Metals accelerate production of the aberrant splicing isoform of the presenilin-2

Oxidative stress is a major risk factor for Alzheimer's disease (AD) and other neurodegenerative disorders. Metals are known to be one of the factors that contribute to oxidative stress. Recently, we reported that the aberrant splicing isoform (PS2V) generated by skipping exon5 of the presenilin-2 (PS2) gene is a diagnostic feature of sporadic AD (SAD). PS2V is inducible by exposure of human neuroblastoma to hypoxia. We examined whether this aberrant splicing was caused by metal-induced oxidative stress, such as exposure to aluminum. As a result, we demonstrated that exposure to aluminum accelerated PS2V production induced by hypoxia. This acceleration of the production of PS2V to hypoxia was caused by chronic aluminum exposure, but was not related to the intracellular content of aluminum. HMGA1a is a mediator of PS2V production, and it was induced by aluminum as well as by hypoxia. Induction of HMGA1a was increased by chronic exposure to aluminum, and a nuclear extract containing HMGA1a bound to a specific sequence on exon5 of PS2 pre-mRNA, as reported previously. Finally, the acceleration of PS2V production induced by aluminum under hypoxic conditions reflected, but has not yet been directly shown to cause, vulnerability to endoplasmic reticulum stress. These results suggest that exposure to some metals can accelerate and enhance PS2V generation, and that hypoxia plus chronic exposure to metals may promote the development of AD.     

The Alzheimer's disease-associated presenilins are differentially phosphorylated proteins located predominantly within the endoplasmic reticulum.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of extracellular senile plaques composed of amyloid beta-peptide (A beta). Whereas most cases of AD occur sporadically, about 10% of AD cases are inherited as a fully penetrant autosomal dominant trait. Mutations in the recently cloned Presenilin genes (PS-1 and PS-2) are by far the most common cause of early onset familial AD. MATERIALS AND METHODS: Cellular expression of endogenous and overexpressed PS proteins was analyzed by immunocytochemistry and metabolic labeling followed by immunoprecipitation. In vivo phosphorylation sites of PS proteins were analyzed by extensive mutagenesis. RESULTS: PS-1 as well as PS-2 proteins were localized predominantly within the endoplasmic reticulum (ER). However, small amounts of the PS proteins were detected within the Golgi compartment, where they colocalize with the beta-amyloid precursor protein (beta APP). The PS-2 protein was found to be highly phosphorylated, whereas very little phosphorylation was observed for PS-1. The selective phosphorylation of PS-2 occurs exclusively on serine residues. In vivo phosphorylation of PS-2 was mapped to serine residues 7, 9, and 19 within an acidic stretch at the N terminus, which is absent in PS-1. casein kinase (CK)-1 and CK-2 were shown to phosphorylate the N terminus of PS-2 in vitro. CONCLUSIONS: The majority of PS proteins were detected in the ER where little if any proteolytic processing of beta APP was reported. ER retention of PS proteins might occur by intramolecular aggregation. Small amounts of PS proteins were also detected in the Golgi where they colocalized with beta APP. This might suggest that potential interactions between PS proteins and beta APP could occur within the Golgi. Selective phosphorylation of PS-2 proteins within the acidic domain missing in PS-1 indicates differences in the biological functions and regulation of the two highly homologous proteins.     

Collapsin response mediator protein-2 hyperphosphorylation is an early event in Alzheimer's disease progression

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that can regulate microtubule assembly in neurons. This function of CRMP2 is regulated by phosphorylation by glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (Cdk5). Here, using novel phosphospecific antibodies, we demonstrate that phosphorylation of CRMP2 at Ser522 (Cdk5-mediated) is increased in Alzheimer's disease (AD) brain, while CRMP2 expression and phosphorylation of the closely related isoform CRMP4 are not altered. In addition, CRMP2 phosphorylation at the Cdk5 and GSK3 sites is increased in cortex and hippocampus of the triple transgenic mouse [presenilin-1 (PS1)(M146V)KI; Thy1.2-amyloid precursor protein (APP)(swe); Thy1.2tau(P301L)] that develops AD-like plaques and tangles, as well as the double (PS1(M146V)KI; Thy1.2-APP(swe)) transgenic mouse. The hyperphosphorylation is similar in magnitude to that in human AD and is evident by 2 months of age, ahead of plaque or tangle formation. Meanwhile, there is no change in CRMP2 phosphorylation in two other transgenic mouse lines that display elevated amyloid beta peptide levels (Tg2576 and APP/amyloid beta-binding alcohol dehydrogenase). Similarly, CRMP2 phosphorylation is normal in hippocampus and cortex of Tau(P301L) mice that develop tangles but not plaques. These observations implicate hyperphosphorylation of CRMP2 as an early event in the development of AD and suggest that it can be induced by a severe APP over-expression and/or processing defect.     

