David E. Kang

A subset of NSAIDs lower amyloidogenic Aβ42 independently of cyclooxygenase activity

Epidemiological studies have documented a reduced prevalence of Alzheimers disease among users of nonsteroidal anti-inflammatory drugs (NSAIDs). It has been proposed that NSAIDs exert their beneficial effects in part by reducing neurotoxic inflammatory responses in the brain, although this mechanism has not been proved. Here we report that the NSAIDs ibuprofen, indomethacin and sulindac sulphide preferentially decrease the highly amyloidogenic Aβ42 peptide (the 42-residue isoform of the amyloid-β peptide) produced from a variety of cultured cells by as much as 80%. This effect was not seen in all NSAIDs and seems not to be mediated by inhibition of cyclooxygenase (COX) activity, the principal pharmacological target of NSAIDs. Furthermore, short-term administration of ibuprofen to mice that produce mutant β-amyloid precursor protein (APP) lowered their brain levels of Aβ42. In cultured cells, the decrease in Aβ42 secretion was accompanied by an increase in the Aβ(1–38) isoform, indicating that NSAIDs subtly alter γ-secretase activity without significantly perturbing other APP processing pathways or Notch cleavage. Our findings suggest that NSAIDs directly affect amyloid pathology in the brain by reducing Aβ42 peptide levels independently of COX activity and that this Aβ42-lowering activity could be optimized to selectively target the pathogenic Aβ42 species.

Genetic association of the low-density lipoprotein receptor-related protein gene (LRP), and apolipoprotein E receptor, with late-onset Alzheimer's disease

The presence of the APOE ϵ4 allele encoding apolipoprotein E4 (apoE4) is the major genetic risk factor for late-onset Alzheimers disease (AD). However, the molecular and cellular mechanisms by which APOE ϵ4 renders AD risk are unclear. In this report, we present genetic evidence that an apoE receptor, LRP, may be associated with the expression of late-onset AD. Using a biallelic genetic marker in exon 3 LRP, late-onset AD cases markedly differed from the control subjects in the distribution of LRP genotypes, and this difference was highly accentuated among AD cases with positive family history of senile dementia. Furthermore, the numbers of neuritic plaques were significantly altered as a consequence of different LRP genotypes in postmortem AD cases. Taken together, our results implicate the pathophysiology of LRP in the expression of late-onset AD.

Modulation of amyloid β-protein clearance and Alzheimer’s disease susceptibility by the LDL receptor–related protein pathway

Susceptibility to Alzheimers disease (AD) is governed by multiple genetic factors. Remarkably, the LDL receptor-related protein (LRP) and its ligands, apoE and alpha2M, are all genetically associated with AD. In this study, we provide evidence for the involvement of the LRP pathway in amyloid deposition through sequestration and removal of soluble amyloid beta-protein (Abeta). We demonstrate in vitro that LRP mediates the clearance of both Abeta40 and Abeta42 through a bona fide receptor-mediated uptake mechanism. In vivo, reduced LRP expression is associated with LRP genotypes and is correlated with enhanced soluble Abeta levels and amyloid deposition. Although LRP has been proposed to be a clearance pathway for Abeta, this work provides the first in vivo evidence that the LRP pathway may modulate Abeta deposition and AD susceptibility by regulating the removal of soluble Abeta.

Presenilin Couples the Paired Phosphorylation of β-Catenin Independent of Axin: Implications for β-Catenin Activation in Tumorigenesis

The Alzheimers disease-linked gene presenilin 1 (PS1) is required for intramembrane proteolysis of APP and Notch. In addition, recent observations strongly implicate PS1 as a negative regulator of the Wnt/beta-catenin signaling pathway, although the mechanism underlying this activity is unknown. Here, we show that presenilin functions as a scaffold that rapidly couples beta-catenin phosphorylation through two sequential kinase activities independent of the Wnt-regulated Axin/CK1alpha complex. Thus, presenilin deficiency results in increased beta-catenin stability in vitro and in vivo by disconnecting the stepwise phosphorylation of beta-catenin, both in the presence and absence of Wnt stimulation. These findings highlight an aspect of beta-catenin regulation outside of the canonical Wnt-regulated pathway and a function of presenilin separate from intramembrane proteolysis.

Subcellular Distribution and Turnover of Presenilins in Transfected Cells

The mechanisms by which mutations in presenilin-1 (PS1) and presenilin-2 (PS2) result in the Alzheimer’s disease phenotype are unclear. Full-length PS1 and PS2 are each processed into stable proteolytic fragments after their biosynthesis in transfected cells. PS1 and PS2 have been localized by immunocytochemistry to the endoplasmic reticulum (ER) and Golgi compartments, but previous studies could not differentiate between the full-length presenilin proteins and their fragments. We carried out subcellular fractionation of cells stably transfected with PS1 or PS2 to determine the localization of full-length presenilins and their fragments. Full-length PS1 and PS2 were principally distributed in ER fractions, whereas the N- and C-terminal fragments were localized predominantly to the Golgi fractions. In cells expressing the PS1 mutant lacking exon 9 (ΔE9), we observed only full-length molecules that were present in the ER and Golgi fractions. The turnover rate was considerably slower for the ΔE9 holoprotein, apparently due to decreased degradation within the ER. Our results suggest that that full-length presenilin proteins are primarily ER resident molecules and undergo endoproteolysis within the ER. The fragments are subsequently transported to the Golgi compartment, where their turnover rate is much slower than that of the full-length presenilin in the ER.

