Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis

One of the pathological hallmarks of Alzheimer disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are primarily composed of aggregated amyloid β peptide (Aβ), a proteolytic fragment of the transmembrane amyloid precursor protein (APP). For APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific protease complexes can cleave APP. Several recent genome-wide association studies have reported that several single nucleotide polymorphisms (SNPs) in the phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) gene were significantly associated with Alzheimer disease, suggesting a role in APP endocytosis and Aβ generation. Here, we show that PICALM co-localizes with APP in intracellular vesicles of N2a-APP cells after endocytosis is initiated. PICALM knockdown resulted in reduced APP internalization and Aβ generation. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM was found to be expressed in neurons and co-localized with APP throughout the cortex and hippocampus in APP/PS1 mice. PICALM expression was altered using AAV8 gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6-month-old APP/PS1 mice. PICALM knockdown decreased soluble and insoluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, PICALM overexpression increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. PICALM contributes to amyloid plaque load in brain likely via its effect on Aβ metabolism.     

Structural basis for increased toxicity of pathological aβ42:aβ40 ratios in Alzheimer disease

The β-amyloid peptide (Aβ) is directly related to neurotoxicity in Alzheimer disease (AD). The two most abundant alloforms of the peptide co-exist under normal physiological conditions in the brain in an Aβ(42):Aβ(40) ratio of ～1:9. This ratio is often shifted to a higher percentage of Aβ(42) in brains of patients with familial AD and this has recently been shown to lead to increased synaptotoxicity. The molecular basis for this phenomenon is unclear. Although the aggregation characteristics of Aβ(40) and Aβ(42) individually are well established, little is known about the properties of mixtures. We have explored the biophysical and structural properties of physiologically relevant Aβ(42):Aβ(40) ratios by several techniques. We show that Aβ(40) and Aβ(42) directly interact as well as modify the behavior of the other. The structures of monomeric and fibrillar assemblies formed from Aβ(40) and Aβ(42) mixtures do not differ from those formed from either of these peptides alone. Instead, the co-assembly of Aβ(40) and Aβ(42) influences the aggregation kinetics by altering the pattern of oligomer formation as evidenced by a unique combination of solution nuclear magnetic resonance spectroscopy, high molecular weight mass spectrometry, and cross-seeding experiments. We relate these observations to the observed enhanced toxicity of relevant ratios of Aβ(42):Aβ(40) in synaptotoxicity assays and in AD patients.     

Focally elevated creatine detected in amyloid precursor protein (APP) transgenic mice and Alzheimer disease brain tissue

The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the beta-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21-89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process.     

Myeloperoxidase targets apolipoprotein A-I, the major high density lipoprotein protein, for site-specific oxidation in human atherosclerotic lesions

Oxidative damage by myeloperoxidase (MPO) has been proposed to deprive HDL of its cardioprotective effects. In vitro studies reveal that MPO chlorinates and nitrates specific tyrosine residues of apoA-I, the major HDL protein. After Tyr-192 is chlorinated, apoA-I is less able to promote cholesterol efflux by the ABCA1 pathway. To investigate the potential role of this pathway in vivo, we used tandem mass spectrometry with selected reaction monitoring to quantify the regiospecific oxidation of apoA-I. This approach demonstrated that Tyr-192 is the major chlorination site in apoA-I in both plasma and lesion HDL of humans. We also found that Tyr-192 is the major nitration site in apoA-I of circulating HDL but that Tyr-18 is the major site in lesion HDL. Levels of 3-nitrotyrosine strongly correlated with levels of 3-chlorotyrosine in lesion HDL, and Tyr-18 of apoA-I was the major nitration site in HDL exposed to MPO in vitro, suggesting that MPO is the major pathway for chlorination and nitration of HDL in human atherosclerotic tissue. These observations may have implications for treating cardiovascular disease, because recombinant apoA-I is under investigation as a therapeutic agent and mutant forms of apoA-I that resist oxidation might be more cardioprotective than the native form.     