Molecular genetics of Alzheimer's disease: presenilin 1 gene analysis in a cohort of patients from the Poznań region

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and personality changes. Pathological hallmarks of AD are: deposition of amyloid plaques and neurofibrillary tangles in the brain, accompanied by neuronal and synaptic loss. The genetic background of AD is heterogeneous and strongly depends on the form of the disease. In most of the families with early-onset AD (EOAD) (10% of the total population of patients), the disease segregates as an autosomal dominant fully penetrant trait. To date, some missense mutations in three genes encoding the amyloid precursor protein, presenilin 1 (PS1) and 2 (PS2) have been found to cause familial EOAD. We screened for mutations in the presenilin genes in a sample of 55 patients with familial or sporadic form of EOAD from the Poznan region. We found 4 missense mutations in the PS1 gene: A246E in exon 7, P267L in exon 8, E318G in exon 9, and L424R in exon 12 among 5 unrelated patients. The frequency of PS1 mutations was 11% (5 of 55) in the whole sample of the patients with EOAD or 50% (3 of 6) if the analysis was restricted to familial cases with a positive history of dementia in the patient's family.     

Presenilin 1 interacts with acetylcholinesterase and alters its enzymatic activity and glycosylation

Presenilin 1 (PS1) plays a critical role in the gamma-secretase processing of the amyloid precursor protein to generate the beta-amyloid peptide, which accumulates in plaques in the pathogenesis of Alzheimer's disease (AD). Mutations in PS1 cause early onset AD, and proteins that interact with PS1 are of major functional importance. We report here the coimmunoprecipitation of PS1 and acetylcholinesterase (AChE), an enzyme associated with amyloid plaques. Binding occurs through PS1 N-terminal fragment independent of the peripheral binding site of AChE. Subcellular colocalization of PS1 and AChE in cultured cells and coexpression patterns of PS1 and AChE in brain sections from controls and subjects with sporadic or familial AD indicated that PS1 and AChE are located in the same intracellular compartments, including the perinuclear compartments. A PS1-A246E pathogenic mutation expressed in transgenic mice leads to decreased AChE activity and alteration of AChE glycosylation and the peripheral binding site, which may reflect a shift in protein conformation and disturbed AChE maturation. In both the transgenic mice and humans, mutant PS1 impairs coimmunoprecipitation with AChE. The results indicate that PS1 can interact with AChE and influence its expression, supporting the notion of cholinergic-amyloid interrelationships.     

The Senescence Hypothesis of Disease Progression in Alzheimer Disease: an Integrated Matrix of Disease Pathways for FAD and SAD

Alzheimer disease (AD) is a progressive, neurodegenerative disease characterised in life by cognitive decline and behavioural symptoms and post-mortem by the neuropathological hallmarks including the microtubule-associated protein tau-reactive tangles and neuritic plaques and amyloid-beta-protein-reactive senile plaques. Greater than 95 % of AD cases are sporadic (SAD) with a late onset and <5 % of AD cases are familial (FAD) with an early onset. FAD is associated with various genetic mutations in the amyloid precursor protein (APP) and the presenilins (PS)1 and PS2. As yet, no disease pathway has been fully accepted and there are no treatments that prevent, stop or reverse the cognitive decline associated with AD. Here, we review and integrate available environmental and genetic evidence associated with all forms of AD. We present the senescence hypothesis of AD progression, suggesting that factors associated with AD can be seen as partial stressors within the matrix of signalling pathways that underlie cell survival and function. Senescence pathways are triggered when stressors exceed the cells ability to compensate for them. The APP proteolytic system has many interactions with pathways involved in programmed senescence and APP proteolysis can both respond to and be driven by senescence-associated signalling. Disease pathways associated with sporadic disease may be different to those involving familial genetic mutations. The interpretation we provide strongly points to senescence as an additional underlying causal process in dementia progression in both SAD and FAD via multiple disease pathways.     

