Metallostasis and amyloid β-degrading enzymes

Amyloid-Beta (Aβ) is a major constituent of senile plaques and one of the principle hallmarks of Alzheimer's disease (AD). The peptide is produced by proteolytic cleavage of the larger amyloid precursor protein (APP). Increased production and aggregation of the peptide are associated with pathology. Emerging evidence suggests that the steady-state levels of Aβ are determined by the balance between its production and degradation. For this reason, the tuning of the activity of enzymes that degrade Aβ may be a promising approach in the development of novel therapeutics aimed at reducing Aβ concentration by enhancing its removal. A great part of Aβ degrading enzymes are known to be metalloproteases. In the last decade increasing evidence supported the idea that metal ion homeostasis is affected in several regions of AD brain and metals play an important role in tuning enzyme activity. There are three main different pathways by which metal ions can affect the proteolytic enzymes responsible for Aβ peptides degradation, as metal ions can: (i) form complexes with Aβ peptides that are not easily degraded; (ii) directly bind to degradative enzymes; (iii) produce signalling cascades that alter enzymes activity involved in Aβ catabolism. In the current literature the three points mentioned above are very often puzzled, resulting in a quite fragmentary scenario. The aim of this work is to find a link between metal ion homeostasis and Aβ degradation by separating and analysing the three different pathways proposed.     

Cudrania cochinchinensis attenuates amyloid β protein-mediated microglial activation and promotes glia-related clearance of amyloid β protein

Background

Microglial inflammation may significantly contribute to the pathology of Alzheimer’s disease. To examine the potential of Cudrania cochinchinensis to ameliorate amyloid β protein (Aβ)-induced microglia activation, BV-2 microglial cell line, and the ramified microglia in the primary glial mixed cultured were employed.

Results

Lipopolysaccharide (LPS), Interferon-γ (IFN-γ), fibrillary Aβ (fAβ), or oligomeric Aβ (oAβ) were used to activate microglia. LPS and IFN-γ, but not Aβs, activated BV-2 cells to produce nitric oxide through an increase in inducible nitric oxide synthase (iNOS) expression without significant effects on cell viability of microglia. fAβ, but not oAβ, enhanced the IFN-γ-stimulated nitric oxide production and iNOS expression.The ethanol/water extracts of Cudrania cochinchinensis (CC-EW) and the purified isolated components (i.e. CCA to CCF) effectively reduced the nitric oxide production and iNOS expression stimulated by IFN-γ combined with fAβ. On the other hand, oAβ effectively activated the ramified microglia in mixed glial culture by observing the morphological alteration of the microglia from ramified to amoeboid. CC-EW and CCB effectively prohibit the Aβ-mediated morphological change of microglia. Furthermore, CC-EW and CCB effectively decreased Aβ deposition and remained Aβ in the conditioned medium suggesting the effect of CC-EW and CCB on promoting Aβ clearance. Results are expressed as mean ± S.D. and were analyzed by ANOVA with post-hoc multiple comparisons with a Bonferroni test.

Conclusions

The components of Cudrania cochinchinensis including CC-EW and CCB are potential for novel therapeutic intervention for Alzheimer’s disease.     

Formation of amyloid fibrils from β-amylase

Fibril formation has been considered a significant feature of amyloid proteins. However, it has been proposed that fibril formation is a common property of many proteins under appropriate conditions. We studied the fibril formation of β-amylase, a non-amyloid protein rich in α-helical structure, because the secondary structure of β-amylase is similar to that of prions. With the conditions for the fibril formation of prions, β-amylase proteins were converted into amyloid fibrils. The features of β-amylase proteins and fibrils are compared to prion proteins and fibrils. Furthermore, the cause of neurotoxicity in amyloid diseases is discussed.     

Surfactant-induced conformational transition of amyloid β-peptide

Accumulating evidence suggests that Aβ_1–42–membrane interactions may play an important role in the pathogenesis of Alzheimer’s disease. However, the mechanism of this structural transition remains unknown. In this work, we have shown that submicellar concentrations of sodium dodecyl sulfate (SDS) can provide a minimal platform for Aβ_1–42 self-assembly. To further investigate the relation between Aβ_1–42 structure and function, we analyzed peptide conformation and aggregation at various SDS concentrations using circular dichroism (CD), Fourier transform infrared spectroscopy, and gel electrophoresis. These aggregates, as observed via atomic force microscopy, appeared as globular particles in submicellar SDS with diameters of 35–60 nm. Upon sonication, these particles increased in disc diameter to 100 nm. Pyrene I ₃/I ₁ ratios and 1-anilinonaphthalene-8-sulfonic acid binding studies indicated that the peptide interior is more hydrophobic than the SDS micelle interior. We have also used Forster resonance energy transfer between N-terminal labeled pyrene and tyrosine (10) of Aβ_1–42 in various SDS concentrations for conformational analysis. The results demonstrate that SDS at submicellar concentrations accelerates the formation of spherical aggregates, which act as niduses to form large spherical aggregates upon sonication. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proteomics of β2-microglobulin amyloid fibrils

Knowledge on the chemical structure of β2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of β2-microglobulin extracted from non-osteotendinous tissues. β2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to β2-microglobulin derived from fibrils but is not observed in the β2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar β2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1–2% of the intact molecule.     

