transgene introduced into these mice originated from genetic studies done on families with early onset AD. ...
We have long promulgated the idea that microglial cells serve an entirely beneficial role in the central nervous system (CNS), not only as immunological sentinels to fend off potentially dangerous infections, but also as constitutively neuroprotective glia that help sustain neuronal function in the normal and especially in the injured CNS when microglia become activated. In recent years, we have reported on the presence of degenerating microglial cells, which are prominent in the brains of aged humans and humans with neurodegenerative diseases, and this has led us to propose a hypothesis stating that loss of microglia and microglial neuroprotective functions could, at least in part, account for aging-related neurodegeneration. In the current review, we sum up the many aspects that characterize microglial activation and compare them to those that characterize microglial senescence and degeneration. We also consider the possible role of oxidative stress as a cause of microglial degeneration. We finish up by discussing the role microglial cells play in terms of amyloid clearance and degradation with the underlying idea that removal of amyloid constitutes a microglial neuroprotective function, which may become compromised during aging.
The Amyloid precursor protein (APP), whose mutations cause Alzheimer's disease, plays an important in vivo role and facilitates transmitter release. Since the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the Ubiquitin-Proteasome-system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2-terminus of the ACR, and CRL4CRBN., which is formed by Cul4a/b, Ddb1 and Crbn, and interacts with the COOH-terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4CRBN, pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues K649/650/651/676/688. Deletion of Crbn reduces ubiquitination of K676 suggesting that K676 is physiologically ubiquitinated by CRL4CRBN. The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely tau and ApoE, interact with and are ubiquitinated via the ACR in vitro. This, and the evidence that CRBN and CUL4B are linked to Intellectual disability, prompts to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well-described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the Ubiquitin-Proteasome-system (UPS) and neurodegeneration make this concept plausible.
This chapter focuses on Alzheimer's disease (AD), the most common age-related dementia, and on the value of experimental models for understanding disease mechanisms and for insights into experimental therapeutics. Characteristic intracellular and extracellular protein aggregates, implicated in pathogenic processes, are critical elements of this pathology. Targeting of genes encoding proteins thought to be implicated in disease pathways has provided new understanding of the roles of specific gene products in AD and the potential of these proteins as therapeutic targets. The value of these targets for new treatment strategies is being tested in model systems and, once safety and efficacy are assured, in human trials. This chapter illustrates the clinical, pathological, biochemical, and genetic features of the human illness. Furthermore, it discusses selected aspects namely, the biology of proteins implicated in pathogenesis of disease, the value of genetically engineered models, the identification of new therapeutic targets, and experimental treatments in models.
A common characteristic of neurodegenerative diseases like Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD) is the accumulation of protein aggregates. This reflects a severe disturbance of protein homeostasis, the proteostasis. Here, we review the involvement of the two major proteolytic machineries, the ubiquitin proteasome system (UPS) and the autophagy/lysosomal system, in the pathogenesis of neurodegenerative diseases. These proteolytic systems cooperate to maintain the proteostasis, as is indicated by intricate cross talk. In addition, the UPS and autophagy are regulated by stress pathways that are activated by disturbed proteostasis, like the unfolded protein response (UPR). We will specifically discuss how these proteolytic pathways are affected in neurodegenerative diseases. We will show that there is a differential involvement of the UPS and autophagy in different neurodegenerative disorders. In addition, the proteolytic impairment may be primary or secondary to the pathology. These differences have important implications for the design of therapeutic strategies. The opportunities and caveats of targeting the UPS and autophagy/lysosomal system as a therapeutic strategy in neurodegeneration will be discussed.
Hyperphosphorylated tau in neurites surrounding beta-amyloid (betaA) deposits, as revealed with phospho-specific anti-tau antibodies, are found in amyloid precursor protein (APP) Tg2576 mice. Because betaA is a source of oxidative stress and may be toxic for cultured cells, the present study examines the expression of phosphorylated (active) stress-activated kinase c-Jun N-terminal kinase (SAPK/JNK-P) and p38 kinase (p38-P), which have the capacity to phosphorylate tau at specific sites, and their specific substrates c-Jun and ATF-2, which are involved in cell death and survival in several paradigms, in Tg2576 mice. The study was planned to shed light about the involvement of these kinases in tau phosphorylation in cell processes surrounding amyloid plaques, as well as in the possible phosphorylation (activation) of c-Jun and activating transcription factor-2 (ATF-2) in relation to betaA deposition. Moderate increase in the expression of phosphorylated mitogen-activated protein kinase and extracelullar signal-regulated kinase (MAPK/ERK-P) occurs in a few amyloid plaques. However, strong expression of SAPK/JNK-P and p38-P is found in the majority of, if not all, amyloid plaques, as seen in serial consecutive sections stained for betaA and stress kinases. Moreover, confocal microscopy reveals colocalization of phospho-tau and SAPK/JNK-P, and phospho-tau and p38-P in many dystrophic neurites surrounding amyloid plaques. Increased expression levels of nonbound tau, SAPK/JNK-P and p38-P are corroborated by Western blots of total cortical homogenate supernatants in Tg2576 mice when compared with age-matched controls. No increase in phosphorylated c-JunSer63 (c-Jun-P) and ATF-2Thr71 (ATF-2-P) is found in association with betaA deposits. In addition, no expression of active (cleaved) caspase-3 (17 kDa) has been found in transgenic mice. Taken together, these observations provide a link between betaA-induced oxidative stress, activation of stress kinases SAPK/JNK and p38, and tau hyperphosphorylation in neurites surrounding amyloid plaques, but activation of these kinases is not associated with accumulation of c-Jun-P and ATF-2-P, nor with activation of active caspase-3 in the vicinity of betaA deposits.