Effects of sodium butyrate on the synthesis of human pregnancy-specific beta 1-glycoprotein

Human pregnancy-specific beta 1-glycoprotein (PS beta G) is a polymorphic placental protein which shows strong sequence similarity with the oncofetal protein, carcinoembryonic antigen. To better understand the role of PS beta G in pregnancy, we examined its synthesis and regulation in placental fibroblasts, which had been shown to express the PS beta G gene. The major placental PS beta G is a 72-kDa glycoprotein, while the major fibroblast PS beta G is a 62-kDa species. Administration of sodium butyrate to these fibroblasts slightly stimulated the synthesis of the 62-kDa species but markedly increased the production of two additional PS beta Gs of 72 and 48 kDa. The similarity between the PS beta Gs synthesized by butyrate-treated fibroblasts and human placenta was confirmed by cell-free protein synthesis. Poly(A)+ RNA from butyrate-treated fibroblasts and placenta directed the synthesis of two polypeptides of 48 and 36 kDa, which form the polypeptide backbone of the 72- and 48-kDa glycoproteins. Moreover, the predicted molecular weights of PS beta Gs encoded by the two types of PS beta G cDNA clones were 48,000 and 36,000. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity at the 5' region (designated PSG-5') but differ in sequences at their 3' regions. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases from placental fibroblasts. Butyrate increased the steady-state levels of all three mRNAs. Ribonuclease protection analysis showed that butyrate increased the PS beta G mRNAs containing the PSG-5' or PSG93-specific sequence to approximately 20% of human placental levels. However, unlike human term placenta, which predominantly expressed PS beta G mRNAs with 3'-sequences similar to PSG16/PSG93, the butyrate-treated fibroblasts expressed roughly equal levels of PS beta G mRNAs with the PSG16/PSG93-3' and PSG95-3' ends. All PS beta G cDNAs identified encode proteins with distinct carboxyl termini, suggesting that the composition of the 72-kDa species in placenta and butyrate-treated fibroblasts is likely to be different. Placental fibroblasts provide a unique model for the study of the mechanisms responsible for the differential expression of the PS beta G gene.     

Interaction between presenilin 1 and ubiquilin 1 as detected by fluorescence lifetime imaging microscopy and a high-throughput fluorescent plate reader

Presenilin 1 (PS1) in its active heterodimeric form is the catalytic center of the gamma-secretase complex, an enzymatic activity that cleaves amyloid precursor protein (APP) to produce amyloid beta (Abeta). Ubiquilin 1 is a recently described PS1 interacting protein, the overexpression of which increases PS1 holoprotein levels and leads to reduced levels of functionally active PS1 heterodimer. In addition, it has been suggested that splice variants of the UBQLN1 gene are associated with an increased risk of developing Alzheimer disease (AD). However, it is still unclear whether PS1 and ubiquilin 1 interact when expressed at endogenous levels under normal physiological conditions. Here, we employ three novel fluorescence resonance energy transfer-based techniques to investigate the interaction between PS1 and ubiquilin 1 in intact cells. We consistently find that the ubiquilin 1 N terminus is in close proximity to several epitopes on PS1. We show that ubiquilin 1 interacts both with PS1 holoprotein and heterodimer and that the interaction between PS1 and ubiquilin 1 takes place near the cell surface. Furthermore, we show that the PS1-ubiquilin 1 interaction can be detected between endogenous proteins in primary neurons in vitro as well as in brain tissue of healthy controls and Alzheimer disease patients, providing evidence of its physiological relevance.     

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.     