Comparative lipidomic analysis of mouse and human brain with Alzheimer disease

Lipids are key regulators of brain function and have been increasingly implicated in neurodegenerative disorders including Alzheimer disease (AD). Here, a systems-based approach was employed to determine the lipidome of brain tissues affected by AD. Specifically, we used liquid chromatography-mass spectrometry to profile extracts from the prefrontal cortex, entorhinal cortex, and cerebellum of late-onset AD (LOAD) patients, as well as the forebrain of three transgenic familial AD (FAD) mouse models. Although the cerebellum lacked major alterations in lipid composition, we found an elevation of a signaling pool of diacylglycerol as well as sphingolipids in the prefrontal cortex of AD patients. Furthermore, the diseased entorhinal cortex showed specific enrichment of lysobisphosphatidic acid, sphingomyelin, the ganglioside GM3, and cholesterol esters, all of which suggest common pathogenic mechanisms associated with endolysosomal storage disorders. Importantly, a significant increase in cholesterol esters and GM3 was recapitulated in the transgenic FAD models, suggesting that these mice are relevant tools to study aberrant lipid metabolism of endolysosomal dysfunction associated with AD. Finally, genetic ablation of phospholipase D(2), which rescues the synaptic and behavioral deficits of an FAD mouse model, fully normalizes GM3 levels. These data thus unmask a cross-talk between the metabolism of phosphatidic acid, the product of phospholipase D(2), and gangliosides, and point to a central role of ganglioside anomalies in AD pathogenesis. Overall, our study highlights the hypothesis generating potential of lipidomics and identifies novel region-specific lipid anomalies potentially linked to AD pathogenesis.     

Amyloidogenic properties of the prion protein fragment PrP(185-208): comparison with Alzheimer's peptide Abeta(1-28), influence of heparin and cell toxicity

Amyloid fibrils are a hallmark of Alzheimer’s and prion diseases. In both pathologies fibrils are found associated to glycosaminoglycans, modulators of the aggregation process. Amyloid peptides and proteins with very poor sequence homologies originate very similar aggregates. This implies the possible existence of a common formation mechanism. A homologous structural motif has recently been described for the Alzheimer’s peptide Aβ(1-28) and the prion protein fragment PrP(185-208). We have studied the influence histidine residues and heparin on the aggregation process of both peptides and determined the possible amyloid characteristics of PrP(185-208), still unknown. The results show that PrP(185-208) forms amyloid aggregates in the presence of heparin. Histidines influence the aggregation kinetics, as in Aβ(1-28), although to a lesser extent. Other spectroscopic properties of the PrP(185-208) fragment are shown to be equivalent to those of other amyloid peptides and PrP(185-208) is shown to be cytotoxic using a neuroblastoma cell line.     

Structural and morphological characterization of aggregated species of α-synuclein induced by docosahexaenoic acid

The interaction of brain lipids with α-synuclein may play an important role in the pathogenesis of Parkinson disease (PD). Docosahexaenoic acid (DHA) is an abundant fatty acid of neuronal membranes, and it is presents at high levels in brain areas with α-synuclein inclusions of patients with PD. In animal models, an increase of DHA content in the brain induces α-synuclein oligomer formation in vivo. However, it is not clear whether these oligomeric species are the precursors of the larger aggregates found in Lewy bodies of post-mortem PD brains. To characterize these species and to define the role of fatty acids in amyloid formation, we investigated the aggregation process of α-synuclein in the presence of DHA. We found that DHA readily promotes α-synuclein aggregation and that the morphology of these aggregates is dependent on the ratio between the protein and DHA. In the presence of a molar ratio protein/DHA of 1:10, amyloid-like fibrils are formed. These fibrils are morphologically different from those formed by α-synuclein alone and have a less packed structure. At a protein/DHA molar ratio of 1:50, we observe the formation of stable oligomers. Moreover, chemical modifications, methionine oxidations, and protein-lipid adduct formations are induced by increasing concentrations of DHA. The extent of these modifications defines the structure and the stability of aggregates. We also show that α-synuclein oligomers are more toxic if generated in the presence of DHA in dopaminergic neuronal cell lines, suggesting that these species might be important in the neurodegenerative process associated with PD.     

Novel Zinc-binding Site in the E2 Domain Regulates Amyloid Precursor-like Protein 1 (APLP1) Oligomerization

The amyloid precursor protein (APP) and the APP-like proteins 1 and 2 (APLP1 and APLP2) are a family of multidomain transmembrane proteins possessing homo- and heterotypic contact sites in their ectodomains. We previously reported that divalent metal ions dictate the conformation of the extracellular APP E2 domain (Dahms, S. O., Könnig, I., Roeser, D., Gührs, K.-H., Mayer, M. C., Kaden, D., Multhaup, G., and Than, M. E. (2012) J. Mol. Biol. 416, 438–452), but unresolved is the nature and functional importance of metal ion binding to APLP1 and APLP2. We found here that zinc ions bound to APP and APLP1 E2 domains and mediated their oligomerization, whereas the APLP2 E2 domain interacted more weakly with zinc possessing a less surface-exposed zinc-binding site, and stayed monomeric. Copper ions bound to E2 domains of all three proteins. Fluorescence resonance energy transfer (FRET) analyses examined the effect of metal ion binding to APP and APLPs in the cellular context in real time. Zinc ions specifically induced APP and APLP1 oligomerization and forced APLP1 into multimeric clusters at the plasma membrane consistent with zinc concentrations in the blood and brain. The observed effects were mediated by a novel zinc-binding site within the APLP1 E2 domain as APLP1 deletion mutants revealed. Based upon its cellular localization and its dominant response to zinc ions, APLP1 is mainly affected by extracellular zinc among the APP family proteins. We conclude that zinc binding and APP/APLP oligomerization are intimately linked, and we propose that this represents a novel mechanism for regulating APP/APLP protein function at the molecular level.     