Hyperphosphorylation and accumulation of tau in neurons (and glial cells) is one the main pathologic hallmarks in Alzheimer's disease (AD) and other tauopathies, including Pick's disease (PiD), progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease and familial frontotemporal dementia and parkinsonism linked to chromosome 17 due to mutations in the tau gene (FTDP-17-tau). Hyperphosphorylation of tau is regulated by several kinases that phosphorylate specific sites of tau in vitro. GSK-3-immunoprecipitated sarcosyl-insoluble fractions in AD have the capacity to phosphorylate recombinant tau. In addition, GSK-3 phosphorylated at Ser9, that inactivates GSK-3, is found in the majority of neurons with neurofibrillary tangles and dystrophic neurites of senile plaques in AD, and in Pick bodies and other phospho-tau-containing neurons and glial cells in other tauopathies. Increased expression of active kinases, including stress-activated kinase, c-Jun N-terminal kinase (SAPK/JNK) and kinase p38 has been found in brain homogenates in all the tauopathies. Strong active SAPK/JNK and p38 immunoreactivity has been observed restricted to neurons and glial cells containing hyperphosphorylated tau, as well as in dystrophic neurites of senile plaques in AD. Moreover, SAPK/JNK- and p38-immunoprecipitated sub-cellular fractions enriched in abnormal hyperphosphorylated tau have the capacity to phosphorylate recombinant tau and c-Jun and ATF-2 which are specific substrates of SAPK/JNK and p38 in AD and PiD. Interestingly, increased expression of phosphorylated (active) SAPK/JNK and p38 and hyperphosphorylated tau containing neurites have been observed around betaA4 amyloid deposits in the brain of transgenic mice (Tg 2576) carrying the double APP Swedish mutation. These findings suggest that betaA4 amyloid has the capacity to trigger the activation of stress kinases which, in turn, phosphorylate tau in neurites surrounding amyloid deposits. Complementary findings have been reported from the autopsy of two AD patients who participated in an amyloid-beta immunization trial and died during the course of immunization-induced encephalitis. The neuropathological examination of the brain showed massive focal reduction of amyloid plaques but not of neurofibrillary degeneration. Activation of SAPK/JNK and p38 were reduced together with decreased tau hyperphosphorylation of aberrant neurites in association with decreased amyloid plaques in both Tg2576 mice and human brains. These findings support the amyloid cascade hypothesis of tau phosphorylation mediated by stress kinases in dystrophic neurites of senile plaques but not that of neurofibrillary tangles and neuropil threads in AD.
Transgenic mice carrying disease-linked forms of genes associated with Alzheimer disease often demonstrate deposition of the beta-amyloid as senile plaques and cerebral amyloid angiopathy. We have characterized the natural history of beta-amyloid deposition in APPswe/PS1dE9 mice, a particularly aggressive transgenic mouse model generated with mutant transgenes for APP (APPswe: KM594/5NL) and PS1 (dE9: deletion of exon 9). Ex vivo histochemistry showed Abeta deposition by 4 months with a progressive increase in plaque number up to 12 months and a similar increase of Abeta levels. In vivo multiphoton microscopy at weekly intervals showed increasing beta-amyloid deposition as CAA and plaques. Although first appearing at an early age, CAA progressed at a significantly slower rate than in the Tg2576 mice. The consistent and early onset of beta-amyloid accumulation in the APPswe/PS1dE9 model confirms its utility for studies of biochemical and pathological mechanisms underlying beta-amyloid deposition, as well as exploring new therapeutic treatments.
Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW) complexes. During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments. Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of β-amyloid precursor protein and Notch. We show that although PS1/PS2 endoproteolysis is not required for inclusion into the higher MW N- and C-terminal fragment-containing complex, aspartate mutant holoprotein presenilins are not incorporated into the high MW complexes. Aspartate mutant presenilin holoproteins also preclude entry of endogenous wild type PS1/PS2 into the high MW complexes but do not affect the incorporation of wild type holoproteins into lower MW holoprotein complexes. These data suggest that the loss of function effects of the aspartate mutants result in altered PS complex maturation and argue that the functional presenilin moieties are contained in the high molecular weight complexes.
Mutations in the presenilin 1 (PS1) and presenilin 2 genes cosegregate with the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. We now document that the Aβ1–42(43)/Aβ1–40 ratio in the conditioned media of independent N2a cell lines expressing three FAD-linked PS1 variants is uniformly elevated relative to cells expressing similar levels of wild-type PS1. Similarly, the Aβ1–42(43)/Aβ1–40 ratio is elevated in the brains of young transgenic animals coexpressing a chimeric amyloid precursor protein (APP) and an FAD-linked PS1 variant compared with brains of transgenic mice expressing APP alone or transgenic mice coexpressing wild-type human PS1 and APP. These studies provide compelling support for the view that one mechanism by which these mutant PS1 cause AD is by increasing the extracellular concentration of Aβ peptides terminating at 42(43), species that foster Aβ deposition.
Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.
Pathogenic mutations in presenilin 1 (PS1) are associated with ≈50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically
cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected
with human PS1, we have found that phorbol 12,13-dibutyrate and forskolin increase the state of phosphorylation of serine
residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single
major species of 18 kDa to a doublet of 20–23 kDa. This mobility shift was also observed with human PS1 that had been transfected
into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage λ protein phosphatase eliminated
the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic
mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column
at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation
of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments
may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.
Mutations in the homologous presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of familial
Alzheimer’s disease. Although PS1 function and dysfunction have been extensively studied, little is known about the function
of PS2 in vivo. To delineate the relationships of PS2 and PS1 activities and whether PS2 mutations involve gain or loss of function, we
generated PS2 homozygous deficient (−/−) and PS1/PS2 double homozygous deficient mice. In contrast to PS1−/− mice, PS2−/− mice are viable and fertile and develop only mild pulmonary fibrosis and hemorrhage with age. Absence of PS2 does not detectably
alter processing of amyloid precursor protein and has little or no effect on physiologically important apoptotic processes,
indicating that Alzheimer’s disease-causing mutations in PS2, as in PS1, result in gain of function. Although PS1+/− PS2 −/− mice survive in relatively good health, complete deletion of both PS2 and PS1 genes causes a phenotype closely resembling
full Notch-1 deficiency. These results demonstrate in vivo that PS1 and PS2 have partially overlapping functions and that PS1 is essential and PS2 is redundant for normal Notch signaling
during mammalian embryological development.
