Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35/Cdk5 and p25/Cdk5

The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APP(beta) (sAPP(beta)) and beta-amyloid peptide (Abeta), the major component of senile plaques in patient brains inflicted with Alzheimer's disease (AD), or soluble APP(alpha) (sAPP(alpha)) and a peptide smaller than Abeta. One of the protein kinases known to phosphorylate APP(Thr668) is cyclin-dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative effects of p35 and p25 on APP(Thr668) phosphorylation and APP processing, however, have not been reported. In this study, we investigated APP(Thr668) phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH-SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APP(Thr668) phosphorylation, specifically, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these differential effects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of Abeta, sAPP(beta), as well as sAPP(alpha). These results confirm the involvement of Cdk5 in APP processing, and suggest that p35- and p25-mediated Cdk5 activities lead to discrete APP phosphorylation.     

The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells.

BACKGROUND: The cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP) is phosphorylated in vitro at Thr654 and Ser655, and both in vitro and in intact cells at Thr668 (numbering for APP695 isoform). MATERIALS AND METHODS: We have developed phosphorylation state-specific antibodies to each of the sites, and we have used these to analyze the phosphorylation of APP in adult rat brain and in cultured cell lines. RESULTS: We demonstrate that all three sites in APP are phosphorylated in adult rat brain. Phosphorylation at Thr654, Ser655, and Thr668 was also observed in several cultured cell lines. In PC12 cells, phosphorylation at Ser655 was increased more than 10-fold by treatment with okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, but was not affected by activators of protein kinase C. In HeLa cells, phosphorylation at Thr668 was regulated in a cell cycle-dependent manner with near-stoichiometric phosphorylation being observed at the G2/M phase of the cell cycle. In general, phosphorylation at Ser655 was found to be highest in mature APP isoforms, whereas phosphorylation of Thr668 was highest in immature APP isoforms in cultured cells. CONCLUSIONS: The results demonstrate that phosphorylation of the cytoplasmic domain of APP occurs at Thr654, Ser655, and Thr668 under physiological conditions. The further characterization of APP phosphorylation using phosphorylation-specific antibodies may help in the elucidation of the biological function of APP.     

Phosphorylation of Amyloid Precursor Protein at Threonine 668 Is Essential for Its Copper-responsive Trafficking in SH-SY5Y Neuroblastoma Cells

Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells.     

NGF controls APP cleavage by downregulating APP phosphorylation at Thr668: relevance for Alzheimer's disease

NGF has been implicated in forebrain neuroprotection from amyloidogenesis and Alzheimer's disease (AD). However, the underlying molecular mechanisms are still poorly understood. Here, we investigated the role of NGF signalling in the metabolism of amyloid precursor protein (APP) in forebrain neurons using primary cultures of septal neurons and acute septo‐hippocampal brain slices. In this study, we show that NGF controls the basal level of APP phosphorylation at Thr668 (T668) by downregulating the activity of the Ser/Thr kinase JNK(p54) through the Tyr kinase signalling adaptor SH2‐containing sequence C (ShcC). We also found that the specific NGF receptor, Tyr kinase A (TrkA), which is known to bind to APP, fails to interact with the fraction of APP molecules phosphorylated at T668 (APP^pT668). Accordingly, the amount of TrkA bound to APP is significantly reduced in the hippocampus of ShcC KO mice and of patients with AD in which elevated APP^pT668 levels are detected. NGF promotes TrkA binding to APP and APP trafficking to the Golgi, where APP–BACE interaction is hindered, finally resulting in reduced generation of sAPPβ, CTFβ and amyloid‐beta (1‐42). These results demonstrate that NGF signalling directly controls basal APP phosphorylation, subcellular localization and BACE cleavage, and pave the way for novel approaches specifically targeting ShcC signalling and/or the APP–TrkA interaction in AD therapy.     

Suppression of the caspase cleavage of beta-amyloid precursor protein by its cytoplasmic phosphorylation

beta-Amyloid precursor protein (APP) is a type I transmembrane protein. Its cleavages by beta- and gamma-secretases yield beta-amyloid, which is the main constituent of senile plaques in Alzheimer's disease (AD). In apoptotic cells and AD brains, APP is alternatively cleaved by caspases in the cytoplasmic region after the Asp664 residue (with respect to the numbering conversion for the APP695 isoform). Caspase-cleaved fragments of APP are cytotoxic and have been implicated in AD pathogenesis; however, the mechanisms regulating the cleavage have not been studied. APP is constitutively phosphorylated at Thr668 in brain. In the present study, we demonstrate that APP phosphorylated at Thr668 is less vulnerable to cytoplasmic cleavage by caspase-3 and caspase-8. This suggests that APP phosphorylation suppresses the generation of caspase-cleaved fragments of APP in the brain and that perturbation of this phosphorylation may be involved in APP-mediated neurotoxicity.     

Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production of beta-amyloid

Neuronal Fe65 is an adapter protein that interacts with the cytoplasmic domain of the beta-amyloid precursor protein (APP). Although the interaction has been reported to occur between the second phosphotyrosine interaction domain of Fe65 and the YENPTY motif in the cytoplasmic domain of APP, the regulatory mechanism and biological function of this interaction remain unknown. We report here that (i) a single amino acid mutation at the Thr-668 residue of APP695, located 14 amino acids toward the amino-terminal end from the (682)YENPTY(687) motif, reduced the interaction between members of the Fe65 family of proteins and APP, whereas interaction of APP with the phosphotyrosine interaction domain of other APP binders such as X11-like and mammalian disabled-1 was not influenced by this mutation; (ii) the phosphorylation of APP at Thr-668 diminished the interaction of APP with Fe65 by causing a conformational change in the cytoplasmic domain that contains the Fe65-binding motif, YENPTY; and (iii) the expression of Fe65 slightly suppressed maturation of APP and decreased production of beta-amyloid (Abeta). Mutation at Thr-668 of APP abolished the effect of Fe65 on APP maturation. This mutation blocked the Fe65-dependent suppression of Abeta production and resulted in the release of increased levels of Abeta in the presence of Fe65. We previously reported that during maturation of APP in neurons, the protein is specifically phosphorylated at Thr-668 and undergoes O-glycosylation. The present results suggest that the phosphorylation of O-glycosylated mature APP at Thr-668 causes a conformational change in its cytoplasmic domain that prevents binding of Fe65 in neurons and may lead to an alteration in the production of Abeta.     

The amyloid-beta precursor protein is phosphorylated via distinct pathways during differentiation, mitosis, stress, and degeneration

Phosphorylation of amyloid-beta precursor protein (APP) at Thr(668) is a normal process linked to neurite extension and anterograde transport of vesicular cargo. By contrast, increased phosphorylation of APP is a pathological trait of Alzheimer's disease. APP is overexpressed in Down's syndrome, a condition that occasionally leads to increased APP phosphorylation, in cultured cells. Whether phosphorylation of APP in normal versus high APP conditions occurs by similar or distinct signaling pathways is not known. Here, we addressed this problem using brainstem-derived neurons (CAD cells). CAD cells that ectopically overexpress APP frequently show features of degenerating neurons. We found that, in degenerating cells, APP is hyperphosphorylated and colocalizes with early endosomes. By contrast, in normal CAD cells, phosphorylated APP (pAPP) is excluded from endosomes, and localizes to the Golgi apparatus and to transport vesicles within the neurites. Whereas the neuritic APP is phosphorylated by c-Jun NH(2)-terminal kinase through a pathway that is modulated by glycogen synthase kinase 3beta, the endosomal pAPP in degenerated CAD cells results from activation of cyclin-dependent kinase 5. Additional signaling pathways, leading to APP phosphorylation, become active during stress and mitosis. We conclude that distinct pathways of APP phosphorylation operate in proliferating, differentiating, stressed, and degenerating neurons.     

Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3)

Threonine(668) (thr(668)) within the carboxy-terminus of the Alzheimer's disease amyloid precursor protein (APP) is a known in vivo phosphorylation site. Phosphorylation of APPthr(668) is believed to regulate APP function and metabolism. Thr(668) precedes a proline, which suggests that it is targeted for phosphorylation by proline-directed kinase(s). We have investigated the ability of four major neuronally active proline-directed kinases, cyclin dependent protein kinase-5, glycogen synthase kinase-3 beta, p42 mitogen-activated protein kinase and stress-activated protein kinase-1b, to phosphorylate APPthr(668) and report here that SAPK1b induces robust phosphorylation of this site both in vitro and in vivo. This finding provides a molecular framework to link cellular stresses with APP metabolism in both normal and disease states.     