Polymorphic triple beta-sheet structures contribute to amide hydrogen/deuterium (H/D) exchange protection in the Alzheimer amyloid beta42 peptide

Characterization of the polymorphic structural range of Aβ oligomers is important to the understanding of the mechanisms of toxicity. Yet for highly polymorphic ensembles, experimental structural elucidation is difficult. Here, we use a combination of NMR solvent protection experiments and computational structural screening to identify major species in the amyloid conformational ensemble. We examined the polymorphic pentamer and fibril seeds of Aβ42 and its mutants and compared the theoretical backbone amide protection obtained from simulations with experimental hydrogen/deuterium (H/D) exchange protection ratio. We observed that highly flexible pentamers do not share structural similarities with fibril seed oligomers, except the turn regions. We found that a novel amyloid structural motif of a triple β-sheet, with the N-terminal residues interacting with the core (Lys(17)-Glu(22)) β-sheet region, correlates with H/D exchange protection. The triple β-sheet Aβ42 oligomer has a minimal exposure of hydrophobic residues and is further stabilized by the E22Q (Dutch) mutation in Alzheimer disease. The experimental H/D exchange solvent protection ratio implies that triple β-sheet fibrils and globulomers could coexist in the Aβ42 ensemble, pointing to a broad heterogeneous aggregate population. Our results suggest that an approach that combines computational modeling with NMR protection data can be a useful strategy for obtaining clues to the preferred conformational species of the assemblies in solution and help in alleviating experimental difficulties and consequently possible errors in the exchange data for Aβ42 fibrils.     

Age-dependent accumulation of soluble amyloid beta (Abeta) oligomers reverses the neuroprotective effect of soluble amyloid precursor protein-alpha (sAPP(alpha)) by modulating phosphatidylinositol 3-kinase (PI3K)/Akt-GSK-3beta pathway in Alzheimer mouse model

Neurotrophins, activating the PI3K/Akt signaling pathway, control neuronal survival and plasticity. Alterations in NGF, BDNF, IGF-1, or insulin signaling are implicated in the pathogenesis of Alzheimer disease. We have previously characterized a bigenic PS1×APP transgenic mouse displaying early hippocampal Aβ deposition (3 to 4 months) but late (17 to 18 months) neurodegeneration of pyramidal cells, paralleled to the accumulation of soluble Aβ oligomers. We hypothesized that PI3K/Akt/GSK-3β signaling pathway could be involved in this apparent age-dependent neuroprotective/neurodegenerative status. In fact, our data demonstrated that, as compared with age-matched nontransgenic controls, the Ser-9 phosphorylation of GSK-3β was increased in the 6-month PS1×APP hippocampus, whereas in aged PS1×APP animals (18 months), GSK-3β phosphorylation levels displayed a marked decrease. Using N2a and primary neuronal cell cultures, we demonstrated that soluble amyloid precursor protein-α (sAPPα), the predominant APP-derived fragment in young PS1×APP mice, acting through IGF-1 and/or insulin receptors, activated the PI3K/Akt pathway, phosphorylated the GSK-3β activity, and in consequence, exerted a neuroprotective action. On the contrary, several oligomeric Aβ forms, present in the soluble fractions of aged PS1×APP mice, inhibited the induced phosphorylation of Akt/GSK-3β and decreased the neuronal survival. Furthermore, synthetic Aβ oligomers blocked the effect mediated by different neurotrophins (NGF, BDNF, insulin, and IGF-1) and sAPPα, displaying high selectivity for NGF. In conclusion, the age-dependent appearance of APP-derived soluble factors modulated the PI3K/Akt/GSK-3β signaling pathway through the major neurotrophin receptors. sAPPα stimulated and Aβ oligomers blocked the prosurvival signaling. Our data might provide insights into the selective vulnerability of specific neuronal groups in Alzheimer disease.     