Genetic linkage studies place a gene causing early onset familial Alzheimer's disease (FAD) on chromosome 14q24.3 (refs 1−4). Five mutations within the S182 (Presenilin 1: PS−1) gene, which maps to this region, have recently been reported in several early onset FAD kindreds5. We have localized the PS-1 gene to a 75 kb region and present the structure of this gene, evidence for alternative splicing and describe six novel mutations in early onset FAD pedigrees all of which alter residues conserved in the STM2
6 (Presenilin 2: PS-2) gene.
Absence of functional presenilin 1 (PS1) protein leads to loss of γ-secretase cleavage of the amyloid precursor protein (βAPP),
resulting in a dramatic reduction in amyloid β peptide (Aβ) production and accumulation of α- or β-secretase-cleaved COOH-terminal
fragments of βAPP (α- or β-CTFs). The major COOH-terminal fragment (CTF) in brain was identified as βAPP-CTF-(11–98), which
is consistent with the observation that cultured neurons generate primarily Aβ-(11–40). In PS1−/− murine neurons and fibroblasts expressing the loss-of-function PS1D385A mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle
differences in the subcellular distribution of CTFs in PS1−/− neurons as compared with PS1D385A mutant fibroblasts. However, there was no obvious redistribution of full-length βAPP or of markers of other organelles in
either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1−/− neurons (as in normal cells) trafficking of βAPP to the Golgi compartment is necessary before α- and β-secretase cleavages
occur. Thus, although we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role
in general protein trafficking. These results are more compatible with a role for PS1 either as the actual γ-secretase catalytic
activity or in other functions indirectly related to γ-secretase catalysis (e.g. an activator of γ-secretase, a substrate adaptor for γ-secretase, or delivery of γ-secretase to βAPP-containing compartments).
Presenilin-1 (PS1) facilitates γ-secretase cleavage of the β-amyloid precursor protein and the intramembraneous cleavage of
Notch1. Although Alzheimer’s disease-associated mutations in the homologous presenilin (PS2) gene elevate amyloid β-peptide
(Aβ42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not
result in a severe phenotype resembling that of Notch-ablated animals. To investigate the amyloidogenic function of PS2 more
directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known
to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic
processing of β-amyloid precursor protein indicating a defect in γ-secretase activity. The reduced γ-secretase activity results
in the almost complete inhibition of Aβ and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing
the wild-type PS2 cDNA produce robust levels of Aβ and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks γ-secretase activity but also inactivates PS2 activity
in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is
functionally involved in Aβ production and Notch signaling by facilitating similar proteolytic cleavages.
Mutations in Cu/Zn superoxide dismutase (SOD1) cause a subset of cases of familial amyotrophic lateral sclerosis. Four lines of mice accumulating one of these mutant proteins (G37R) develop severe, progressive motor neuron disease. At lower levels of mutant accumulation, pathology is restricted to lower motor neurons, whereas higher levels cause more severe abnormalities and affect a variety of other neuronal populations. The most obvious cellular abnormality is the presence in axons and dendrites of membrane-bounded vacuoles, which appear to be derived from degenerating mitochondria. Since multiple lines of mice expressing wild-type human SOD1 at similar and higher levels do not show disease, the disease in mice expressing the G37R mutant SOD1 must arise from the acquisition of an adverse property by the mutant enzyme, rather than elevation or loss of SOD1 activity.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the extracellular deposition of amyloid beta-protein (A beta), a molecule produced by post-translational processing of the beta-amyloid precursor protein (beta APP). Mutations within the gene encoding beta APP have been linked to early onset forms of AD, but the pathogenetic mechanism(s) producing the phenotype are unknown. We analyzed the effects on beta APP processing in vitro of a naturally occurring Ala-->Gly mutation at position 692 of beta APP770 (A692G) (Hendriks, L., Duijin, C., Cras, P., Cruts, M., van Hul, W., van Harskamp, F., Warren, A., McInnis, M., Antonarakis, S., Martin, J.-J., Hofman, A., and van Broeckhoven, C. (1992) Nature Genet. 1, 218-221), as well as the effects of five genetically engineered mutations at or near this site. Substitution of glycine or proline for Ala692, or for Phe690, produced relative increases in secretion of A beta and relative decreases in secretion of the p3 peptide(s) arising after alpha-secretase generation of soluble APP (APPs). The Phe690-->Pro substitution also resulted in the synthesis of truncated APPs molecules. The structurally conservative substitutions Ala692-->Val and Phe690-->Tyr did not exhibit these effects. Certain of the substitutions also resulted in the production of a minor peptides, previously undescribed in vitro, beginning at Ala2, Lys16, and Phe19 of A beta. These data show that beta APP mutations carboxyl-terminal to alpha-secretase and beta-secretase cleavage sites can exert strong control over beta APP processing. Increased secretion of A beta may accelerate amyloidogenesis by providing more precursors for aggregation. It is also possible that truncated A beta peptides resulting from several of these mutations may accelerate amyloidogenesis through self-aggregation and/or seeding the fibrillogenesis of longer, more abundant A beta species.
Missense mutations in the presenilin 2 (PS-2) gene on chromosome 1 were sought by direct nucleotide sequence analysis of the open reading frame of 60 pedigrees with familial Alzheimer's disease (FAD). In the majority of these pedigrees, PS-1 and beta-amyloid precursor protein (beta APP) gene mutations had been excluded. While no additional PS-2 pathogenic mutations were detected, four silent nucleotide substitutions and alternative splicing of nucleotides 1338-1340 (Glu325) were observed. Analysis of additional members of a pedigree known to segregate a Met239Val mutation in PS-2 revealed that the age of onset of symptoms is highly variable (range 45-88 years). This variability is not attributable to differences in ApoE genotypes. These results suggest (i) that, in contrast to mutations in PS-1, mutations in PS-2 are a relatively rare cause of FAD; (ii) that other genetic or environmental factor modify the AD phenotype associated with PS-2 mutations; and (iii) that still other FAD susceptibility genes remain to be identified.
Mutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the "loop," N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm.
