Familial Alzheimer’s disease mutations in the presenilin 1 gene reduce cell-cell adhesion in transfected fibroblasts

Experimental evidence has been obtained that mutations in the presenilin 1 (PS1) gene in familial Alzheimer’s disease can lead to the disturbance of cell adhesion in model cell cultures. It was shown that, in L fibroblasts of mice with stable expression of GFP-PS1 cDNA containing G209V or E319G mutations, cell-cell interactions and the accumulation of GFP-PS1 cDNA in intercellular contacts are disturbed. Similar results were obtained in transfected human epithelial HEp2 cells. It is assumed that mutations in familial Alzheimer’s disease lead to the disturbance of the functions of presenelin 1 in cell adhesion.     

The role of Alzheimer's disease-related presenilin 1 in intercellular adhesion

Most cases of familial early-onset Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene. However, the cellular functions of PS1 are unknown. We showed predominant localization of PS1 to cell-cell contacts of the plasma membrane in human prostate epithelial tissue and in a human epithelial cell line HEp2 stably transfected with an inducible PS1 construct. PS1 co-immunoprecipitated with beta-catenin from cell lysates of stable transfectants. Conversely, PS1 lacking the PS1-beta-catenin interaction site did not co-immunoprecipitate with beta-catenin and was not recruited to the cell-cell contacts. L cells, which do not form tight intercellular contacts, formed clusters of adhered cells after stable transfection with GFP-PS1 cDNA and demonstrated a clear preference for independent aggregation in the mixed cultures. However, L cells transfected with mutant GFP-PS1 constructs, which had a truncated N-terminus of PS1 or deleted PS1-beta-catenin interaction site, failed to form intercellular contacts. In addition, in primary cultures of mouse cortical neurons PS1 was highly concentrated on the surface of extended growth cones. Taken together, our results suggest an important role of PS1 in intercellular adhesion in epithelial cells and neurons.     

Potential role of presenilin 1 in regulation of synaptic function

One of the earliest neuropathological symptoms of Alzheimer's disease is the loss of synapses, which preceed the formation of amyloidosis and neurodegeneration. Although most cases of early-onset familial Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene, the functions of PS1 and its role in synaptic disfunction are not yet completely understood. In this paper we analysed of the intracellular and extracellular distribution of PS1 in the cultures of mouse cortical embryonic neurons. We found that PS1 is concentrated on the surface of the growth cone and at neurite contact sites. PS1 was also found in synapses where it is co-localized with synaptophysin. Independent evidense of involvement of PS1 in synaptic function we obtained by transfection of neurons with GFP-PS1 cDNA. GFP was colocalized with synaptophysin in transfected cultures. GFP-immunoprecepitates from transfected neurons contained processed N-cadherin. This result presents an additional proof of involvment PS1 in synapse formation. To evaluate the role of PS1 inactivation in the synaptic functions, we compare synaptic density in neuronal cell cultures from PS1 knockout mice PS1 (-/-) and wild type mice PS1 (+/+). Our results clearly show that PS1 (-/-) displayed a low number of morphological synapses in comparing with wild type culture PS1 (+/+). In summary, our results indicate a role of PS1 in synaptic function.     

PS1/GFP融合蛋白对PS1的亚细胞定位与功能的初步研究

PS1基因突变与早发家族性老年痴呆有密切联系。本文构建pEGFP-C1-PS1以及pEGFP-N2-PS1融合基因表达载体,于HEK293和CHO细胞系中表达PS1/GFP融合蛋白,以GFP绿色荧光作为PS1的亚细胞定位信号,通过SPOTII以及CONFOCAL显微镜进行观察,初步获得PS1全长蛋白在细胞中定位的部分信息,即本实验条件下,PS1定位于细胞核膜,细胞质内有不均匀的分布,少量存在于细胞-细胞接触处的细胞膜上。     

Expression of presenilin 1 on the cell surface in motile polarized cells

Most cases of familial early-onset Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene. However, the cellular functions of PS1 are not yet completely understood. We showed that endogenous PS1 and the adhesion protein CD44 are redistributed on the surface of cell projections (lamellipodia) in polarized T- lymphocytes (Jurkat cells) after the adhesion to a collagen matrix. This effect was not observed for another surface protein of T lymphocytes, which is not involved in cell adhesion processes, the T cell receptor. In primary cultures of mouse cortical neurons, PS1 was concentrated at the surface of extended growth cones and at the sites of neurite contacts. The concentration of PS1 at the surface of cellular structures that promote cell motility and cell contacts suggests an important role of PSI in cell adhesion in motile polarized cells.     

Subcellular localization of PS1 based on PS1/GFP fusing protein

Mutations in presenilin 1 (PS1) gene are closely associated with the early onset of familial Alzheimer's disease (EOFAD). The fusion genes, GFP-PS1 (recombinant plasmid pEGFP-C1-PS1) and PS1-GFP (recombinant plasmid pEGFP-N2-PS1) were constructed to study the subcellular localization of PS1 holoprotein. Recombinant plasmids were transiently transfected into two cell lines, HEK293 and CHO, respectively, using the green fluorescence from GFP (green fluorescence protein) as the PS1 localization signal. Then, we observed green fluorescence with a SPOT Ⅱ (Olympus, BH2) and CONFOCAL microscope (Olympus, FV300) under 488 nm. The results show that PS1 located on the nuclear envelope. A few can be found on the cellular membrane and in the cytosol in a non-homogeneous distribution.     

Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.     

The potential role of presenilin 1 in regulation of synaptic function

One of the earliest neuropathological symptoms of Alzheimer’s disease is the loss of synapses that precedes the formation of amyloid plaques and neurodegeneration. Although most cases of early-onset familial Alzheimer’s disease are caused by mutations in the presenilin 1 (PS1) gene, the functions of PS1 and its role in synaptic dysfunction are not yet completely understood. In this paper, we analyzed PS1 intra- and extracellular distribution in cultures of mouse cortical embryonic neurons. We found that PS1 was concentrated on the surface of the growth cone and neurite contact sites. PS1 was also found in synapses where it was colocalized with synaptophysin. We obtained independent evidence of PS1 involvement in synaptic function by transfection of neurons with GFP-PS1cDNA. GFP was colocalized with synaptophysin in transfected cultures. GFP-immunoprecepitates from transfected neurons contained N-cadherin. This finding represents additional evidence of PS1 participation in the synapse formation. To evaluate the role of PS1 inactivation in the synaptic functions, we compare the synaptic density in neuronal cell cultures from knockout mice PS1 (−/−) and wild type mice PS1 (+/+). Our results obviously show that PS1 (−/−) cultures displayed lower number of morphological synapses compared to wild type culture PS1 (+/+). In summary, our findings show the role of PS1 in synaptic function.     

Protein kinase C and amyloid precursor protein processing in skin fibroblasts from sporadic and familial Alzheimer's disease cases

Non-amyloidogenic alpha-secretase processing of amyloid precursor protein (APP) is stimulated by protein kinase C (PKC). Levels and activity of PKC are decreased in sporadic Alzheimer's disease skin fibroblasts. We investigated whether alterations in PKC and PKC-mediated APP processing occur also in fibroblasts established from individuals with familial Alzheimer's disease APP KM670/671NL, PS1 M146V and H163Y mutations. These pathogenic mutations are known to alter APP metabolism to increase Abeta. PKC activities, but not levels, were decreased by 50% in soluble fractions from sporadic Alzheimer's disease cases. In contrast, familial Alzheimer's disease fibroblasts showed no significant changes in PKC enzyme activity. Fibroblasts bearing the APP KM670/671NL mutation showed no significant differences in either PKC levels or PKC-mediated soluble APP (APPs) secretion, compared to controls. Fibroblasts bearing PS1 M146V and H163Y mutations showed a 30% increase in soluble PKC levels and a 40% decrease in PKC-mediated APPs secretion. These results indicate that PKC deficits are unlikely to contribute to increased Abeta seen with APP and PS1 mutations, and also that PS1 mutations decrease alpha-secretase derived APPs production independently of altered PKC activity.     

Presenilin 1 expression on the cell surface in motile polarized cells

Most cases of early-onset familial Alzheimer’s disease are caused by mutations in the presenilin 1 gene. Nonetheless, the function of presenilin 1 are not yet completely understood. It was shown that endogenous presenilin 1, as well as the adhesion protein CD44, is concentrated on the surface of lamellipodia of polarized T cells (Jurkat cells) after adhesion to a collagen matrix. This phenomenon was not observed for another surface protein of T cells, T cell receptor, which is not involved in cell adhesion processes. In cultures of primary mouse cortical neurons, presenilin 1 was concentrated on the surface of the growth cone and at neurite contact sites. The concentration of presenilin 1 on the surface of structures that determine cell motility and intercellular contacts suggests that presenilin 1 plays an important role in cell adhesion in motile polarized cells.     

The first proline of PALP motif at the C terminus of presenilins is obligatory for stabilization, complex formation, and gamma-secretase activities of presenilins.

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins, resulting in failure of Abeta42 overproduction on familial Alzheimer's disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities.     

Akt/GSK3beta serine/threonine kinases: evidence for a signalling pathway mediated by familial Alzheimer's disease mutations

Although Alzheimer's disease pathologically affects the brain, familial Alzheimer's disease associated mutations of beta-amyloid precursor protein and presenilin are ubiquitously expressed and therefore aberrant intracellular signals, separate from but similar to, the brain may be expected. Here, we report selective down regulation of the serine/threonine kinase, Akt/PKB, concurrent with elevated endogenous GSK3beta kinase activity in familial Alzheimer's disease beta-amyloid precursor protein expressing human embryonic kidney (HEK) and familial Alzheimer's disease presenilin lymphoblast cells. Further, familial Alzheimer's disease presenilin in the human lymphoblast was associated with beta-catenin destabilization. Moreover, limited immunohistochemistry analysis reveals Akt/PKB in a subset of neurofibrillary tangles where GSK3beta and tau have been reported to co-localize, suggesting a possible Akt/GSK3beta and tau interaction in vivo. Our data suggest that familial Alzheimer's disease mutants of beta-amyloid precursor protein and presenilin signal, at least in part, through the Akt/GSKbeta pathway and that Akt/GSK3beta-mediated signalling may contribute to the underlying Alzheimer's disease pathogenesis induced by familial Alzheimer's disease mutants.     

