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The mechanism of γ-Secretase dysfunction in familial Alzheimer disease

Mutations in presenilin (PSEN) and amyloid precursor protein (APP) cause dominant early-onset Alzheimer's disease (AD), but the mechanism involved is debated. Here, such mutations are shown to alter γ-secretase activity, leading to changes in Aβ peptide cleavage patterns.

The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid β (Aβ)42 relative to Aβ40 by an unknown, possibly gain-of-toxic-function, mechanism. However, many PSEN mutations paradoxically impair γ-secretase and ‘loss-of-function' mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aβ generation via three different mechanisms, resulting in qualitative changes in the Aβ profiles, which are not limited to Aβ42. Loss of ɛ-cleavage function is not generally observed among FAD mutants. On the other hand, γ-secretase inhibitors used in the clinic appear to block the initial ɛ-cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase-like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aβ products, and suggest fundamental improvements for current drug development efforts.

Alzheimer’s disease (AD) is an insidious and progressive disease with a genetically complex and heterogenous etiology. More than 200 fully penetrant mutations in the amyloid β-protein precursor (APP), presenilin 1 (or PSEN1), and presenilin 2 (PSEN2) have been linked to early-onset familial AD (FAD). 177 PSEN1 FAD mutations have been identified so far and account for more than ~80% of all FAD mutations. All PSEN1 FAD mutations can increase the Aβ42:Aβ40 ratio with seemingly different and incompletely understood mechanisms. A recent study has shown that the 286 amino acid N-terminal fragment of APP (N-APP), a proteolytic product of β-secretase-derived secreted form of APP (sAPPβ), could bind the death receptor, DR6, and lead to neurodegeneration. Here we asked whether PSEN1 FAD mutations lead to neurodegeneration by modulating sAPPβ levels. All four different PSEN1 FAD mutations tested (in three mammalian cell lines) did not alter sAPPβ levels. Therefore PS1 mutations do not appear to contribute to AD pathogenesis via altered production of sAPPβ.

Background: Autosomal dominant early onset Alzheimer's disease (ADEOAD) is genetically heterogeneous. Mutations of the amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) genes have been identified.

Objective: To further clarify the respective contribution of these genes to ADEOAD.

Methods: 31 novel families were investigated. They were ascertained using stringent criteria (the occurrence of probable or definite cases of Alzheimer's disease with onset before 60 years of age in three generations). All cases fulfilled the NINCDS-ADRDA criteria for probable or definite Alzheimer's disease. The entire coding regions of PSEN1 and PSEN2 genes and exons 16 and 17 of APP gene were sequenced from genomic DNA

Results: PSEN1 mutations, including eight previously unreported mutations, were detected in 24 of the 31 families, and APP mutations were found in five families. In this sample, the mean ages of disease onset in PSEN1 and APP mutation carriers were 41.7 and 51.2 years, respectively.

Conclusions: Combining these data with previously published data, yielding 65 ADEOAD families, 66% of the cases were attributable to PSEN1 mutations and 16% to APP mutations, while 18% remained unexplained.

Mutations in PSEN1 and PSEN2 genes account for the majority of cases of early-onset familial Alzheimer disease. Since the first prediction of a genetic link between PSEN1 and PSEN2 with Alzheimer's disease, many research groups from both academia and pharmaceutical industry have sought to unravel how pathogenic mutations in PSEN cause presenile dementia. PSEN genes encode polytopic membrane proteins termed presenilins (PS1 and PS2), which function as the catalytic subunit of γ-secretase, an intramembrane protease that has a wide spectrum of type I membrane protein substrates. Sequential cleavage of amyloid precursor protein by BACE and γ-secretase releases highly fibrillogenic β-amyloid peptides, which accumulate in the brains of aged individuals and patients with Alzheimer's disease. Familial Alzheimer's disease-associated presenilin variants are thought to exert their pathogenic function by selectively elevating the levels of highly amyloidogenic Aβ42 peptides. In addition to Alzheimer's disease, several recent studies have linked PSEN1 to familiar frontotemporal dementia. Here, we review the biology of PS1, its role in γ-secretase activity, and discuss recent developments in the cell biology of PS1 with respect to Alzheimer's disease pathogenesis.