To gain insights into the significance of presenilins (PS) in the pathogenetic mechanisms of early-onset familial Alzheimer disease (FAD), we expressed cDNAs for wild-type PS2 and PS2 with the Volga German (N141I) mutation in cultured cells and then examined the metabolism of the transfected proteins and their effect on the C-terminal properties of secreted amyloid beta protein (A beta). PS2 was identified as a 50- to 55-kDa protein, which was cleaved to produce N-terminal fragments of 35-40 kDa and C-terminal fragments of 19-23 kDa. The Volga German (N141I) mutation did not cause any significant change in the metabolism of PS2. COS-1 cells doubly transfected with cDNAs for N141I mutant PS2 and human beta-amyloid precursor protein (betaAPP) or a C-terminal fragment thereof, as well as mouse Neuro2a neuroblastoma cells stably transfected with N141I mutant PS2 alone, secreted 1.5- to 10-fold more A beta ending at residues 42 (or 43) [A beta42(43)] compared with those expressing the wild-type PS2. These results strongly suggest that the PS2 mutation (N141I) linked to FAD alters the metabolism of A beta/betaAPP to foster the production of the form of A beta that most readily deposits in amyloid plaques. Thus, mutant PS2 may lead to AD by altering the metabolism of A beta/betaAPP.
Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes can cause Alzheimer's disease in affected members of the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. PS1 encodes an ubiquitously expressed, eight transmembrane protein. PS1 is endoproteolytically processed to an amino-terminal derivative (approximately 27-28 kDa) and a carboxy-terminal derivative (approximately 17-18 kDa). These polypeptides accumulate to saturable levels in the brains of transgenic mice, independent of the expression of PS1 holoprotein. We now document that, in the brains of transgenic mice, the absolute amounts of accumulated N- and C-terminal derivatives generated from the FAD-linked PS1 variants in which Glu replaces Ala at codon 246 (A246E) or Leu replaces Met at codon 146 (M146L) accumulate to a significantly higher degree (approximately 40-50%) than the fragments derived from wild-type PS1. Moreover, the FAD-linked deltaE9 PS1 variant, a polypeptide that is not subject to endoproteolytic cleavage in vivo, also accumulates in greater amounts than the fragments generated from wild-type human PS1. Thus, the metabolism of PS1 variants linked to FAD is fundamentally different from that of wild-type PS1 in vivo.
Presenilin 1 (PS1), mutated in pedigrees of early-onset familial Alzheimer’s disease, is a polytopic integral membrane protein
that is endoproteolytically cleaved into 27-kDa N-terminal and 17-kDa C-terminal fragments. Although these fragments are the
principal PS1 species found in normal mammalian brain, the role of endoproteolysis in the maturation of PS1 has been unclear.
The present study, which uses stably transfected mouse neuroblastoma N2a cells, demonstrates that full-length polypeptides,
derived from either wild-type or A246E FAD-mutant human (hu) PS1, are relatively short-lived (t½ 1.5 h) proteins that give rise to the N- and C-terminal PS1 fragments, which are more stable (t½ ∼ 24 h). N-terminal fragments, generated artificially by engineering a stop codon at amino acid 306 (PS1–306) of wild-type
huPS1, were short-lived, whereas an FAD-linked variant that lacked exon 9 (ΔE9) and was not endoproteolytically cleaved exhibited
a long half-life. These observations suggest that endoproteolytic cleavage and stability are not linked, leading us to propose
a model in which wild-type full-length huPS1 molecules are first stabilized then subsequently endoproteolytically cleaved
to generate the N- and C-terminal fragments. These fragments appear to represent the mature and functional forms of wild-type
huPS1.
Mutations in two related genes, PS1and PS2, account for the majority of early onset cases of familial Alzheimer’s disease. PS1 and PS2 are homologous polytopic membrane
proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a
neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice
expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease
in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished
in cultured cells overexpressing human PS2. To define the minimal sequence requirements for “replacement” we expressed familial
Alzheimer’s disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation
of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic
cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately
by competition for limiting cellular factor(s).
Missense mutations in two related genes, termed presenilin 1 (PS1) and presenilin 2 (PS2), cause dementia in a subset of early-onset familial Alzheimer's disease (FAD) pedigrees. In a variety of experimental in vitro and in vivo settings, FAD-linked presenilin variants influence the processing of the amyloid precursor protein (APP), leading to elevated levels of the highly fibrillogenic Abeta1-42 peptides that are preferentially deposited in the brains of Alzheimer Disease (AD) patients. In this report, we demonstrate that transgenic animals that coexpress a FAD-linked human PS1 variant (A246E) and a chimeric mouse/human APP harboring mutations linked to Swedish FAD kindreds (APP swe) develop numerous amyloid deposits much earlier than age-matched mice expressing APP swe and wild-type Hu PS1 or APP swe alone. These results provide evidence for the view that one pathogenic mechanism by which FAD-linked mutant PS1 causes AD is to accelerate the rate of beta-amyloid deposition in brain.
Point mutations in the presenilin-1 gene (PS1) are a major cause of familial Alzheimer's disease. They result in a selective increase in the production of the amyloidogenic peptide amyloid-beta(1-42) by proteolytic processing of the amyloid precursor protein (APP). Here we investigate whether PS1 is also involved in normal APP processing in neuronal cultures derived from PS1-deficient mouse embryos. Cleavage by alpha- and beta-secretase of the extracellular domain of APP was not affected by the absence of PS1, whereas cleavage by gamma-secretase of the transmembrane domain of APP was prevented, causing carboxyl-terminal fragments of APP to accumulate and a fivefold drop in the production of amyloid peptide. Pulse-chase experiments indicated that PS1 deficiency specifically decreased the turnover of the membrane-associated fragments of APP. As in the regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, PS1 appears to facilitate a proteolytic activity that cleaves the integral membrane domain of APP. Our results indicate that mutations in PS1 that manifest clinically cause a gain of function and that inhibition of PS1 activity is a potential target for anti-amyloidogenic therapy in Alzheimer's disease.
We have examined the trafficking and metabolism of the beta-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Abeta, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.
Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signalling.