Gain-of-Function Enhancement of IP3 Receptor Modal Gating by Familial Alzheimer’s Disease–Linked Presenilin Mutants in Human Cells and Mouse Neurons

By altering IP3 receptor gating, presenilin mutations associated with familial Alzheimer’s disease increase Ca2+ release from the endoplasmic reticulum. Opening the Calcium Floodgates? Alzheimer’s disease (AD), which is the most common cause of dementia, is a neurodegenerative disorder that affects some 5 million Americans. Although most cases of AD are sporadic, an early-onset form of familial AD (FAD) has been linked to mutations in the presenilins (PSs), transmembrane proteins localized to the endoplasmic reticulum (ER). Cheung et al. investigated the effects of wild-type and mutant forms of PS on inositol trisphosphate receptor (IP3R)–mediated Ca2+ release from the ER in various different cellular systems, including human lymphoblasts derived from individuals with FAD and cortical neurons from a mouse model of FAD. They found that FAD-linked PS mutants enhanced Ca2+ release by modulating IP3R channel gating through a gain-of-function mechanism, consistent with the autosomal-dominant inheritance of FAD. FAD-linked PS mutants, but not PS mutants associated with another form of dementia, shifted IP3R channel gating to a mode in which the probability that individual channels were open after stimulation was increased, leading to exaggerated Ca2+ signals. Familial Alzheimer’s disease (FAD) is caused by mutations in amyloid precursor protein or presenilins (PS1 and PS2). Many FAD-linked PS mutations affect intracellular calcium (Ca2+) homeostasis by mechanisms proximal to and independent of amyloid production, although the molecular details are controversial. We found that several FAD-causing PS mutants enhance gating of the inositol trisphosphate receptor (IP3R) Ca2+ release channel by a gain-of-function effect that mirrored the genetics of FAD and was independent of secretase activity. In contrast, wild-type PS or PS mutants that cause frontotemporal dementia had no such effect. FAD-causing PS mutants altered the modes in which the IP3R channel gated. Recordings of endogenous IP3R in lymphoblasts derived from individuals with FAD or cortical neurons of asymptomatic PS1-AD mice revealed that they were more likely than IP3R in cells with wild-type PS to dwell in a high open-probability burst mode, resulting in enhanced Ca2+ signaling. These results indicate that exaggerated Ca2+ signaling through IP3R-PS interaction is a disease-specific and robust proximal mechanism in FAD.

Perturbed neurogenesis in the adult hippocampus associated with presenilin-1 A246E mutation.

In addition to its well-established role in gamma-secretase cleavage, presenilin (PS) also plays a role in regulating the stability of cytosolic beta-catenin, a protein involved in Wnt signaling. Several familial Alzheimers disease-associated PS1 mutations have been shown to increase the stability of the signaling pool of beta-catenin, correlating with enhanced cell proliferation. Accordingly, we hypothesized that in the setting of PS1 mutations, abnormal activation of Wnt/beta-catenin signaling leads to increased cell division. We tested this hypothesis by examining whether there is evidence of increased neurogenesis in the hippocampus of adult transgenic mice that overexpress the PS1 A246E mutation. In PS1/PS2-deficient fibroblasts, expression of PS1 A246E Familial AD mutation failed to restore the rapid turnover of beta-catenin compared with wild-type PS1. We then examined whether the same mutation enhanced neurogenesis in vivo in adult hippocampus of PS1-deficient mice when restored by wild-type human PS1 (PS1(-/-)WT) or A246E PS1 mutation (PS1(-/-)AE). The PS1 A246E mutation stimulated the proliferation of progenitor cells in the dentate gyrus of adult mice, as assessed by 5-bromo-2-deoxyuridine incorporation, but did not influence their survival or differentiation. These observations suggest that the PS1 A246E mutation influences cell growth putatively via abnormal beta-catenin signaling in vivo.

Low-density lipoprotein receptor-related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway

The major defining pathological hallmark of Alzheimers disease (AD) is the accumulation of amyloid β protein (Aβ), a small peptide derived from β‐ and γ‐secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that β‐ and γ‐secretase activities of BACE1 and prese‐nilin, respectively, are concentrated in intracellular lipid raft microdomains. However, the manner in which APP normally traffics to lipid rafts is unknown. In this study, using transient transfection and immuno‐precip‐itation assays, we show that the cytoplasmic domain of low‐density lipoprotein receptor‐related protein (LRP) interacts with APP and increases Aβ secretion and APP β‐CTF (C‐terminal fragment) generation by promoting BACE1‐APP interaction. We also employed discontinuous sucrose density gradient ultracentrifugation to show that the LRP cytoplasmic domain‐mediated effect was accompanied by greatly increased localization of APP and BACE1 to lipid raft membranes, where β‐ and γ‐secretase activities are highly enriched. Moreover, we provide evidence that endogenous LRP is required for the normal delivery of APP to lipid rafts and Aβ generation primarily in the endocytic but not secretory pathway. These results may provide novel insights to block Aβ generation by targeting LRP‐mediated delivery of APP to raft microdomains.—Yoon I.‐S., Chen, E., Busse, T., Repetto, E., Lakshmana, M. K., Koo, E. H., Kang D. E. Low‐density lipoprotein receptor‐related protein promotes amyloid precursor protein trafficking to lipid rafts in the endocytic pathway. FASEB J. 21, 2742–2752 (2007)

Novel Role of RanBP9 in BACE1 Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation

Accumulation of the amyloid β (Aβ) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted Aβ generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and Aβ generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced β-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced Aβ generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease.

Presenilin 1 Regulates Epidermal Growth Factor Receptor Turnover and Signaling in the Endosomal-Lysosomal Pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Aβ production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/β-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS-/-) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS+/+ cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS-/- cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.