Although mutations in three genes, amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2), have been identified as genetic causes of early-onset Alzheimer's disease (EOAD), there has been a single report on a PSEN1 mutation in Koreans. In the present study, we performed a genetic analysis of six Korean patients with EOAD. Direct sequencing analysis of the APP, PSEN1 and PSEN2 genes revealed two different mutations of the PSEN1 gene (G206S and M233T) and one mutation of the APP gene (V715M) in three patients with age-at-onset of 34, 35, and 42 yr, respectively. In addition, two patients with age-at-onset of 55 and 62 yr, respectively, were homozygous for APOE ε4 allele. One woman had no genetic alterations. These findings suggest that PSEN1 and APP gene mutations may not be uncommon in Korean patients with EOAD and that genetic analysis should be provided to EOAD patients not only for the identification of their genetic causes but also for the appropriate genetic counseling.

Late-onset Alzheimer’s disease is a common complex disorder of old age. Though these types of disorders can be highly heritable, they differ from single-gene (Mendelian) diseases in that their causes are often multifactorial with both genetic and environmental components. Genetic risk factors that have been firmly implicated in the cause are mutations in the amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes, which are found in large multi-generational families with an autosomal dominant pattern of disease inheritance, the apolipoprotein E (APOE)ε4 allele and the sortilin-related receptor (SORL1) gene. Environmental factors that have been associated with late-onset Alzheimer’s disease include depressive illness, various vascular risk factors, level of education, head trauma and estrogen replacement therapy. This complexity may help explain their high prevalence from an evolutionary perspective, but the etiologic complexity makes identification of disease-related genes much more difficult. The “endophenotype” approach is an alternative method for measuring phenotypic variation that may facilitate the identification of susceptibility genes for complexly inherited traits. The usefulness of endophenotypes in genetic analyses of normal brain morphology and, in particular for Alzheimer’s disease will be reviewed as will the implications of these findings for models of disease causation. Given that the pathways from genotypes to end-stage phenotypes are circuitous at best, identifying endophenotypes more proximal to the effects of genetic variation may expedite the attempts to link genetic variants to disorders.

Background

Mutations in either Aβ Precursor protein (APP) or genes that regulate APP processing, such as BRI2/ITM2B and PSEN1/PSEN2, cause familial dementias. Although dementias due to APP/PSEN1/PSEN2 mutations are classified as familial Alzheimer disease (FAD) and those due to mutations in BRI2/ITM2B as British and Danish dementias (FBD, FDD), data suggest that these diseases have a common pathogenesis involving toxic APP metabolites. It was previously shown that FAD mutations in APP and PSENs promote activation of caspases leading to the hypothesis that aberrant caspase activation could participate in AD pathogenesis.

Results

Here, we tested whether a similar mechanism applies to the Danish BRI2/ITM2B mutation. We have generated a genetically congruous mouse model of FDD, called FDDKI, which presents memory and synaptic plasticity deficits. We found that caspase-9 is activated in hippocampal synaptic fractions of FDDKI mice and inhibition of caspase-9 activity rescues both synaptic plasticity and memory deficits.

Conclusion

These data directly implicate caspase-9 in the pathogenesis of Danish dementia and suggest that reducing caspase-9 activity is a valid therapeutic approach to treating human dementias.

Background

Mutations in presenilin-1 (Psen1) cause familial Alzheimer's disease (FAD). Both hypoxia and ischemia have been implicated in the pathological cascade that leads to amyloid deposition in AD. Here we investigated whether Psen1 might regulate hypoxic responses by modulating induction of the transcription factor hypoxia inducible factor 1-α (HIF-1α).

Results

In fibroblasts that lack Psen1 induction of HIF-1α was impaired in response to the hypoxia mimetic cobalt chloride, as well as was induction by insulin and calcium chelation. Reintroduction of human Psen1 using a lentiviral vector partially rescued the responsiveness of Psen1-/- fibroblasts to cobalt chloride induction. HIF-1α induction did not require Psen1's associated γ-secretase activity. In addition, the failure of insulin to induce HIF-1α was not explicable on the basis of failed activation of the phosphatidylinositol 3-kinase (PI3K/Akt) pathway which activated normally in Psen1-/- fibroblasts. Rather we found that basal levels of HIF-1α were lower in Psen1-/- fibroblasts and that the basis for lower constitutive levels of HIF-1α was best explained by accelerated HIF-1α degradation. We further found that Psen1 and HIF-1α physically interact suggesting that Psen1 may protect HIF-1α from degradation through the proteasome. In fibroblasts harboring the M146V Psen1 FAD mutation on a mouse Psen1 null background, metabolic induction of HIF-1α by insulin was impaired but not hypoxic induction by cobalt chloride. Unlike Psen1-/- fibroblasts, basal levels of HIF-1α were normal in FAD mutant fibroblasts but activation of the insulin-receptor pathway was impaired. Interestingly, in Psen1-/- primary neuronal cultures HIF-1α was induced normally in response to cobalt chloride but insulin induction of HIF-1α was impaired even though activation of the PI3K/Akt pathway by insulin proceeded normally in Psen1-/- neuronal cultures. Basal levels of HIF-1α were not significantly different in Psen1-/- neurons and HIF-1α levels were normal in Psen1-/- embryos.