G protein betagamma complex-mediated apoptosis by familial Alzheimer''s disease mutant of APP.

In familial Alzheimer's disease (FAD), three missense mutations, V642I, V642F and V642G, that co-segregate with the disease phenotype have been discovered in the 695 amino acid form of the amyloid precursor protein APP. Expression of these mutants causes a COS cell NK1 clone to undergo pertussis toxin-sensitive apoptosis in an FAD trait-linked manner by activating the G protein Go, which consists of G alpha(o) and G betagamma subunits. We investigated which subunit was responsible for the induction of apoptosis by V642I APP in NK1 cells. In the same system, expression of mutationally activated G alpha(o) or G alpha(i) induced little apoptosis. Apoptosis by V642I APP was antagonized by the overexpression of the carboxy-terminal amino acids 495-689 of the beta-adrenergic receptor kinase-1, which blocks the specific functions of G betagamma. Co-transfection of G beta2gamma2 cDNAs, but not that of other G beta(x)gamma(z) (x = 1-3; z = 2, 3), induced DNA fragmentation in a manner sensitive to bcl-2. These data implicate G betagamma as a cell death mediator for the FAD-associated mutant of APP.     

Abnormal tau-containing filaments in neurodegenerative diseases

It has been known for some time that the neurofibrillary pathology in Alzheimer's disease consists of so-called paired helical and straight filaments made up of the microtubule-associated protein tau. The degree of dementia observed in the disease correlates better with the extent of neurofibrillary pathology than with the Abeta amyloid deposits, the other characteristic defining pathological fibrous deposit in Alzheimer's disease. However, no familial cases of Alzheimer's disease have been genetically linked to the tau protein locus. Recently a group of frontotemporal dementias with parkinsonism linked to chromosome 17 has been shown to be caused by mutations in the tau gene. Some are missense mutations giving altered tau proteins, whereas others affect the splicing of the pre-mRNA and change the balance between different tau isoforms. Histologically these diseases are all characterised by various kinds of filamentous tau protein deposits, mostly in the complete absence of Abeta deposits. The abnormal tau filaments show different morphologies, depending on the nature of the tau mutation. These diseases show that tau mutations can be a prime cause of inherited dementing illness and may throw some light on the pathological process in the much larger number of sporadic cases of Alzheimer's disease.     

gamma-cleavage-independent functions of presenilin, nicastrin, and Aph-1 regulate cell-junction organization and prevent tau toxicity in vivo

Genetic analysis of familial Alzheimer's disease has revealed that mutations in the gamma-secretase enzyme presenilin promote toxic Abeta secretion; however, presenilin mutations might also influence tau hyperphosphorylation and neurodegeneration through gamma-secretase-independent mechanisms. To address this possibility and determine whether other components of the gamma-secretase complex possess similar regulatory functions, we analyzed the roles of presenilin, nicastrin, and aph-1 in a Drosophila model for tau-induced neurodegeneration. Here, we show that presenilin and nicastrin prevent tau toxicity by modulating the PI3K/Akt/GSK3beta phosphorylation pathway, whereas aph-1 regulates aPKC/PAR-1 activities. Moreover, we found that these transmembrane proteins differentially regulate the intracellular localization of GSK3beta and aPKC at cell junctions. Inhibition of gamma-secretase activity neither interfered with these kinase pathways nor induced aberrant tau phosphorylation. These results establish new in vivo molecular functions for the three components of the gamma-secretase complex and reveal a different mechanism that might contribute to neuronal degeneration in Alzheimer's disease.     

Expression of Presenilin 1 mRNA in Rat Peripheral Organs and Brain

At least 50 different mutations in the presenilin 1 gene have been shown to cause early onset familial Alzheimer's disease. Although presenilin 1 has an obvious role in the pathogenesis of Alzheimer's disease, its function is still unknown. In the present study, the occurrence and distribution of presenilin 1 mRNA was examined in rat peripheral organs as well as in the brain by in situ hybridization histochemistry, using a radiolabelled oligonucleotide probe. In comparison to the brain, a high presenilin 1 mRNA expression was found in the testis, kidney, spleen, adrenal gland and thymus. It was also observed in skeletal muscle, liver, small intestine and lung, whereas no presenilin 1 could be detected in the heart, spinal cord and pancreas. Since presenilin 1 mRNA was found to be abundant in peripheral tissues which apparently are not affected in Alzheimer's disease, additional functions of presenilin 1 are suggested, unrelated to its role in the pathological processes of the disease.