Effects of human presenilin 1 isoforms on proliferation and survival of rat pheochromocytoma cell line PC12

Missense mutations in human presenilin 1 gene (hPS1) cause an autosomal dominant, early onset form of Alzheimer's disease (AD). To study effects of mutant presenilin on processes of cell growth, differentiation, and susceptibility to apoptotic signals, we produced a series of rat pheochromocytoma PC12 poly- and monoclonal cell lines stably expressing wild type hPS1 and hPS1 with mutations in amino (N-) and carboxyl (C-) terminal regions of the PS1 protein. Employing a heterologous rat PC12 cell system, we demonstrated that: 1) AD mutations inhibit, in part, processing of hPS1 holoprotein; 2) negative selection against highly expressed hPS1 may occur in polyclonal cell cultures; 3) expression of N-terminus mutant (M146V) hPS1 increases susceptibility to apoptosis in differentiated neuronal PC12 cells under deprivation conditions; 4) monoclones with hPS1 C-terminal AD mutation (C410Y) have lower proliferation rates than monoclones expressing wild type hPS1 under deprivation conditions and during NGF-induced neuronal differentiation. The data demonstrate deleterious effect of PS1 AD mutations. The effect depends on the level of expression of the hPS1 isoforms, the number of passages, and trophic and differentiation conditions used for growing PC12 cells.     

Identification of caspases that cleave presenilin-1 and presenilin-2. Five presenilin-1 (PS1) mutations do not alter the sensitivity of PS1 to caspases

Mutations in the presenilin (PS) genes PSI and PS2 are involved in Alzheimer's disease (AD). Recently, apoptosis-associated cleavage of PS proteins was identified. Here we demonstrate that PS1 as well as PS2 are substrates for different members of the caspase protein family. Remarkably, the caspases acting on PS1 could be subdivided in two groups. One group, containing caspase-8, -6 and -11, cleaved PSI after residues ENDD329 and to a lesser extent after residues AQRD341. A second group consisting of caspase-3, -7 and -1 acted uniquely on AQRD341. Importantly, these two cleavage sites were also recognized by caspases in the C-terminal PS1 fragment produced by constitutive proteolysis. In decreasing order of activity, caspase-8, -3, -1, -6 and -7 proteolysed PS2 at the recognition site D326SYD329. Caspase-8 and -3 exhibited the highest proteolytic activity on both PS1 and PS2. PS1 and PS2 were not hydrolyzed by caspase-2 and PS2 also not by caspase-11. None of five missense mutations affected the sensitivity of PSI to caspase-mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase-dependent processing.     

Characterization of the immune response to a secondary encephalitogenic epitope of basic protein in Lewis rats. I. T cell receptor peptide regulation of T cell clones expressing cross-reactive V beta genes.

In Lewis rats, immunization with myelin basic protein induces two distinct encephalitogenic T cell populations, those responding to the immunodominant 72-89 epitope and those specific for a secondary epitope including residues 87-99. The 72-89 specific T cells were I-A restricted and preferentially expressed V beta 8.2 in their TCR. To determine the fine specificity, MHC restriction, and TCR V beta gene use in T cells reactive to the secondary epitope, we characterized 23 T cell clones from the lymph nodes (LN) and spinal cords (SC) of rats immunized with either whole basic protein or synthetic peptides S85-99 and S87-99 that were found to be functionally similar. The S85-99/S87-99 specific clones from LN and SC were all encephalitogenic despite differences in recognition of intact basic protein and class II MHC restriction. Unlike LN clones that overexpressed V beta 8 (46%+) and V beta 6 (31%+), however, SC clones were strongly biased (86%+) in their expression of V beta 6. This V gene bias raised the possibility of TCR peptide therapy using V beta 6 peptides. The V beta 6 sequence was similar to V beta 8.2 in the CDR2 region, and the corresponding peptides from this region were found to be cross-reactive in vivo. Moreover, both peptides were effective in the treatment of EAE induced with either S85-99, biased in V beta 6+ and V beta 8+ T cells, or guinea pig basic protein, biased only in V beta 8+ T cells. These data demonstrate the presence of common immunogenic epitopes among subsets of TCR V region gene families that possess important regulatory activity on effector T cell function.     

Brain expression of presenilins in sporadic and early-onset, familial Alzheimer's disease