Conclusions

Collectively these studies show that Psen1 regulates induction of HIF-1α although they indicate that cell type specific differences exist in the effect of Psen1 on induction. They also show that the M146V Psen1 FAD mutation impairs metabolic induction of HIF-1α, an observation that may have pathophysiological significance for AD.

Beta amyloid peptides (Aβ) play a key role in the pathogenesis of Alzheimer disease (AD). Presenilins (PS) function as the catalytic subunits of γ-secretase, the enzyme that releases Aβ from ectodomain cleaved amyloid precursor protein (APP) by intramembrane proteolysis. Familial Alzheimer disease (FAD)-linked PSEN mutations alter APP processing in a manner that increases the relative abundance of longer Aβ42 peptides to that of Aβ40 peptides. The mechanisms by which Aβ40 and Aβ42 peptides are produced in a ratio of ten to one by wild type presenilin (PS) and by which Aβ42 is overproduced by FAD-linked PS variants are not completely understood. We generated chimeras of the amyloid precursor protein C-terminal fragment (C99) and PS to address this issue. We found a chimeric protein where C99 is fused to the PS1 N-terminus undergoes in cis processing to produce Aβ and that a fusion protein harboring FAD-linked PS1 mutations overproduced Aβ42. To change the molecular interactions within the C99-PS1 fusion protein, we made sequential deletions of the junction between C99 and PS1. We found differential effects of deletion in C99-PS1 on Aβ40 and 42 production. Deletion of the junction between APP CTF and PS1 in the fusion protein decreased Aβ40, while it did not decrease Aβ42 production in the presence or absence of FAD-linked PS1 mutation. These results are consistent with the idea that the APP/PS interaction is differentially regulated during Aβ40 and 42 production.

Background

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.

Methodology/Principal Findings

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.

Conclusion/Significance

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.

Early-onset dominantly inherited forms of Alzheimer's disease (AD) are rare, but studies of such cases have revealed important information about the disease mechanisms. Importantly, mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) and PSEN2, alter the APP processing and lead to an increased amyloid β-peptide (Aβ) 42/40 ratio. This, together with other studies on pathogenic mechanisms, show that Aβ42 is a major player in the etiology of AD. Here, we present a clinical and neuropathological description of a Swedish family with an I143T mutation in the PSEN1 gene, which gives rise to a severe form of AD. We also performed an extensive investigation on the concentration and distribution of Aβ species of different lengths in six brain regions from two mutation carriers. Our study showed that Aβ42 and a longer peptide, Aβ43, were present both in plaque cores and in total amyloid preparations, and were each clearly more frequent than Aβ40 in all examined regions, as shown by both mass spectrometry and immunohistochemistry.

Objective

Early onset familial Alzheimer’s disease (EOFAD) can be caused by mutations in genes for amyloid precursor protein (APP), presenilin 1 (PSEN1) or presenilin 2 (PSEN2). There is considerable phenotypic variability in EOFAD, including some patients with spastic paraparesis. The objective is to describe clinical and neuropathologic features of a family with a PSEN1 mutation that has been reported previously, without autopsy confirmation, in a single Greek family whose affected members presented with memory loss in their thirties, as well as variable limb spasticity and seizures.

Methods

We prospectively evaluated two children (son and daughter) with EOFAD and reviewed medical records on their mother. Archival material from the autopsy of the mother was reviewed and postmortem studies were performed on the brain of the daughter.

Results

All three individuals in this family had disease onset in their thirties, with cognitive deficits in multiple domains, including memory, language and attention, as well as less common features such as spastic dysarthria, limb spasticity and seizures. At autopsy both the mother and her daughter had pathologic findings of AD, as well as histological evidence of corticospinal tract degeneration. Genetic studies revealed a mutation in PSEN1 leading to an asparagine to serine substitution at amino acid residue 135 (N135S) in presenilin-1.