Statins Reduce Amyloid β-Peptide Production by Modulating Amyloid Precursor Protein Maturation and Phosphorylation Through a Cholesterol-Independent Mechanism in Cultured Neurons

Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been reported to attenuate amyloid-β peptide (Aβ) production in various cellular models. However, the mechanisms by which statins affect neuronal Aβ production have not yet been clarified. Here, we investigated this issue in rat primary cortical neurons using two statins, pitavastatin (PV) and atorvastatin (AV). Treatment of neurons with 0.2–2.5 μM PV or AV for 4 days induced a concentration- and time-dependent reduction in the secretion of both Aβ40 and Aβ42. Moreover, Western blot analyses of cell lysates showed that treatment with PV or AV significantly reduced expression levels of the mature form of amyloid precursor protein (APP) and Thr668-phosphorylated APP (P-APP), but not immature form of APP; the decreases in P-APP levels were more notable than those of mature APP levels. The statin treatment did not alter expression of BACE1 (β-site APP-cleaving enzyme 1) or γ-secretase complex proteins (presenilin 1, nicastrin, APH-1, and PEN-2). In neurons overexpressing APP via recombinant adenoviruses, PV or AV similarly reduced Aβ secretion and the levels of mature APP and P-APP. Statins also markedly reduced cellular cholesterol content in neurons in a concentration-dependent manner. Co-treatment with mevalonate reversed the statin-induced decreases in Aβ secretion and mature APP and P-APP levels, whereas co-treatment with cholesterol did not, despite recovery of cellular cholesterol levels. Finally, cell-surface biotinylation experiments revealed that both statins significantly reduced the levels of cell-surface P-APP without changing those of cell surface mature APP. These results suggest that statins reduce Aβ production by selectively modulating APP maturation and phosphorylation through a mechanism independent of cholesterol reduction in cultured neurons.     

Membrane-microdomain localization of amyloid β-precursor protein (APP) C-terminal fragments is regulated by phosphorylation of the cytoplasmic Thr668 residue

Amyloid β-precursor protein (APP) is primarily cleaved by α- or β-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr(668) and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648-695)) with Thr(P)(668) did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aβ generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aβ in Alzheimer disease.     

APP processing is regulated by cytoplasmic phosphorylation

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.     

Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation

Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser(8) and Thr(138) as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr(138) was dephosphorylated in adult rat, although both Ser(8) and Thr(138) were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser(8) p35 to dual phospho-Ser(8)/Thr(138) p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr(138) predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration.     

Facilitation of stress-induced phosphorylation of beta-amyloid precursor protein family members by X11-like/Mint2 protein

Beta-amyloid precursor protein (APP) is the precursor of beta-amyloid (Abeta), which is implicated in Alzheimer's disease pathogenesis. APP complements amyloid precursor-like protein 2 (APLP2), and together they play essential physiological roles. Phosphorylation at the Thr(668) residue of APP (with respect to the numbering conversion for the APP 695 isoform) and the Thr(736) residue of APLP2 (with respect to the numbering conversion for the APLP2 763 isoform) in their cytoplasmic domains acts as a molecular switch for their protein-protein interaction and is implicated in neural function(s) and/or Alzheimer's disease pathogenesis. Here we demonstrate that both APP and APLP2 can be phosphorylated by JNK at the Thr(668) and Thr(736) residues, respectively, in response to cellular stress. X11-like (X11L, also referred to as X11beta and Mint2), which is a member of the mammalian LIN-10 protein family and a possible regulator of Abeta production, elevated APP and APLP2 phosphorylation probably by facilitating JNK-mediated phosphorylation, whereas other members of the family, X11 and X11L2, did not. These observations revealed an involvement of X11L in the phosphorylation of APP family proteins in cellular stress and suggest that X11L protein may be important in the physiology of APP family proteins as well as in the regulation of Abeta production.     

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.     