Breaking the light and heavy chain linkage of human immunoglobulin G1 (IgG1) by radical reactions

We report that the production of hydrogen peroxide by radical chain reductions of molecular oxygen into water in buffers leads to hinge degradation of a human IgG1 under thermal incubation conditions. The production of the hydrogen peroxide can be accelerated by superoxide dismutase or redox active metal ions or inhibited by free radical scavengers. The hydrogen peroxide production rate correlates well with the hinge cleavage. In addition to radical reaction mechanisms described previously, new degradation pathways and products were observed. These products were determined to be generated via radical reactions initiated by electron transfer and addition to the interchain disulfide bond between Cys(215) of the light chain and Cys(225) of the heavy chain. Decomposition of the resulting disulfide bond radical anion breaks the C-S bond at the side chain of Cys, converting it into dehydroalanine and generating a sulfur radical adduct at its counterpart. The hydrolysis of the unsaturated dehydropeptides removes Cys and yields an amide at the C terminus of the new fragment. Meanwhile, the competition between the carbonyl (-C(α)ONH-) and the side chain of Cys allows an electron transfer to the α carbon, forming a new intermediate radical species (-(·)C(α)(O(-))NH-) at Cys(225). Dissociative deamidation occurs along the N-C(α) bond, resulting in backbone cleavage. Given that hydrogen peroxide is a commonly observed product of thermal stress and plays a role in mediating the unique degradation of an IgG1, strategies for improving stability of human antibody therapeutics are discussed.     

Acetylation of retinal histones in diabetes increases inflammatory proteins: effects of minocycline and manipulation of histone acetyltransferase (HAT) and histone deacetylase (HDAC)

Histone acetylation was significantly increased in retinas from diabetic rats, and this acetylation was inhibited in diabetics treated with minocycline, a drug known to inhibit early diabetic retinopathy in animals. Histone acetylation and expression of inflammatory proteins that have been implicated in the pathogenesis of diabetic retinopathy were increased likewise in cultured retinal Müller glia grown in a diabetes-like concentration of glucose. Both the acetylation and induction of the inflammatory proteins in elevated glucose levels were significantly inhibited by inhibitors of histone acetyltransferase (garcinol and antisense against the histone acetylase, p300) or activators of histone deacetylase (theophylline and resveratrol) and were increased by the histone deacetylase inhibitor, suberolylanilide hydroxamic acid. We conclude that hyperglycemia causes acetylation of retinal histones (and probably other proteins) and that the acetylation contributes to the hyperglycemia-induced up-regulation of proinflammatory proteins and thereby to the development of diabetic retinopathy.     

Biophysical Studies on Interactions and Assembly of Full-size E3 Ubiquitin Ligase: SUPPRESSOR OF CYTOKINE SIGNALING 2 (SOCS2)-ELONGIN BC-CULLIN 5-RING BOX PROTEIN 2 (RBX2)*

The multisubunit cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes, and cancer and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, among their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5^SOCS2), binds phosphorylated growth hormone receptor as its main substrate. Here, we demonstrate that the components of CRL5^SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated growth hormone receptor peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in Escherichia coli and in Sf21 insect cells, respectively, were used to reconstitute neddylated and unneddylated CRL5^SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike other E3 ligases, CRL5^SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multiangle static light scattering and native MS. Affinities of the protein-protein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for full-size neddylated and unneddylated CRL5^SOCS2 complexes is supported by traveling wave ion mobility mass spectrometry data.     

Unambiguous characterization of site-specific phosphorylation of leucine-rich repeat Fli-I-interacting protein 2 (LRRFIP2) in Toll-like receptor 4 (TLR4)-mediated signaling

In the TLR4 signaling pathways, we previously characterized a signal regulator, LRRFIP2, that modulates the time course-dependent changes in NF-κB activity through its dynamic interaction with the TLR adaptor protein, MyD88. However, little is known about the driving force behind the LPS-inducible dynamics between LRRFIP2 and MyD88. We have therefore designed a multiplex label-free quantitative proteomics method to investigate dynamic changes of LRRFIP2 phosphorylation upon LPS stimulation. Given our observation that LRRFIP2 binds to MyD88 through its serine-rich domain in which most of serine residues have the propensity to be phosphorylated, we used collision-activated dissociation- and electron transfer dissociation-based methods in a complementary manner to unambiguously localize phosphorylation sites in the peptides constituting the serine-rich domain. Among 23 phosphorylation sites identified and first quantified by the label-free approach and then verified by the AACT/SILAC (amino acid-coded tagging/stable isotope labeling in cell culture)-based quantitation method, phosphorylation at serine 202 showed a significant LPS-induced dynamic change during the full-course cellular response to LPS stimulation. The substitution of serine 202 with nonphosphorylated residues by site-directed mutagenesis resulted in a weakened LRRFIP2-MyD88 interaction and a concurrently reduced activity in downstream NF-κB. Taking these results together, phosphorylation at serine 202 was found to regulate the dynamics of the LRRFIP2-MyD88 interaction, which in turn modulated the strength and duration of TLR4 signaling. Strategically, we have demonstrated the importance of precise identification of the biologically relevant phosphorylation site(s) using comprehensive mass spectrometry-based quantitative proteomics approaches in guiding downstream biological characterization experiments, which could otherwise be both time- and cost-consuming for a large number of phosphorylation possibilities.