Conclusions

This is the first description of neuropathologic findings in EOFAD due to N135S PSEN1 mutation. The clinical phenotype was remarkable for spastic dysarthria, limb spasticity and seizures, in addition to more typical features of EOFAD.

Background

Mutations in the presenilin (PSEN) genes are associated with early-onset familial Alzheimer's disease (FAD). Biochemical characterizations and comparisons have revealed that many PSEN mutations alter γ-secretase activity to promote accumulation of toxic Aβ42 peptides. In this study, we compared the histopathologic and biochemical profiles of ten FAD cases expressing independent PSEN mutations and determined the degradation patterns of amyloid-β precursor protein (AβPP), Notch, N-cadherin and Erb-B4 by γ-secretase. In addition, the levels of Aβ40/42 peptides were quantified by ELISA.

Results

We observed a wide variation in type, number and distribution of amyloid deposits and neurofibrillary tangles. Four of the ten cases examined exhibited a substantial enrichment in the relative proportions of Aβ40 over Aβ42. The AβPP N-terminal and C-terminal fragments and Tau species, assessed by Western blots and scanning densitometry, also demonstrated a wide variation. The Notch-1 intracellular domain was negligible by Western blotting in seven PSEN cases. There was significant N-cadherin and Erb-B4 peptide heterogeneity among the different PSEN mutations.

Conclusion

These observations imply that missense mutations in PSEN genes can alter a range of key γ-secretase activities to produce an array of subtly different biochemical, neuropathological and clinical manifestations. Beyond the broad common features of dementia, plaques and tangles, the various PSEN mutations resulted in a wide heterogeneity and complexity and differed from sporadic AD.

Disturbances in intracellular calcium homeostasis are likely prominent and causative factors leading to neuronal cell death in Alzheimer's disease (AD). Familial AD (FAD) is early-onset and exhibits autosomal dominant inheritance. FAD-linked mutations have been found in the genes encoding the presenilins and amyloid precursor protein (APP). Several studies have shown that mutated presenilin proteins can directly affect calcium release from intracellular stores independently of Aβ production. Although less well established, there is also evidence that APP may directly modulate intracellular calcium homeostasis. Here, we directly examined whether overexpression of FAD-linked APP mutants alters intracellular calcium dynamics. In contrast to previous studies, we found that overexpression of mutant APP has no effects on basal cytosolic calcium, ER calcium store size or agonist-induced calcium release and subsequent entry. Thus, we conclude that mutated APP associated with FAD has no direct effect on intracellular calcium homeostasis independently of Aβ production.

Mutations in presenilin-1 (PS1) and presenilin-2 (PS2) cause familial Alzheimer’s disease (FAD). Presenilins influence multiple molecular pathways and are best known for their role in the γ-secretase cleavage of type I transmembrane proteins including the amyloid precursor protein (APP). PS1 and PS2 FAD mutant transgenic mice have been generated using a variety of promoters. PS1-associated FAD mutations have also been knocked into the endogenous mouse gene. PS FAD mutant mice consistently show elevations of Aβ42 with little if any effect on Aβ40. When crossed with plaque forming APP FAD mutant lines, the PS1 FAD mutants cause earlier and more extensive plaque deposition. Although single transgenic PS1 or PS2 mice do not form plaques, they exhibit a number of pathological features including age-related neuronal and synaptic loss as well as vascular pathology. They also exhibit increased susceptibility to excitotoxic injury most likely on the basis of exaggerated calcium release from the endoplasmic reticulum. Electrophysiologically long-term potentiation in the hippocampus is increased in young PS1 FAD mutant mice but this effect appears to be lost with aging. In most studies neurogenesis in the adult hippocampus is also impaired by PS1 FAD mutants. Mice in which PS1 has been conditionally knocked out in adult forebrain on a PS2 null background (PS1/2 cDKO) develop a striking neurodegeneration that mimics AD neuropathology in being associated with neuronal and synaptic loss, astrogliosis and hyperphosphorylation of tau, although it is not accompanied by plaque deposits. The relevance of PS transgenic mice as models of AD is discussed.

Background

Both familial and sporadic Alzheimer's disease (AD) result in progressive cortical and subcortical atrophy. Familial autosomal dominant AD (FAD) allows us to study AD brain changes presymptomatically.