Can serum amyloid A or macrophage colony stimulating factor serve as marker of amyloid formation process?

Amyloid formation depends on amyloid precursor production and is influenced by the activity of the underlying disorder and mediated by some proinflammatory cytokines. In this pilot study we tried to find some specific markers that could establish the activity of the disease. We investigated 45 samples of sera and 38 samples of urine from patients (pts) with secondary amyloidosis (AA), primary amyloidosis (AL), systemic autoimmune diseases with renal impairment (Vasc) and healthy controls (Co). Pts with AA had increased plasma levels of TNF alpha (9.97 +/- 4.22 vs. 2.63 +/- 1.34 pg/mL, p < 0.001) and SAA (43.14 +/- 16.0 vs. 3.42 +/- 0.7 ng/mL, p < 0.05) in comparison with Co. Plasma levels of M-CSF in the AA group were significantly increased in comparison with Co (1077.34 +/- 238.6 vs. 137.71 +/- 19.6, pg/mL, p < 0.001) and also in comparison with Vasc (482.24 +/- 86.7 pg/mL, p < 0.05). Urinary excretions of TNF alpha (8.92 +/- 8.1 vs. 0.17 +/- 0.11 microgram/mol creatinine, p < 0.01), sIL-6R (1.39 +/- 1.14 vs. 0.07 +/- 0.05 g/mol creatinine, p < 0.01) and M-CSF (650.2 +/- 153.7 vs. 33.3 +/- 8.6 micrograms/mol creatinine, p < 0.01) in AA were significantly increased in comparison with Co. Pts with AL had increased plasma levels of M-CSF (819.83 +/- 264.2 vs. 137.71 +/- 19.6 pg/mL, p < 0.05) and urinary excretion of M-CSF (865.0 +/- 188.4 vs. 33.3 +/- 8.6 micrograms/mol creatinine, p < 0.01) in comparison with Co. SAA has a low specificity for amyloidosis but is a sensitive acute phase reactant. TNF alpha, a proinflammatory cytokine, may reflect the activity of the underlying diseases in secondary amyloidosis. M-CSF was increased both in plasma and urine in amyloidosis groups and seems to be the most promising (possibly specific) marker of amyloidosis.     

Pyroglutamate amyloid β (Aβ) aggravates behavioral deficits in transgenic amyloid mouse model for Alzheimer disease

Pyroglutamate-modified Aβ peptides at amino acid position three (Aβ(pE3-42)) are gaining considerable attention as potential key players in the pathogenesis of Alzheimer disease (AD). Aβ(pE3-42) is abundant in AD brain and has a high aggregation propensity, stability and cellular toxicity. The aim of the present work was to study the direct effect of elevated Aβ(pE3-42) levels on ongoing AD pathology using transgenic mouse models. To this end, we generated a novel mouse model (TBA42) that produces Aβ(pE3-42). TBA42 mice showed age-dependent behavioral deficits and Aβ(pE3-42) accumulation. The Aβ profile of an established AD mouse model, 5XFAD, was characterized using immunoprecipitation followed by mass spectrometry. Brains from 5XFAD mice demonstrated a heterogeneous mixture of full-length, N-terminal truncated, and modified Aβ peptides: Aβ(1-42), Aβ(1-40), Aβ(pE3-40), Aβ(pE3-42), Aβ(3-42), Aβ(4-42), and Aβ(5-42). 5XFAD and TBA42 mice were then crossed to generate transgenic FAD42 mice. At 6 months of age, FAD42 mice showed an aggravated behavioral phenotype compared with single transgenic 5XFAD or TBA42 mice. ELISA and plaque load measurements revealed that Aβ(pE3) levels were elevated in FAD42 mice. No change in Aβ(x)(-42) or other Aβ isoforms was discovered by ELISA and mass spectrometry. These observations argue for a seeding effect of Aβ(pE-42) in FAD42 mice.     

Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid?