BACKGROUND: Mutations in the presenilin proteins cause early-onset, familial Alzheimer's disease (FAD). MATERIALS AND METHODS: We characterized the cellular localization and endoproteolysis of presenilin 2 (PS2) and presenilin 1 (PS1) in brains from 25 individuals with presenilin-mutations causing FAD, as well as neurologically normal individuals and individuals with sporadic Alzheimer's disease (AD). RESULTS: Amino-terminal antibodies to both presenilins predominantly decorated large neurons. Regional differences between the broad distributions of the two presenilins were greatest in the cerebellum, where most Purkinje cells showed high levels of only PS2 immunoreactivity. PS2 endoproteolysis in brain yielded multiple amino-terminal fragments similar in size to the PS1 amino-terminal fragments detected in brain. In addition, two different PS2 amino-terminal antibodies also detected a prominent 42 kDa band that may represent a novel PS2 form in human brain. Similar to PS1 findings, neither amino-terminal nor antiloop PS2 antibodies revealed substantial full-length PS2 in brain. Immunocytochemical examination of brains from individuals with the N141I PS2 mutation or eight different PS1 mutations, spanning the molecule from the second transmembrane domain to the large cytoplasmic loop domain, revealed immunodecoration of no senile plaques and only neurofibrillary tangles in the M139I PS1 mutation stained with PS1 antibodies. CONCLUSIONS: Overall presenilin expression and the relative abundance of full-length and amino-terminal fragments in presenilin FAD cases were similar to control cases and sporadic AD cases. Thus, accumulation of full-length protein or other gross mismetabolism of neither PS2 nor PS1 is a consequence of the FAD mutations examined.     

Genetic study of familial cases of Alzheimer's disease

A small number (1-5%) of Alzheimer's disease (AD) cases associated with the early-onset form of the disease (EOAD) appears to be transmitted as a pure genetic, autosomal dominant trait. To date, three genes responsible for familial EOAD have been identified in the human genome: amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2). Mutations in these genes account for a significant fraction (18 to 50%) of familial cases of early onset AD. The mutations affect APP processing causing increased production of the toxic Abeta42 peptide. According to the "amyloid cascade hypothesis", aggregation of the Abeta42 peptide in brain is a primary event in AD pathogenesis. In our study of twenty AD patients with a positive family history of dementia, 15% (3 of 20) of the cases could be explained by coding sequence mutations in the PS1 gene. Although a frequency of PS1 mutations is less than 2% in the whole population of AD patients, their detection has a significant diagnostic value for both genetic counseling and treatment in families with AD.     

Amyloid beta.

Amyloid beta (A beta) is a 39-43 residue amyloidogenic peptide that is deposited into the extracellular amyloid plaques which characterize an Alzheimer's disease (AD) brain. A beta is derived from the amyloid precursor protein (APP) and undergoes a toxic conformational change (gain of toxic function). The length of the A beta peptide dramatically influences its properties with the longer 42 and 43 residue species being more amyloidogenic. The genetics of familial AD (FAD) supports a central role for A beta in AD since mutations in the FAD causing genes APP and the presenilins (PS1 and PS2) increase the formation of A beta 42,43. Considerable activity is directed towards A beta as a therapeutic target. These strategies aim to inhibit A beta synthesis, A beta fibril formation, its toxic actions on cells or promote its clearance from the brain.     

Genetic factors and a polygenic model of Alzheimer's disease

Genetic factors are responsible, to a certain degree, for many, if not all, Alzheimer's disease (AD) cases. A certain proportion of early-onset (below 65 years of age) AD cases follows an autosomal dominant mode of inheritance. Three genes were identified whose mutations account for 50-70% of early-onset monogenic AD cases in AD pedigrees. These are the genes of the amyloid precursor protein (APP) and two presenilins (PS I and PS II). The polymorphic variant of apolipoprotein E, APOE epsilon 4, is a genetic causative factor in familial and sporadic cases of various early- and late-onset AD forms (it is found, in general, in 20-50% of all AD cases). The action of the epsilon 4 allele is codominant, with the AD risk increased in homozygotes (epsilon 4/epsilon 4 > epsilon 4 > epsilon 3 or epsilon 2). In contrast to the mutations in the PS I and APP genes, the APOE epsilon 4 allele is not a necessary and sufficient condition for AD development. Mutations in these genes have not been found in a proportion of familial early-onset AD cases and are not causative factors in the majority of late-onset familial and sporadic forms. The genes determining AD are evolutionarily conservative and are expressed in all human tissues as early as at initial ontogenetic stages. This raises the question as to why AD is a progressive disorder affecting certain cerebral regions only at middle or old age. A hypothesis and model are suggested to explain the interaction between evolutionary, ontogenetic, and epigenetic factors of the development of the central nervous system and the products of genes whose mutations result in AD. Findings of different mutant genes indicate that AD is a set of genetic disorders (ADs) with a common pathological manifestation.