Methods

33 subjects at risk for FAD (25 for PSEN1 and 8 for APP mutations; 22 mutation carriers and 11 controls) and 3 demented PSEN1 mutation carriers underwent T1-weighted MPRAGE 1.5T MRI. Using the hippocampal radial distance and cortical pattern matching techniques, we investigated the effects of carrier status and dementia diagnosis on cortical and hippocampal atrophy. All analyses were corrected for age and relative age (years to median age of disease onset in the family).

Results

The dementia cases had pronounced cortical atrophy in the lateral and medial parietal, posterior cingulate and frontal cortices and hippocampal atrophy bilaterally relative to both nondemented carriers and controls. Nondemented carriers did not show significant cortical thinning or hippocampal atrophy relative to controls.

Conclusions

FAD is associated with thinning of the posterior association and frontal cortices and hippocampal atrophy. Larger sample sizes may be necessary to reliably identify cortical atrophy in presymptomatic carriers.

The neurodegenerative disorder Alzheimer’s disease (AD) is the 6th leading cause of death in the USA. In addition to neurological and psychiatric symptoms, AD is characterized by deficiencies in S–adenylmethionine (SAM), vitamin B12, and folate. Deficiency in these nutrients has been shown to result in gene promoter methylation with upregulation of AD–associated genes. While some cases of AD are due to specific mutations in genes such as presenilin 1 (PSEN) and the amyloid–β peptide precursor protein (APP), these familial AD (FAD) cases account for a minority of cases. The majority of genetic contribution consists of risk factors with incomplete penetrance. Several environmental risk factors, such as cholesterol and diet, head trauma, and reduced levels of exercise, have also been determined for AD. Nevertheless, the majority of risk for AD appears to be established early in life. We propose to explain this via the LEARn (Latent Early–life Associated Regulation) model. LEARn-AD (LAD) would be a “two-hit” disorder, wherein the first hit would occur due to environmental stress within the regulatory sequences of AD–associated genes, maintained by epigenetic changes such as in DNA methylation. This hit would most likely come in early childhood. The second hit could consist of further stress, such as head trauma, poor mid–life diet, or even general changes in expression of genes that occur later in life independent of any pathogenesis. Given that the primary risk for LAD would be maintained by DNA (hypo)methylation, we propose that long–term nutritional remediation based on the LEARn model, or LEARn–based nutritional gain (LEARnING), beginning early in life, would significantly reduce risk for AD late in life.

Alzheimer's disease is the leading cause of cognitive loss and neurodegeneration in the developed world. Although its genetic and environmental causes are not generally known, familial forms of the disease (FAD) are due to mutations in a single copy of the Presenilin (PS) and Amyloid Precursor Protein (APP) genes. The dominant inheritance pattern of FAD indicates that it may be due to gain or change of function mutations. Studies of FAD-linked forms of presenilin in model organisms, however, indicate that they are loss of function, leading to the possibility that a reduction in PS activity might contribute to FAD and that proper psn levels are important for maintaining normal cognition throughout life. To explore this issue further, we have tested the effect of reducing psn activity during aging in Drosophila melanogaster males. We have found that flies in which the dosage of psn function is reduced by 50% display age-onset impairments in learning and memory. Treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium during the aging process prevented the onset of these deficits, and treatment of aged flies reversed the age-dependent deficits. Genetic reduction of DmGluRA, the inositol trisphosphate receptor (InsP3R) or IPPase also prevented these age-onset cognitive deficits. These findings suggest that reduced psn activity may contribute to the age onset cognitive loss observed with FAD. They also indicate that enhanced mGluR signaling and calcium release regulated by InsP3R as underlying causes of the age-dependent cognitive phenotypes observed when psn activity is reduced.