Amyloid Precursor Protein Enhances Nav1.6 Sodium Channel Cell Surface Expression

Amyloid precursor protein (APP) is commonly associated with Alzheimer disease, but its physiological function remains unknown. Nav1.6 is a key determinant of neuronal excitability in vivo. Because mouse models of gain of function and loss of function of APP and Nav1.6 share some similar phenotypes, we hypothesized that APP might be a candidate molecule for sodium channel modulation. Here we report that APP colocalized and interacted with Nav1.6 in mouse cortical neurons. Knocking down APP decreased Nav1.6 sodium channel currents and cell surface expression. APP-induced increases in Nav1.6 cell surface expression were G_o protein-dependent, enhanced by a constitutively active G_o protein mutant, and blocked by a dominant negative G_o protein mutant. APP also regulated JNK activity in a G_o protein-dependent manner. JNK inhibition attenuated increases in cell surface expression of Nav1.6 sodium channels induced by overexpression of APP. JNK, in turn, phosphorylated APP. Nav1.6 sodium channel surface expression was increased by T668E and decreased by T668A, mutations of APP695 mimicking and preventing Thr-668 phosphorylation, respectively. Phosphorylation of APP695 at Thr-668 enhanced its interaction with Nav1.6. Therefore, we show that APP enhances Nav1.6 sodium channel cell surface expression through a G_o-coupled JNK pathway.     

Regulation and role of cyclin-dependent kinase activity in neuronal survival and death

Cyclin-dependent kinase (Cdk)5 is a proline-directed Ser/Thr protein kinase that functions mainly in neurons and is activated by binding to a regulatory subunit, p35 or p39. Kinase activity is mainly determined by the amount of p35 available, which is controlled by a balance between synthesis and degradation. Kinase activity is also regulated by Cdk5 phosphorylation, but the activity of phosphorylated Cdk5 is in contrast to that of cycling Cdks. Cdk5 is a versatile protein kinase that regulates multiple neuronal activities including neuronal migration and synaptic signaling. Further, Cdk5 plays a role in both survival and death of neurons. Long-term inactivation of Cdk5 triggers cell death, and the survival activity of Cdk5 is apparent when neurons suffer from stress. In contrast, hyper-activation of Cdk5 by p25 promotes cell death, probably by reactivating cell-cycle machinery in the nucleus. The pro-death activity is suppressed by membrane association of Cdk5 via myristoylation of p35. Appropriate activity, localization, and regulation of Cdk5 may be critical for long-term survival of neurons, which is more than 80 years in the case of humans.     

Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein.

Accumulation of the amyloid A beta peptide, which is derived from a larger precursor protein (APP), and the formation of plaques, are major events believed to be involved in the etiology of Alzheimer's disease. Abnormal regulation of the metabolism of APP may contribute to the deposition of plaques. APP is an integral membrane protein containing several putative phosphorylation sites within its cytoplasmic domain. We report here that APP is phosphorylated at Thr668 by p34cdc2 protein kinase (cdc2 kinase) in vitro, and in a cell cycle-dependent manner in vivo. At the G2/M phase of the cell cycle, when APP phosphorylation is maximal, the levels of mature APP (mAPP) and immature APP (imAPP) do not change significantly. However, imAPP is altered qualitatively. Furthermore, the level of the secreted extracellular N-terminal domain (APPS) is decreased and that of the truncated intracellular C-terminal fragment (APPCOOH) is increased. These findings suggest the possibility that phosphorylation-dependent events occurring during the cell cycle affect the metabolism of APP. Alterations in these events might play a role in the pathogenesis of Alzheimer's disease.     

Dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A-mediated phosphorylation of amyloid precursor protein: evidence for a functional link between Down syndrome and Alzheimer's disease

Most individuals with Down Syndrome (DS) show an early-onset of Alzheimer's disease (AD), which potentially results from the presence of an extra copy of a segment of chromosome 21. Located on chromosome 21 are the genes that encode β-amyloid (Aβ) precursor protein ( APP ), a key protein involved in the pathogenesis of AD, and dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A ( DYRK1A ), a proline-directed protein kinase that plays a critical role in neurodevelopment. Here, we describe a potential mechanism for the regulation of AD pathology in DS brains by DYRK1A-mediated phosphorylation of APP. We show that APP is phosphorylated at Thr668 by DYRK1A in vitro and in mammalian cells. The amounts of phospho-APP and Aβ are increased in the brains of transgenic mice that over-express the human DYRK1A protein. Furthermore, we show that the amounts of phospho-APP as well as those of APP and DYRK1A are elevated in human DS brains. Taken together, these results reveal a potential regulatory link between APP and DYRK1A in DS brains, and suggest that the over-expression of DYRK1A in DS may play a role in accelerating AD pathogenesis through phosphorylation of APP.     

Reduced amyloidogenic processing of the amyloid β-protein precursor by the small-molecule Differentiation Inducing Factor-1

The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Aβ properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid β-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Aβ40 and Aβ42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Aβ42 to Aβ40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Aβ. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a γ-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.