In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-32. However, soluble amyloid oligomers of lysozyme caused death of all these cell types, as estimated by flow-cytometry counting dead cells stained with ethidium bromide. The primary cell cultures appeared to be more sensitive to amyloid than neuroblastoma cell line IMR-32. Amyloid cytotoxicity depends on the size of oligomeric particles: samples containing 20-mers formed at pH 4.5 were more toxic than tetramers and octamers present in the solution at pH 2.0. Soluble amyloid oligomers can self-assemble into doughnut-like structures; however, no correlation was observed between the amount of the doughnut-like structures in the sample and its cytotoxicity. The fact that the intermediate oligomers of such an abundant protein as lysozyme display cytotoxicity confirms a hypothesis that cytotoxicity is a common feature of protein amyloid. Inhibition of intermediate oligomer formation is crucial in preventing amyloid pathogeneses.     

Pharmacological removal of serum amyloid P component from intracerebral plaques and cerebrovascular Aβ amyloid deposits in vivo

Human amyloid deposits always contain the normal plasma protein serum amyloid P component (SAP), owing to its avid but reversible binding to all amyloid fibrils, including the amyloid β (Aβ) fibrils in the cerebral parenchyma plaques and cerebrovascular amyloid deposits of Alzheimer''s disease (AD) and cerebral amyloid angiopathy (CAA). SAP promotes amyloid fibril formation in vitro, contributes to persistence of amyloid in vivo and is also itself directly toxic to cerebral neurons. We therefore developed (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a drug that removes SAP from the blood, and thereby also from the cerebrospinal fluid (CSF), in patients with AD. Here we report that, after introduction of transgenic human SAP expression in the TASTPM double transgenic mouse model of AD, all the amyloid deposits contained human SAP. Depletion of circulating human SAP by CPHPC administration in these mice removed all detectable human SAP from both the intracerebral and cerebrovascular amyloid. The demonstration that removal of SAP from the blood and CSF also removes it from these amyloid deposits crucially validates the strategy of the forthcoming ‘Depletion of serum amyloid P component in Alzheimer''s disease (DESPIAD)’ clinical trial of CPHPC. The results also strongly support clinical testing of CPHPC in patients with CAA.     

Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson’s disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.     

Interactions between amyloid β peptide and lipid membranes

The presence of amyloid plaques in the brain is a typical characteristic of Alzheimer's disease (AD). Amyloid plaques are formed from the deposits of aggregated amyloid β peptide (Aβ). The toxicity induced by Aβ aggregates is correlated with Aβ-membrane interactions. The mutual influences between aggregation and membranes are complicated and unclear. In recent years advanced experiments and findings are emerging to give us more detailed information on Aβ-membrane interactions. In this review, we mainly focus on the Aβ-membrane interactions and membrane-induced Aβ structures. The mechanism of Aβ-membrane interactions is also summarized, which provides insights into the prevention and treatment of AD.     

Molecular biology of Alzheimer's amyloid—Dutch variant

Hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D) (or familial cerebral amyloid angiopathy) and familial Alzheimer's disease (FAD) share several properties. Both are autosomal dominant forms of cerebral amyloidosis characterized by beta-amyloid (A beta) deposition. In HCHWA-D the A beta is predominantly found in blood vessels and in early parenchymal plaques, whereas in AD parenchymal A beta deposits in the form of senile plaques and neurofibrillary tangles are a more prominent finding. Point mutations in the amyloid precursor protein (APP) have recently been described, in both conditions. A G to C transversion at codon 618 (extracellular portion of APP695), producing a single amino acid substitution of glutamine instead of glutamine acid, occurs in HCHWA-D; whereas mutations at codon 642 in the intramembrane region of APP695 (phenylalanine, isoleucine, or glycine instead of valine) are associated with early onset FAD. This suggests that the site of particular mutations in the APP gene and the type of amino acid substitution in the APP holoprotein are more important in determining clinicopathological phenotype and age at which A beta is deposited. Thus FAD and HCHWA-D can be regarded as two sides of the same coin.     

The neurotransmitter serotonin interrupts α-synuclein amyloid maturation

Indolic derivatives can affect fibril growth of amyloid forming proteins. The neurotransmitter serotonin (5-HT) is of particular interest, as it is an endogenous molecule with a possible link to neuropsychiatric symptoms of Parkinson disease. A key pathomolecular mechanism of Parkinson disease is the misfolding and aggregation of the intrinsically unstructured protein α-synuclein. We performed a biophysical study to investigate an influence between these two molecules. In an isolated in vitro system, 5-HT interfered with α-synuclein amyloid fiber maturation, resulting in the formation of partially structured, SDS-resistant intermediate aggregates. The C-terminal region of α-synuclein was essential for this interaction, which was driven mainly by electrostatic forces. 5-HT did not bind directly to monomeric α-synuclein molecules and we propose a model where 5-HT interacts with early intermediates of α-synuclein amyloidogenesis, which disfavors their further conversion into amyloid fibrils.     

Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?

In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 degrees C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.     

Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid β protein

Background  

The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by α-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of β- and γ-secretases generates the amyloid β protein (Aβ). In this study, we investigated the effects of modulators of endocytosis on APP processing.