Overexpression of amyloid precursor protein (APP), as well as mutations in the APP and presenilin genes, causes rare forms of Alzheimer’s disease (AD). These genetic changes have been proposed to cause AD by elevating levels of amyloid-β peptides (Aβ), which are thought to be neurotoxic. Since overexpression of APP also causes defects in axonal transport, we tested whether defects in axonal transport were the result of Aβ poisoning of the axonal transport machinery. Because directly varying APP levels also alters APP domains in addition to Aβ, we perturbed Aβ generation selectively by combining APP transgenes in Drosophila and mice with presenilin-1 (PS1) transgenes harboring mutations that cause familial AD (FAD). We found that combining FAD mutant PS1 with FAD mutant APP increased Aβ42/Aβ40 ratios and enhanced amyloid deposition as previously reported. Surprisingly, however, this combination suppressed rather than increased APP-induced axonal transport defects in both Drosophila and mice. In addition, neuronal apoptosis induced by expression of FAD mutant human APP in Drosophila was suppressed by co-expressing FAD mutant PS1. We also observed that directly elevating Aβ with fusions to the Familial British and Danish Dementia-related BRI protein did not enhance axonal transport phenotypes in APP transgenic mice. Finally, we observed that perturbing Aβ ratios in the mouse by combining FAD mutant PS1 with FAD mutant APP did not enhance APP-induced behavioral defects. A potential mechanism to explain these findings was suggested by direct analysis of axonal transport in the mouse, which revealed that axonal transport or entry of APP into axons is reduced by FAD mutant PS1. Thus, we suggest that APP-induced axonal defects are not caused by Aβ.

Background

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD). Some instances of FAD have been linked to mutations in the β-amyloid protein precursor (APP). Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated.

Results

Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Aβ)40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Aβ42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP) in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP.

Conclusion

This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue-specific expression in the two transgenic rat lines and in wild-type rats contradicts our current understanding of APP gene regulation. Determination of the elements that are responsible for tissue-specific expression of APP may enable new treatment options for AD.

Autosomal dominant familial Alzheimer's disease (FAD) of young onset due to alterations in the PSEN1, APP, and PSEN2 genes is a fully-penetrant and devastating condition. As the subsequent development of AD in persons inheriting such genes is essentially certain, the condition provides a unique opportunity to perform informative studies of interventions with potential for preventing the disease. Though feasible, there are many challenges to such an endeavor including the fact that most persons at-risk for FAD do not desire to know their genetic status. Other challenges include the time course over which a preventative treatment would need to be administered and potential limitations to the degree to which the knowledge gained might be validly generalized to the more common late-onset AD. In this paper we discuss issues of study design including power estimates, protocols in which subjects' genetic status is not revealed to them, and the advantage of one-time interventions such as vaccinations. Though addressed in the context of FAD, many of the issues discussed are relevant to other fully-penetrant autosomal dominant degenerative illnesses such as Huntington's disease. We also discuss important next steps including the performance of pre-clinical studies in model systems appropriate for FAD and the recently funded international Dominantly Inherited Alzheimer Network (DIAN). The goals of the DIAN are to characterize the natural history of FAD and to establish the infrastructure that would be required to perform meaningful studies in this rare, widely dispersed, but informative population.

Alzheimer’s disease (AD) can be divided into sporadic AD (SAD) and familial AD (FAD). Most AD cases are sporadic and may result from multiple etiologic factors, including environmental, genetic and metabolic factors, whereas FAD is caused by mutations of presenilins or amyloid-β (Aβ) precursor protein (APP). A commonly used mouse model for AD is 3xTg-AD mouse, which is generated by over-expression of mutated presenilin 1, APP and tau in the brain and thus represents a mouse model of FAD. A mouse model generated by intracerebroventricular (icv) administration of streptozocin (STZ), icv-STZ mouse, shows many aspects of SAD. Despite the wide use of these two models for AD research, differences in gene expression between them are not known. Here, we compared the expression of 84 AD-related genes in the hippocampus and the cerebral cortex between icv-STZ mice and 3xTg-AD mice using a custom-designed qPCR array. These genes are involved in APP processing, tau/cytoskeleton, synapse function, apoptosis and autophagy, AD-related protein kinases, glucose metabolism, insulin signaling, and mTOR pathway. We found altered expression of around 20 genes in both mouse models, which affected each of above categories. Many of these gene alterations were consistent with what was observed in AD brain previously. The expression of most of these altered genes was decreased or tended to be decreased in the hippocampus of both mouse models. Significant diversity in gene expression was found in the cerebral cortex between these two AD mouse models. More genes related to synaptic function were dysregulated in the 3xTg-AD mice, whereas more genes related to insulin signaling and glucose metabolism were down-regulated in the icv-STZ mice. The present study provides important fundamental knowledge of these two AD mouse models and will help guide future studies using these two mouse models for the development of AD drugs.

