A delta-catenin signaling pathway leading to dendritic protrusions

Delta-catenin is a synaptic adherens junction protein pivotally positioned to serve as a signaling sensor and integrator. Expression of delta-catenin induces filopodia-like protrusions in neurons. Here we show that the small GTPases of the Rho family act coordinately as downstream effectors of delta-catenin. A dominant negative Rac prevented delta-catenin-induced protrusions, and Cdc42 activity was dramatically increased by delta-catenin expression. A kinase dead LIMK (LIM kinase) and a mutant Cofilin also prevented delta-catenin-induced protrusions. To link the effects of delta-catenin to a physiological pathway, we noted that (S)-3,5-dihydroxyphenylglycine (DHPG) activation of metabotropic glutamate receptors induced dendritic protrusions that are very similar to those induced by delta-catenin. Furthermore, delta-catenin RNA-mediated interference can block the induction of dendritic protrusions by DHPG. Interestingly, DHPG dissociated PSD-95 and N-cadherin from the delta-catenin complex, increased the association of delta-catenin with Cortactin, and induced the phosphorylation of delta-catenin within the sites that bind to these protein partners.     

Oxidative Stress in Synaptosomal Proteins from Mutant Presenilin-1 Knock-in Mice: Implications for Familial Alzheimer's Disease

Presenilin-1 (PS-1) is a transmembrane protein that may be involved in the processing of amyloid precursor protein (APP). Mutations in PS-1 are the major cause of familial Alzheimer's disease (AD). AD brain is under significant oxidative stress, including protein oxidation. In the present study, protein oxidation was compared in synaptosomes from knock-in mice expressing mutant human PS-1 (M146V mutation) and from wild-type mice expressing non-mutant human PS-1. Synaptosomal membrane protein conformational alterations associated with oxidative stress were measured using electron paramagnetic resonance (EPR) in conjunction with a protein-specific spin-label. Direct synaptosomal protein oxidation was assessed by a carbonyl detection assay. Synaptosomal proteins from PS-1 mutant mice displayed increased oxidative stress as measured by both techniques, compared with synaptosomal proteins from wild type mice. These data suggest that PS-1 mutations cause oxidative alterations in synaptosomal membrane protein structure and oxidative modification of synaptosomal proteins. Our findings suggest that familial AD may be associated with oxidative stress that may play a pivotal role in neuronal dysfunction and death.     

Delta-catenin-induced dendritic morphogenesis. An essential role of p190RhoGEF interaction through Akt1-mediated phosphorylation

Delta-catenin was first identified through its interaction with Presenilin-1 and has been implicated in the regulation of dendrogenesis and cognitive function. However, the molecular mechanisms by which delta-catenin promotes dendritic morphogenesis were unclear. In this study, we demonstrated delta-catenin interaction with p190RhoGEF, and the importance of Akt1-mediated phosphorylation at Thr-454 residue of delta-catenin in this interaction. We have also found that delta-catenin overexpression decreased the binding between p190RhoGEF and RhoA, and significantly lowered the levels of GTP-RhoA but not those of GTP-Rac1 and -Cdc42. Delta-catenin T454A, a defective form in p190RhoGEF binding, did not decrease the binding between p190RhoGEF and RhoA. Delta-catenin T454A also did not lower GTP-RhoA levels and failed to induce dendrite-like process formation in NIH 3T3 fibroblasts. Furthermore, delta-catenin T454A significantly reduced the length and number of mature mushroom shaped spines in primary hippocampal neurons. These results highlight signaling events in the regulation of delta-catenin-induced dendrogenesis and spine morphogenesis.     