Mutations in genes encoding presenilins (PS1 and PS2) cosegregate with the majority of early onset cases of familial Alzheimer’s
disease. PS1 and PS2 are polytopic membrane proteins that undergo endoproteolytic cleavage to generate stable NH2- and COOH-terminal derivatives (NTF and CTF, respectively). Several lines of evidence suggest that the endoproteolytic derivatives
are likely the functional units of PSin vivo. In the present report, we examine the disposition of PS NTF and CTF assemblies in stable mouse N2a neuroblastoma cell lines
expressing human PS polypeptides. We show that exogenous expression of PS1 NTFs neither assemble with endogenous CTF nor exhibit
dominant negative inhibitory effects on the endogenous PS1 cleavage and the accumulation of derivatives. In cells co-expressing
PS1 and PS2, PS1- and PS2-derived fragments do not form mixed assemblies. In contrast, cells expressing a chimeric PS1/PS2
polypeptide form stable PS1 NTF-PS2 CTF assemblies. Moreover, expression of chimeric PS1/PS2 polypeptides harboring a familial
early onset AD-linked mutation (M146L) elevates the production of Aβ42 peptides. Our results provide evidence that assembly
of structural domains contained within NH2- and COOH-terminal regions of PS occur prior to endoproteolytic cleavage.
Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). To identify a PS subdomain responsible for overproduction of Abeta42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in Abeta42 secretion, and Abeta42 overproduction was dependent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of Abeta42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.
Dentatorubral and pallidoluysian atrophy (DRPLA) is a member of a family of progressive neurodegenerative diseases caused by polyglutamine repeat expansion. Transgenic mice expressing full-length human atrophin-1 with 65 consecutive glutamines exhibit ataxia, tremors, abnormal movements, seizures, and premature death. These mice accumulate atrophin-1 immunoreactivity and inclusion bodies in the nuclei of multiple populations of neurons. Subcellular fractionation revealed 120 kDa nuclear fragments of mutant atrophin-1, whose abundance increased with age and phenotypic severity. Brains of DRPLA patients contained apparently identical 120 kDa nuclear fragments. By contrast, mice overexpressing atrophin-1 with 26 glutamines were phenotypically normal and did not accumulate the 120 kDa fragments. We conclude that the evolution of neuropathology in DRPLA involves proteolytic processing of mutant atrophin-1 and nuclear accumulation of truncated fragments.
Missense mutations in the presenilin 2 (PS-2) gene on chromosome 1 were sought by direct nucleotide sequence analysis of the open reading frame of 60 pedigrees with familial Alzheimer's disease (FAD). In the majority of these pedigrees, PS-1 and β-amyloid precursor protein (βAPP) gene mutations had been excluded. While no additional PS-2 pathogenic mutations were detected, four silent nucleotide substitutions and alternative splicing of nucleotides 1338-1340 (Glu325) were observed. Analysis of additional members of a pedigree known to segregate a Met239Val mutation in PS-2 revealed that the age of onset of symptoms is highly variable (range 45-88 years). This variability is not attributable to differences in ApoE genotypes. These results suggest (i) that, in contrast to mutations in PS-1, mutations in PS-2 are a relatively rare cause of FAD ; (ii) that other genetic or environmental factors modify the AD phenotype associated with PS-2 mutations ; and (iii) that still other FAD susceptibility genes remain to be identified.
. Si crystals were implanted with 2.0- MeV Er+ at the doses of 51012 ions/cm2, 11014 ions/cm2, 51014ions/cm2, 11015 ions/cm2 and 2.51015 ions/cm2. Conventional furnace thermal annealing was carried out in the temperature range from 600 C to 1150 C. The depth distribution
of Er, associated damage profiles and annealing behavioar were investigated using the Rutherford backscattering spectrometry
and channelling (RBS/C) technique. A proper convolution program was used to extract the distribution of Er from the experimental
RBS spectrum. The obtained distribution parameters, projected range Rp, projected range straggling ΔRp and skewness SK were compared with those of TRIM96 calculation.The experimental Rp and SK values agree well with the simulated values, while the experimental ΔRp is larger than TRIM 96 simulated value by a factor of 18%. The damage profile of silicon crystal induced by 2.0-MeV Er+ at a dose of 11014 ions/cm2 was extracted using the multiple-scattering dechannelling model based on Feldman’s method, which is in a good agreement with
the TRIM96 calculation. For the samples with dose of 51014 ions/cm2 and more, an abnormal annealing behavioar was found and a qualitative explaination has been given.
Mutations in the presenilin-1 (PS1) gene are associated with
Alzheimer's disease and cause increased secretion of the neurotoxic
amyloid- peptide (A). Critical intramembraneous aspartates
at residues 257 and 385 are required for the function of PS1 protein. Here
we investigate the biological function of a naturally occurring PS1 splice
variant (PS1 exon 8), which lacks the critical aspartate 257. Cell
lines that stably express PS1 exon 8 or a PS1 protein in which aspartate
residue 257 is mutated secrete significant levels of A, whereas A
generation is severely reduced in cells transfected with PS1 containing a
mutation of aspartate 385. In contrast, endoproteolytic processing of Notch
is almost completely inhibited in cell lines expressing any of the PS1 variants
that lack one of the critical aspartates. These data indicate that PS1 may
differentially facilitate -secretase-mediated generation of A and
endoproteolysis of Notch.
The mechanism by which mutations in the presenilin (PS) genes cause the most aggressive form of early-onset Alzheimer's disease (AD) is unknown, but fibroblasts from mutation carriers secrete increased levels of the amyloidogenic A42 peptide, the main component of AD plaques. We established transfected cell and transgenic mouse models that coexpress human PS and amyloid -protein precursor (APP) genes and analyzed quantitatively the effects of PS expression on APP processing. In both models, expression of wild-type PS genes did not alter APP levels, - and -secretase activity and A production. In the transfected cells, PS1 and PS2 mutations caused a highly significant increase in A42 secretion in all mutant clones. Likewise, mutant but not wild-type PS1 transgenic mice showed significant overproduction of A42 in the brain, and this effect was detectable as early as 2−4 months of age. Different PS mutations had differential effects on A generation. The extent of A42 increase did not correlate with presenilin expression levels. Our data demonstrate that the preseniiin mutations cause a dominant gain of function and may induce AD by enhancing A42 production, thus promoting cerebral -amyloidosis.