β-amyloid precursor protein (APP) and presenilins mutations cause early-onset familial Alzheimer’s disease (FAD). Some FAD-based mouse models produce amyloid plaques, others don’t. β-Amyloid (Aβ) deposition can manifest as compact and diffuse plaques; it is unclear why the same Aβ molecules aggregate in different patterns. Is there a basic cellular process governing Aβ plaque pathogenesis? We showed in some FAD mouse models that compact plaque formation is associated with a progressive axonal pathology inherent with increased expression of β-secretase (BACE1), the enzyme initiating the amyloidogenic processing of APP. A monoclonal Aβ antibody, 3D6, visualized distinct axon terminal labeling before plaque onset. The present study was set to understand BACE1 and axonal changes relative to diffuse plaque development and to further characterize the novel axonal Aβ antibody immunoreactivity (IR), using triple transgenic AD (3xTg-AD) mice as experimental model. Diffuse-like plaques existed in the forebrain in aged transgenics and were regionally associated with increased BACE1 labeled swollen/sprouting axon terminals. Increased BACE1/3D6 IR at axon terminals occurred in young animals before plaque onset. These axonal elements were also co-labeled by other antibodies targeting the N-terminal and mid-region of Aβ domain and the C-terminal of APP, but not co-labeled by antibodies against the Aβ C-terminal and APP N-terminal. The results suggest that amyloidogenic axonal pathology precedes diffuse plaque formation in the 3xTg-AD mice, and that the early-onset axonal Aβ antibody IR in transgenic models of AD might relate to a cross-reactivity of putative APP β-carboxyl terminal fragments.

The contribution of mutations in amyloid precursor protein (APP) and presenilin (PSEN) to familial Alzheimer's disease (AD) is well established. However, little is known about the molecular mechanisms leading to amyloid beta (Aβ) generation in sporadic AD. Increased brain ceramide levels have been associated with sporadic AD, and are a suggested risk factor. Serine palmitoyltransferase (SPT) is the first rate limiting enzyme in the de novo ceramide synthesis. However, the regulation of SPT is not yet understood. Evidence suggests that it may be post-transcriptionally regulated. Therefore, we investigated the role of miRNAs in the regulation of SPT and amyloid beta (Aβ) generation. We show that serine palmitoyltransferase (SPT) is upregulated in a subgroup of sporadic AD patient brains. This is further confirmed in mouse model studies of risk factors associated with AD. We identified that the loss of miR-137, -181c, -9 and 29a/b-1 increases SPT and in turn Aβ levels, and provides a mechanism for the elevated risk of AD associated with age, high saturated fat diet and gender. Finally, these results suggest SPT and the respective miRNAs may be potential therapeutic targets for sporadic AD.

Background

Aggregatable amyloid β-peptide (Aβ) and non-aggregatable p3-Alcα are metabolic products of the γ-secretase cleavage of amyloid β-protein precursor (APP) and Alcadeinα (Alcα), respectively. Familial AD (FAD) -linked mutations in the presenilin 1 or 2 (PS1 or PS2) component of γ-secretase can cause alternative intramembranous processing of APP and Alcα, leading to a coordinated generation of variants of both Aβ and p3-Alcα. Variant Alcα peptides have been observed in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment and sporadic Alzheimer's disease (AD). Since, like APP, Alcα is largely expressed in brain, one might predict that alternative processing of Alcα would be reflected in body fluids of some AD patients. These patients with misprocessing of multiple γ-secretase substrates might define an endophenotype of p3-Alcα, in whom AD is due either to dysfunction of γ-secretase or to a disorder of the clearance of hydrophobic peptides such as those derived from transmembrane domains.

Results

We developed a simple procedure for extraction of p3-Alcα from plasma and for analyzing this extract in a sensitive, p3-Alcα-specific sandwich enzyme-linked immunosorbent assay (ELISA) system. Plasma p3-Alcα levels and Aβ40 levels were examined in sporadic AD subjects from two independent Japanese cohorts. In some of these patients, levels of plasma p3-Alcα were significantly higher, and were accompanied by parallel changes in Aβ40 levels. This AD-related difference was more marked in female subjects, but this phenomenon was not observed in subjects with frontotemporal lobar degeneration (FTLD).

Conclusion

Reagents and procedures have been established that enable extraction of p3-Alcα from plasma and for quantification of plasma p3-Alcα levels by ELISA. Some populations of AD subjects apparently show increased levels of both p3-Alcα and Aβ40. Quantification of p3-Alcα level may be useful as a readily accessible biomarker for a population of sporadic AD patients in which disease pathogenesis is associated with either dysfunction of γ-secretase or with a disorder of the clearance of transmembrane domain-derived peptides.