Deletion of the neuron-specific protein delta-catenin leads to severe cognitive and synaptic dysfunction

Delta-catenin (delta-catenin) is a neuron-specific catenin, which has been implicated in adhesion and dendritic branching. Moreover, deletions of delta-catenin correlate with the severity of mental retardation in Cri-du-Chat syndrome (CDCS), which may account for 1% of all mentally retarded individuals. Interestingly, delta-catenin was first identified through its interaction with Presenilin-1 (PS1), the molecule most frequently mutated in familial Alzheimer's Disease (FAD). We investigated whether deletion of delta-catenin would be sufficient to cause cognitive dysfunction by generating mice with a targeted mutation of the delta-catenin gene (delta-cat(-/-)). We observed that delta-cat(-/-) animals are viable and have severe impairments in cognitive function. Furthermore, mutant mice display a range of abnormalities in hippocampal short-term and long-term synaptic plasticity. Also, N-cadherin and PSD-95, two proteins that interact with delta-catenin, are significantly reduced in mutant mice. These deficits are severe but specific because delta-cat(-/-) mice display a variety of normal behaviors, exhibit normal baseline synaptic transmission, and have normal levels of the synaptic adherens proteins E-cadherin and beta-catenin. These data reveal a critical role for delta-catenin in brain function and may have important implications for understanding mental retardation syndromes such as Cri-du-Chat and neurodegenerative disorders, such as Alzheimer's disease, that are characterized by cognitive decline.     

Quantitative Proteomic Analysis of Mitochondria in Aging PS-1 Transgenic Mice

Accumulating evidence suggests mitochondrial alterations are intimately associated with the pathogenesis of Alzheimer’s disease (AD). In order to determine if mutations of presenilin-1 (PS-1) affect levels of mitochondrial proteins at different ages we enriched mitochondrial fractions from 3-, 6-, 12-month-old knock-in mice expressing the M146V PS-1 mutation and identified, and quantified proteins using cleavable isotope-coded affinity tag labeling and two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS). Using this approach, 165 non-redundant proteins were identified with 80 of them present in all three age groups. Specifically, at young ages (3 and 6 months), Na⁺/K⁺ ATPase and several signal transduction proteins exhibited elevated levels, but dropped dramatically at 12 months. In contrast, components of the oxidative phosporylation pathway (OXPHOS), the mitochondrial permeability transition pore (MPTP), and energy metabolism proteins remained unchanged at 3 months but significantly increased with age. We propose that alterations in calcium homeostasis induced by the PS-1 mutation have a major impact in young animals by inhibiting the function of relevant proteins and inducing compensatory changes. However, in older mice combination of the PS-1 mutation and accumulated oxidative damage results in a functional suppression of OXPHOS and MPTP proteins requiring a compensatory increase in expression levels. In contrast, signal transduction proteins showed decreased levels due to a break down in the compensatory mechanisms. The dysfunction of Na⁺/K⁺ ATPase and signal transduction proteins may induce impaired cognition and memory before neurodegeneration occurs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Profiling Reveals Diversity of Stress Signal Transduction Cascades in Highly Penetrant Alzheimer's Disease Human Skin Fibroblasts

The serious and growing impact of the neurodegenerative disorder Alzheimer''s disease (AD) as an individual and societal burden raises a number of key questions: Can a blanket test for Alzheimer''s disease be devised forecasting long-term risk for acquiring this disorder? Can a unified therapy be devised to forestall the development of AD as well as improve the lot of present sufferers? Inflammatory and oxidative stresses are associated with enhanced risk for AD. Can an AD molecular signature be identified in signaling pathways for communication within and among cells during inflammatory and oxidative stress, suggesting possible biomarkers and therapeutic avenues? We postulated a unique molecular signature of dysfunctional activity profiles in AD-relevant signaling pathways in peripheral tissues, based on a gain of function in G-protein-coupled bradykinin B2 receptor (BKB2R) inflammatory stress signaling in skin fibroblasts from AD patients that results in tau protein Ser hyperphosphorylation. Such a signaling profile, routed through both phosphorylation and proteolytic cascades activated by inflammatory and oxidative stresses in highly penetrant familial monogenic forms of AD, could be informative for pathogenesis of the complex multigenic sporadic form of AD. Comparing stimulus-specific cascades of signal transduction revealed a striking diversity of molecular signaling profiles in AD human skin fibroblasts that express endogenous levels of mutant presenilins PS-1 or PS-2 or the Trisomy 21 proteome. AD fibroblasts bearing the PS-1 M146L mutation associated with highly aggressive AD displayed persistent BKB2R signaling plus decreased ERK activation by BK, correctible by gamma-secretase inhibitor Compound E. Lack of these effects in the homologous PS-2 mutant cells indicates specificity of presenilin gamma-secretase catalytic components in BK signaling biology directed toward MAPK activation. Oxidative stress revealed a JNK-dependent survival pathway in normal fibroblasts lost in PS-1 M146L fibroblasts. Complex molecular profiles of signaling dysfunction in the most putatively straightforward human cellular models of AD suggest that risk ascertainment and therapeutic interventions in AD as a whole will likely demand complex solutions.     