DNA damage induces apoptosis through a signalling pathway that can be suppressed
by the BCL-2 protein, but the mechanism by which DNA damage does this is unknown.
Here, using yeast two-hybrid and co-immunoprecipitation studies, we show that
RAD9, a human protein involved in the control of a cell-cycle checkpoint,
interacts with the anti-apoptotic Bcl-2-family proteins BCL-2 and BCL-x
L, but not with the pro-apoptotic BAX and BAD. When overexpressed in
mammalian cells, RAD9 induces apoptosis that can be blocked by BCL-2 or BCL-x
L. Conversely, antisense RAD9 RNA suppresses cell death induced
by methyl methanesulphonate. These findings indicate that RAD9 may have a
new role in regulating apoptosis after DNA damage, in addition to its previously
described checkpoint-control and other radioresistance-promoting functions.
The prevalence of severe dementia in the United States is about 1.3 million cases, of which at least 50 to 60% are of the Alzheimer type. Severe dementia of the Alzheimer type is found rarely in a clearly dominant pattern, although often one or more relatives are affected. Down's syndrome in adults is often associated with Alzheimer changes. The diagnosis is a clinicopathological one; there is a considerable error rate in the clinical diagnosis early in the course of the disease, especially in regard to dementia in depression. The differential diagnosis involves a great many disorders, including multi-infarct dementia, tumors, subdural hematomas, and others. Physiological aspects of Alzheimer's disease include a diffusely slow electroencephalogram, reduced cerebral blood flow, and particular patterns noted on positron emission tomographic scanning. The latter technique has also demonstrated that oxygen extraction is normal in Alzheimer's disease, thus excluding ischemia from possible pathogenetic factors. Morphological changes, that is, the presence of plaques and tangles, are widely distributed in neocortex, paleocortex, and many deep gray areas down through the pontine tegmentum, but largely exclude the basal ganglia, thalamus, and substantia nigra. Numerous plaques without neocortical tangles are found in many demented persons older than 75 years. A severe loss of large neocortical neurons is characteristic of the disease. The chemical nature of the paired helical filaments that make up the neurofibrillary tangle has not yet been ascertained. Neurons are markedly deficient in the basal forebrain nuclei, and this deficiency may account for the severe diminution of choline acetyltransferase and acetylcholine in the neocortex and palecortex. Muscarinic cholinergic receptors are present in normal amounts. Norepinephrine is reduced in some cases, and somatostatin in most. Substance P is low in severe cases. The etiology of the disorder is unknown and the role of aluminum is disputed. Management of patients with Alzheimer's disease is difficult, and neuroleptics are to be used with great caution because of their side effects. Substrate therapy has not been effective; physostigmine improves memory but is not suitable for general use. Trophic factors, gangliosides, and aluminum chelation are being investigated for use in pharmacological intervention.
The 4-kilodalton (39 to 43 amino acids) amyloid beta protein (beta AP), which is deposited as amyloid in the brains of patients
with Alzheimer's diseases, is derived from a large protein, the amyloid beta protein precursor (beta APP). Human mononuclear
leukemic (K562) cells expressing a beta AP-bearing, carboxyl-terminal beta APP derivative released significant amounts of
a soluble 4-kilodalton beta APP derivative essentially identical to the beta AP deposited in Alzheimer's disease. Human neuroblastoma
(M17) cells transfected with constructs expressing full-length beta APP and M17 cells expressing only endogenous beta APP
also released soluble 4-kilodalton beta AP, and a similar, if not identical, fragment was readily detected in cerebrospinal
fluid from individuals with Alzheimer's disease and normal individuals. Thus cells normally produce and release soluble 4-kilodalton
beta AP that is essentially identical to the 4-kilodalton beta AP deposited as insoluble amyloid fibrils in Alzheimer's disease.
Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.
To study the putative precursor proteins (PreA4(695), PreA4(751), and PreA4(770] of Alzheimer's disease A4 amyloid protein, polyclonal and monoclonal antibodies were raised against a recombinant bacterial PreA4(695) fusion protein. These antibodies were used to identify the precursors in different cell lines as well as in human brain homogenates and cerebrospinal fluid (CSF). The precursors are tyrosine-sulfated, O- and N-glycosylated membrane proteins and have half-lives of 20-30 min in cells. Cells express the polypeptides at their surface but also secrete C-terminal truncated proteins into the medium. These proteins are also found in CSF of both Alzheimer's disease patients and normal individuals. The proteins are derived from their cognate membrane-associated forms by proteolysis and have apparently lost the cytoplasmic and the transmembrane domains. Since the latter contributes to the A4 amyloid sequence, it seems possible that this proteolytic cleavage represents the first step in the formation of A4 amyloid deposits.
Some cases of Alzheimer's disease are inherited as an autosomal dominant trait. Genetic linkage studies have mapped a locus (AD3) associated with susceptibility to a very aggressive form of Alzheimer's disease to chromosome 14q24.3. We have defined a minimal cosegregating region containing the AD3 gene, and isolated at least 19 different transcripts encoded within this region. One of these transcripts (S182) corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. Five different missense mutations have been found that cosegregate with early-onset familial Alzheimer's disease. Because these changes occurred in conserved domains of this gene, and are not present in normal controls, they are likely to be causative of AD3.
During the past year, apoptosis has been recognized as a process that is perpetually poised to be initiated--often from the cytoplasm rather than from the nucleus--unless it is suppressed by survival factors. Suspected mediators of apoptosis that have recently been investigated include the cysteine protease interleukin-1 beta-converting enzyme, free radicals and cell cycle kinases. Known inhibitors of programmed cell death, such as Bcl-2 and its homologues, have been further studied, and the results suggest that cell death may be regulated by multiple pathways. With the recent identification of the Drosophila gene reaper, which appears to play a role in the initiation of apoptosis, another genetic system for studying cell death has become available.
Normal processing of the amyloid beta protein precursor (beta APP) results in secretion of a soluble 4-kilodalton protein
essentially identical to the amyloid beta protein (A beta) that forms insoluble fibrillar deposits in Alzheimer's disease.