N141I mutant presenilin-2 gene enhances neuronal cell death and decreases bcl-2 expression

A missense mutation (N1411) in Presenilin-2 (PS-2) gene is associated with early-onset familial Alzheimer's disease. In this study, SK-N-SH human neuroblastoma cells were transfected with wild-type and mutant PS-2 gene to examine presenilin-2 effects on apoptosis. Serum deprivation resulted in enhanced apoptosis in mutant PS-2 comparing with wild-type PS-2. Similarly, mutant PS-2 induced lactate dehydrogenase release to greater extent than wild-type PS-2. Time course experiment demonstrated that the increase in caspase-3-like activity was more pronounced and accelerated in mutant PS-2, compared to wild-type PS-2. While a significant decrease in bcl-2, an anti-apoptotic molecule, occurred in the cells overexpressing mutant PS-2, no significant change was observed in bax, a pro-apoptotic molecule, as compared with the cells overexpressing wild-type PS-2. Our study demonstrated that mutant PS-2 induces apoptosis accompanied by increased caspase-3-like activity and decreased bcl-2 expression in neuronal cells after serum-deprivation.     

Endogenous expression of FAD-linked PS1 impairs proliferation,neuronal differentiation and survival of adult hippocampal progenitors

Background

Alzheimer’s disease (AD) is characterized by progressive memory loss and impaired cognitive function. Early-onset familial forms of the disease (FAD) are caused by inheritance of mutant genes encoding presenilin 1 (PS1) variants. We have demonstrated that prion promoter (PrP)-driven expression of human FAD-linked PS1 variants in mice leads to impairments in environmental enrichment (EE)-induced adult hippocampal neural progenitor cell (AHNPC) proliferation and neuronal differentiation, and have provided evidence that accessory cells in the hippocampal niche expressing PS1 variants may modulate AHNPC phenotypes, in vivo. While of significant interest, these latter studies relied on transgenic mice that express human PS1 variant transgenes ubiquitously and at high levels, and the consequences of wild type or mutant PS1 expressed under physiologically relevant levels on EE-mediated AHNPC phenotypes has not yet been tested.

Results

To assess the impact of mutant PS1 on EE-induced AHNPC phenotypes when expressed under physiological levels, we exposed adult mice that constitutively express the PSEN1 M146V mutation driven by the endogenous PSEN1 promoter (PS1 M146V “knock-in” (KI) mice) to standard or EE-housed conditions. We show that in comparison to wild type PS1 mice, AHNPCs in mice carrying homozygous (PS1 ^M146V/M146V ) or heterozygous (PS1 ^M146V/+ ) M146V mutant alleles fail to exhibit EE-induced proliferation and commitment towards neurogenic lineages. More importantly, we report that the survival of newborn progenitors are diminished in PS1 M146V KI mice exposed to EE-conditions compared to respective EE wild type controls.

Conclusions

Our findings reveal that expression at physiological levels achieved by a single PS1 M146V allele is sufficient to impair EE-induced AHNPC proliferation, survival and neuronal differentiation, in vivo. These results and our finding that microglia expressing a single PS1 M146V allele impairs the proliferation of wild type AHNPCs in vitro argue that expression of mutant PS1 in the AHNPC niche impairs AHNPCs phenotypes in a dominant, non-cell autonomous manner.