Human neuroblastoma (M17) cells transfected with constructs expressing wild-type beta APP or the beta APP717 mutants linked
to familial Alzheimer's disease were compared by (i) isolation of metabolically labeled 4-kilodalton A beta from conditioned
medium, digestion with cyanogen bromide, and analysis of the carboxyl-terminal peptides released, or (ii) analysis of the
A beta in conditioned medium with sandwich enzyme-linked immunosorbent assays that discriminate A beta 1-40 from the longer
A beta 1-42. Both methods demonstrated that the 4-kilodalton A beta released from wild-type beta APP is primarily but not
exclusively A beta 1-40. The beta APP717 mutations, which are located three residues carboxyl to A beta 43, consistently caused
a 1.5- to 1.9-fold increase in the percentage of longer A beta generated. Long A beta (for example, A beta 1-42) forms insoluble
amyloid fibrils more rapidly than A beta 1-40. Thus, the beta APP717 mutants may cause Alzheimer's disease because they secrete
increased amounts of long A beta, thereby fostering amyloid deposition.
The 4-kilodalton amyloid beta protein (A beta), which forms fibrillar deposits in Alzheimer's disease (AD), is derived from a large protein referred to as the amyloid beta protein precursor (beta APP). Human neuroblastoma (M17) cells transfected with constructs expressing wild-type beta APP or a mutant, beta APP delta NL, recently linked to familial AD were compared. After continuous metabolic labeling for 8 hours, cells expressing beta APP delta NL had five times more of an A beta-bearing, carboxyl terminal, beta APP derivative than cells expressing wild-type beta APP and they released six times more A beta into the medium. Thus this mutant beta APP may cause AD because its processing is altered in a way that releases increased amounts of A beta.
The majority of early-onset cases of familial Alzheimer's disease (FAD) are linked to mutations in two related genes, PS1 and PS2, located on chromosome 14 and 1, respectively. Using two highly specific antibodies against nonoverlapping epitopes of the PS1-encoded polypeptide, termed presenilin 1 (PS1), we document that the preponderant PS1-related species that accumulate in cultured mammalian cells, and in the brains of rodents, primates, and humans are approximately 27-28 kDa N-terminal and approximately 16-17 kDa C-terminal derivatives. Notably, a FAD-linked PS1 variant that lacks exon 9 is not subject to endoproteolytic cleavage. In brains of transgenic mice expressing human PS1, approximately 17 kDa and approximately 27 kDa PS1 derivatives accumulate to saturable levels, and at approximately 1:1 stoichiometry, independent of transgene-derived mRNA. We conclude that PS1 is subject to endoproteolytic processing in vivo.
Mutations in the genes encoding amyloid-beta precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2) are known to cause early-onset, autosomal dominant Alzheimer's disease. Studies of plasma and fibroblasts from subjects with these mutations have established that they all alter amyloid beta-protein (beta APP) processing, which normally leads to the secretion of amyloid-beta protein (relative molecular mass 4,000; M(r) 4K; approximately 90% A beta1-40, approximately 10% A beta1-42(43)), so that the extracellular concentration of A beta42(43) is increased. This increase in A beta42(43) is believed to be the critical change that initiates Alzheimer's disease pathogenesis because A beta42(43) is deposited early and selectively in the senile plaques that are observed in the brains of patients with all forms of the disease. To establish that the presenilin mutations increase the amount of A beta42(43) in the brain and to test whether presenilin mutations act as true (gain of function) dominants, we have now constructed mice expressing wild-type and mutant presenilin genes. Analysis of these mice showed that overexpression of mutant, but not wild-type, PS1 selectively increases brain A beta42(43). These results indicate that the presenilin mutations probably cause Alzheimer's disease through a gain of deleterious function that increases the amount of A beta42(43) in the brain.
Mutations in the presenilin 1 (PS1) and presenilin 2 genes cosegregate with the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. We now document that the Abeta1-42(43)/Abeta1-40 ratio in the conditioned media of independent N2a cell lines expressing three FAD-linked PS1 variants is uniformly elevated relative to cells expressing similar levels of wild-type PS1. Similarly, the Abeta1-42(43)/Abeta1-40 ratio is elevated in the brains of young transgenic animals coexpressing a chimeric amyloid precursor protein (APP) and an FAD-linked PS1 variant compared with brains of transgenic mice expressing APP alone or transgenic mice coexpressing wild-type human PS1 and APP. These studies provide compelling support for the view that one mechanism by which these mutant PS1 cause AD is by increasing the extracellular concentration of Abeta peptides terminating at 42(43), species that foster Abeta deposition.
An expression plasmid (MoPrP.Xho), for use in transgenic mice, was developed from the promoter, 5' intronic, and 3' untranslated sequences of the murine prion protein gene. Analyses of mice harboring the MoPrP.Xho construct with cDNA genes encoding the amyloid precursor protein (APP) and human presenilin 1 demonstrated that this vector provides relatively high levels of transgene-encoded polypeptides in brains and hearts of transgenic mice. The MoPrP.Xho vector should be very useful in strategies designed to overexpress a variety of wild-type and disease related mutant transgenes in the heart and brain.
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.
Genetic causes of Alzheimer's disease (AD) include mutations in the amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2) genes. The mutant APP(K670N,M671L) transgenic line, Tg2576, shows markedly elevated amyloid beta-protein (A beta) levels at an early age and, by 9-12 months, develops extracellular AD-type A beta deposits in the cortex and hippocampus. Mutant PS1 transgenic mice do not show abnormal pathology, but do display subtly elevated levels of the highly amyloidogenic 42- or 43-amino acid peptide A beta42(43). Here we demonstrate that the doubly transgenic progeny from a cross between line Tg2576 and a mutant PS1M146L transgenic line develop large numbers of fibrillar A beta deposits in cerebral cortex and hippocampus far earlier than their singly transgenic Tg2576 littermates. In the period preceding overt A beta deposition, the doubly transgenic mice show a selective 41% increase in A beta42(43) in their brains. Thus, the development of AD-like pathology is substantially enhanced when a PS1 mutation, which causes a modest increase in A beta42(43), is introduced into Tg2576-derived mice. Remarkably, both doubly and singly transgenic mice showed reduced spontaneous alternation performance in a "Y" maze before substantial A beta deposition was apparent. This suggests that some aspects of the behavioral phenotype in these mice may be related to an event that precedes plaque formation.
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.
Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp --> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 --> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 --> Ala mutation still inhibited gamma-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease.
Families bearing mutations in the presenilin 1 (PS1) gene develop early onset familial Alzheimer's disease (FAD). Further, some PS1 mutants enhance secretion of the longer form of amyloid beta protein (Abeta42). We constructed cDNAs encoding human PS1 harboring 28 FAD-linked mutations, and examined the effects of the expressed PS1 mutants on Abeta42 secretion in beta amyloid precursor producing COS-1 cells. All the mutants significantly enhanced the ratio of Abeta42 to total Abeta compared with wild-type PS1. However, the increase in Abeta42 ratio in cells with each PS1 mutation did not correlate with the reported age of onset of FAD caused by that mutation. These results suggest that increased Abeta42 secretion is important for the development of Alzheimer's disease (AD), but may not be the only factor contributing to the onset of AD.
Mutations of presenilin (PS)-1, an endoplasmic reticulum/Golgi transmembrane protein, have been associated with early-onset familial Alzheimer's disease (FAD). In mammalian brain, PS1 exists primarily as its processed fragments; however, the role of this cleavage event in PS1 function remains unclear. Although some investigators have shown that mutant PS1 processing is unaltered (with the exception of PS1-deltaE9, which lacks the cleavage site) in stably transfected cells and PS1-FAD transgenic mice, other investigators have reported altered FAD mutant PS1 and PS2 protein processing in transiently transfected cells and human FAD patients. The present study uses recombinant replication-defective adenoviral vectors to transiently express wild-type (WT) or mutant PS1 in various cells, including primary cultured hippocampal neurons. We show that in contrast to PS1-WT, overexpression of mutant PS1 results in an increased ratio of mutant holoprotein to endoproteolytic products that is dependent on cell type and differentiation state. In addition, mutant PS1 overexpression leads to an increase in caspase-type protease derived fragments above that seen with PS1-WT overexpression. Furthermore, overexpression of at least one mutant significantly alters the processing of coexpressed PS1-WT, suggesting that mutant PS1 may affect PS1-WT function. These findings suggest that a defect in PS1 holoprotein stability may be a general defect seen in cells expressing mutant PS1, especially neuronal cells, and may play a critical role in the pathogenesis of FAD.
1. Full-length form of human presenilin 1 (PS1) is processed and an N-terminal fragment (28 KD) and C-terminal fragment (19 KD) are generated. To elucidate the possible role of presenilin mutations in Alzheimer's disease (AD), the authors analyze the effects of AD-linked mutations on PS1 processing in cultured cells. 2. Complementary DNAs encoding genes for human PS1 harboring twenty-nine missense mutations linked with familial Alzheimer's disease (FAD) were introduced into PC12 cells. Human PS1 exogenously expressed in the cells was detected by immunoblotting using a monoclonal antibody that recognized the N-terminal region of human PS1. The amounts of full-length form (48 KD) and N-terminal fragment (28 KD) of PS1 was quantified by densitometrical analysis. 3. The ratio of the N-terminal fragment to total PS1 was reduced by twenty-nine mutations. The specific effects on PS1 processing varied according to mutation. 4. These results suggest that AD-linked missense mutations of PS1 are involved in neurodegeneration via inhibition of PS1 processing.
Alzheimer's disease (AD) is characterized by the invariant accumulation of senile plaques predominantly composed of the pathologically relevant 42-amino acid amyloid beta-peptide (Abeta42). The presenilin (PS) proteins play a key role in Abeta generation. FAD-associated mutations in PS1 and PS2 enhance the production of Abeta42, and PS1 is required for physiological Abeta production, since a gene knockout of PS1 and dominant negative mutations of PS1 abolish Abeta generation. PS proteins undergo endoproteolytic processing, and current evidence indicates that fragment formation may be required for the amyloidogenic function of PS. We have now determined the sequence requirements for endoproteolysis of PS1. Mutagenizing amino acids at the previously determined major cleavage site (amino acid 298) had no effect on PS1 endoproteolysis. In contrast, mutations or deletions at the additional cleavage site around amino acid 292 blocked endoproteolysis. The uncleavable PS1 derivatives accumulated as full-length proteins and replaced the endogenous PS1 proteins. In contrast to the previously described aspartate mutations within transmembrane domains 6 and 7, the uncleaved PS1 variants do not act as dominant negative inhibitors of Abeta production. Moreover, when a FAD-associated mutation (M146L) was combined with a mutation blocking endoproteolysis, Abeta42 production still reached pathological levels. These data therefore demonstrate that endoproteolysis of presenilins is not an absolute prerequisite for the amyloidogenic function of PS1. These data also show that accumulation of the PS1 holoprotein is not associated with the pathological activity of PS1 mutations as suggested previously.
We have investigated the subcellular distribution of presenilin-1 (PS1) and presenilin-2 (PS2) in a variety of mammalian cell lines. In Iodixanol-based density gradients, PS1 derivatives show a biphasic distribution, cofractionating with membranes containing ER-resident proteins and an additional population of membranes with low buoyant density that do not contain markers of the Golgi complex, ERGIC, COP II vesicles, ER exit compartment, COP II receptor, Golgi SNARE, trans-Golgi network, caveolar membranes, or endocytic vesicles. Confocal immunofluorescence and immunoelectron microscopy studies fully supported the fractionation studies. These data suggest that PS1 fragments accumulate in a unique subcompartment(s) of the ER or ER to Golgi trafficking intermediates. Interestingly, the FAD-linked PS1 variants show a marked redistribution toward the heavier region of the gradient. Finally, and in contrast to PS1, PS2 fragments are detected preponderantly in more densely sedimenting membranes, suggesting that the subcellular compartments in which these molecules accumulate are distinct.