     

Wild-type presenilin 1 protects against Alzheimer disease mutation-induced amyloid pathology

Mutations in presenilin 1 (PS1) lead to dominant inheritance of early onset familial Alzheimer disease (FAD). These mutations are known to alter the gamma-secretase cleavage of the amyloid precursor protein, resulting in increased ratio of Abeta42/Abeta40 and accelerated amyloid plaque pathology in transgenic mouse models. To investigate the factors that drive the Abeta42/Abeta40 ratio and amyloid pathogenesis and to investigate the possible interactions between wild-type and FAD mutant PS1, which are co-expressed in transgenic animals, we expressed the PS1 M146V knock-in allele either on wild-type PS1 (PS1M146V/+) or PS1 null (PS1M146V/-) background and crossed these alleles with the Tg2576 APP transgenic mice. Introduction of the PS1 M146V mutation on Tg2576 background resulted in earlier onset of plaque pathology. Surprisingly, removing the wild-type PS1 in the presence of the PS1 M146V mutation (PS1M146V/-) greatly exacerbated the amyloid burden; and this was attributed to a reduction of gamma-secretase activity rather than an increase in Abeta42. Our findings establish a protective role of the wild-type PS1 against the FAD mutation-induced amyloid pathology through a partial loss-of-function mechanism.     

Genetic and cytological analyses of a partial-female-sterile mutant (PS-1) in soybean (Glycine max; Leguminosae)

Soybean partial-female-sterile mutant 1 (PS-1) was recovered from a gene-tagging study. The objectives were to study the inheritance, linkage, allelism, and certain aspects of the reproductive biology of the PS-1 mutant. For inheritance and linkage tests, PS-1 was crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient mutant CD-1, also recovered from the gene-tagging study. For allelism tests, reciprocal crosses were made with PS-1 and three other partial-sterile mutants (PS-2, PS-3, and PS-4) recovered from the same gene-tagging study. The PS-1 mutant is inherited in a 3:1 ratio and is a single recessive gene. Linkage results indicated that the gene for partial sterility in PS-1 is not linked either to the w4 locus or to the CD-1 locus. Allelism tests showed that the gene in PS-1 is nonallelic to the gene in PS-2, PS-3, and PS-4. Investigations of developing and mature pollen indicated no differences in morphology, stainability, or fluorescence between normal and partial-sterile genotypes. The PS-1 mutant is completely male fertile. Confocal scanning laser microscopy was used to determine that early embryo abortion in PS-1 is due indirectly to abnormal migration of the fused polar nucleus, which prevented it from being fertilized. Subsequent absence of endosperm development leads directly to abortion of the proembryo.     

Presenilin-1 D257A and D385A mutants fail to cleave Notch in their endoproteolyzed forms, but only presenilin-1 D385A mutant can restore its gamma-secretase activity with the compensatory overexpression of normal C-terminal fragment

The enzyme gamma-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the gamma-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other gamma-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of gamma-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their gamma-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349-467). However, the defective PS-1 D257A mutant could not restore their gamma-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the gamma-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.     

Familial Alzheimer's disease-linked presenilin-1 mutation M146V affects store-operated calcium entry: Does gain look like loss?

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to neuron death and synapse loss in the hippocampus and cortex, with consequent cognitive disability and dementia. Mutations in the presenilin-1 (PS1) gene lead to familial Alzheimer's disease (FAD). Here, we report that the expression of FAD-linked PS1 M146V mutant affects store-operated calcium channel activity (Isoc) in human neuroblastoma SK–N–SH cells. Electrophysiological measurements and calcium imaging experiments have revealed the emergent role of calcium sensor STIM2 in the inhibition of calcium release-activated calcium channel activity (Icrac) and enhancement of intracellular Ca²⁺ stores content due to PS1 M146V mutant expression. In general, the results of this study suggest that the pathological inhibition of one type of store-operated calcium channels caused by FAD PS1 mutant expression may be accounted for by preceding gain of spontaneous activity of store-operated calcium channels driven by STIM2.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

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.     

Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor channel gating

Mutations in presenilins (PS) are the major cause of familial Alzheimer's disease (FAD) and have been associated with calcium (Ca2+) signaling abnormalities. Here, we demonstrate that FAD mutant PS1 (M146L)and PS2 (N141I) interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and exert profound stimulatory effects on its gating activity in response to saturating and suboptimal levels of InsP3. These interactions result in exaggerated cellular Ca2+ signaling in response to agonist stimulation as well as enhanced low-level Ca2+signaling in unstimulated cells. Parallel studies in InsP3R-expressing and -deficient cells revealed that enhanced Ca2+ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP3R stimulates amyloid beta processing,an important feature of AD pathology. These observations provide molecular insights into the "Ca2+ dysregulation" hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention.