The multiple mechanisms of amyloid deposition: The role of parkin

Amyloid deposition is one of the central neuropathological abnormalities in Alzheimer disease (AD) but it also takes places in many neurodegenerative diseases such as prionic disorders, Huntington''s disease (HD) and others. Up to very recently amyloid formation was considered a very slow process of deposition of an abnormal protein due to genetic abnormalities or post-translational modification of the deposited protein. Recent data suggest that the process of amyloidogenesis may be much more rapid in many cases and due to multiple mechanisms.We have found a mouse model of progressive neurodegeneration that resemble motor, behavioral and pathological hallmarks of parkinsonism and tauopathies, but surprisingly, also present amyloid deposits in brain and peripheral organs. Here we review some of these recent works which may provide new insight into the process of formation of amyloid and, perhaps, new ideas for its treatment.Key words: □-amyloid, Alzheimer disease, chaperones, fronto-temporal dementia, parkin, Parkinson disease, PSP, proteosome, tau protein, tauopathies, autophagy, transgenic mice     

In silico prediction of novel residues involved in amyloid primary nucleation of human I56T and D67H lysozyme

Background

Amyloidogenic proteins are most often associated with neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, but there are more than two dozen human proteins known to form amyloid fibrils associated with disease. Lysozyme is an antimicrobial protein that is used as a general model to study amyloid fibril formation. Studies aimed at elucidating the process of amyloid formation of lysozyme tend to focus on partial unfolding of the native state due to the relative instability of mutant amyloidogenic variants. While this is well supported, the data presented here suggest the native structure of the variants may also play a role in primary nucleation.

Results

Three-dimensional structural analysis identified lysozyme residues 21, 62, 104, and 122 as displaced in both amyloidogenic variants compared to wild type lysozyme. Residue interaction network (RIN) analysis found greater clustering of residues 112–117 in amyloidogenic variants of lysozyme compared to wild type. An analysis of the most energetically favored predicted dimers and trimers provided further evidence for a role for residues 21, 62, 104, 122, and 112–117 in amyloid formation.

Conclusions

This study used lysozyme as a model to demonstrate the utility of combining 3D structural analysis with RIN analysis for studying the general process of amyloidogenesis. Results indicated that binding of two or more amyloidogenic lysozyme mutants may be involved in amyloid nucleation by placing key residues (21, 62, 104, 122, and 112–117) in proximity before partial unfolding occurs. Identifying residues in the native state that may be involved in amyloid formation could provide novel drug targets to prevent a range of amyloidoses.

     

Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts

Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.     

Dependency of Glucose Transport on d-Glucoside 3-Dehydrogenase as a Conversion System of Glucose to 3-Ketoglucose in Agrobacterium tumefaciens

Alzheimer’s disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.     

The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice

Accumulation of cerebral amyloid beta-protein (Abeta) is believed to be part of the pathogenic process in Alzheimer's disease. Abeta is derived by proteolytic cleavage from a precursor protein, the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein, and its carboxyl-terminal intracellular domain binds to X11beta, a neuronal adaptor protein. X11beta has been shown to inhibit the production of Abeta in transfected non-neuronal cells in culture. However, whether this is also the case in vivo in the brain and whether X11beta can also inhibit the deposition of Abeta as amyloid plaques is not known. Here we show that transgenic overexpression of X11beta in neurons leads to a decrease in cerebral Abeta levels in transgenic APPswe Tg2576 mice that are a model of the amyloid pathology of Alzheimer's disease. Moreover, overexpression of X11beta retards amyloid plaque formation in these APPswe mice. Our findings suggest that modulation of X11beta function may represent a novel therapeutic approach for preventing the amyloid pathology of Alzheimer's disease.     

Strong inhibition of peptide amyloid formation by a fatty acid

The aggregation of peptides into amyloid fibrils is associated with several diseases, including Alzheimer’s and Parkinson’s disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic molecules, such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68–78 of the protein α-synuclein involved in Parkinson’s disease. Based primarily on circular dichroism measurements, we found that even a very small amount of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the observed inhibitory effect on fibrillation is due to co-association of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that associate with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.     

Potential roles of abundant extracellular chaperones in the control of amyloid formation and toxicity

The in vivo formation of fibrillar proteinaceous deposits called amyloid is associated with more than 40 serious human diseases, collectively referred to as protein deposition diseases. In many cases the amyloid deposits are extracellular and are found associated with newly identified abundant extracellular chaperones (ECs). Evidence is presented suggesting an important regulatory role for ECs in amyloid formation and disposal in the body. A model is presented which proposes that, under normal conditions, ECs stabilize extracellular misfolded proteins by binding to them, and then guide them to specific cell receptors for uptake and subsequent degradation. Thus ECs and their receptors may be critical parts of a quality control system to protect the body against dangerously hydrophobic proteins/peptides. However, it also appears possible that in the presence of a high molar excess of misfolded protein, such as might occur during disease, the limited amounts of ECs available may actually exacerbate pathology. Further advances in understanding of the mechanisms that control extracellular protein folding are likely to identify new strategies for effective disease therapies.     

Sporadic Alzheimer’s Disease Begins as Episodes of Brain Ischemia and Ischemically Dysregulated Alzheimer’s Disease Genes

The study of sporadic Alzheimer’s disease etiology, now more than ever, needs an infusion of new concepts. Despite ongoing interest in Alzheimer’s disease, the basis of this entity is not yet clear. At present, the best-established and accepted “culprit” in Alzheimer’s disease pathology by most scientists is the amyloid, as the main molecular factor responsible for neurodegeneration in this disease. Abnormal upregulation of amyloid production or a disturbed clearance mechanism may lead to pathological accumulation of amyloid in brain according to the “amyloid hypothesis.” We will critically review these observations and highlight inconsistencies between the predictions of the “amyloid hypothesis” and the published data. There is still controversy over the role of amyloid in the pathological process. A question arises whether amyloid is responsible for the neurodegeneration or if it accumulates because of the neurodegeneration. Recent evidence suggests that the pathophysiology and neuropathology of Alzheimer’s disease comprises more than amyloid accumulation, tau protein pathology and finally brain atrophy with dementia. Nowadays, a handful of researchers share a newly emerged view that the ischemic episodes of brain best describe the pathogenic cascade, which eventually leads to neuronal loss, especially in hippocampus, with amyloid accumulation, tau protein pathology and irreversible dementia of Alzheimer type. The most persuasive evidences come from investigations of ischemically damaged brains of patients and from experimental ischemic brain studies that mimic Alzheimer-type dementia. This review attempts to depict what we know and do not know about the triggering factor of the Alzheimer’s disease, focusing on the possibility that the initial pathological trigger involves ischemic episodes and ischemia-induced gene dysregulation. The resulting brain ischemia dysregulates additionally expression of amyloid precursor protein and amyloid-processing enzyme genes that, in addition, ultimately compromise brain functions, leading over time to the complex alterations that characterize advanced sporadic Alzheimer’s disease. The identification of the genes involved in Alzheimer’s disease induced by ischemia will enable to further define the events leading to sporadic Alzheimer’s disease-related abnormalities. Additionally, knowledge gained from the above investigations should facilitate the elaboration of the effective treatment and/or prevention of Alzheimer’s disease.     

Gut power: Modulation of human amyloid formation by amyloidogenic proteins in the gastrointestinal tract

Protein assembly into amyloid fibers underlies many neurodegenerative disorders. In Parkinson's disease, amyloid formation of α-synuclein is linked to brain cell death. The gut–brain axis plays a key role in Parkinson's disease, and initial α-synuclein amyloid formation may occur distant from the brain. Because different amyloidogenic proteins can cross-seed, and α-synuclein is expressed outside the brain, amyloids present in the gut (from food products and secreted by microbiota) may modulate α-synuclein amyloid formation via direct interactions. I here describe existing such data that only began to appear in the literature in the last few years. The striking, but limited, data set—spanning from acceleration to inhibition—calls for additional investigations that may unravel disease mechanisms as well as new treatments.     

The secondary structure of human amyloid deposits as determined by circular dichroism spectroscopy

The deposition of amyloid fibrils has been associated with a diversity of pathologies including plasma cell dyscrasias, chronic inflammatory diseases, and several types of neurological diseases including Alzheimer's disease. Using circular dichroism spectroscopy, the secondary structure of a human amyloid protein deposit from a patient with plasma cell (light chain)-associated amyloidosis (amyloidosis AL) has been determined. The protein contains 52% beta structure, which is consistent with these depositions arising from the aberrant catabolism of immunoglobulin light chains, which are rich in beta sheets. The protein was also found to contain 20% alpha-helix, suggesting that partial refolding may occur during amyloidogenesis.     

The bad seed in Alzheimer's disease

In this issue of Neuron, McGowan et al. report on a new mouse model of amyloid deposition as occurs in Alzheimer's disease. Unlike previous models in which overexpression of the amyloid precursor protein results in amyloid plaque formation, McGowan et al. have produced mice that overexpress only Abeta40 or Abeta42 and prove that Abeta42 is critical for the formation of amyloid deposits in vivo.     

The sulfate moieties of glycosaminoglycans are critical for the enhancement of beta-amyloid protein fibril formation

Our previous studies have demonstrated that perlecan and perlecan-derived glycosaminoglycans (GAGs) not only bind beta-amyloid protein (Abeta) 1-40 and 1-42, but are also potent enhancers of Abeta fibril formation and stabilize amyloid fibrils once formed. However, it was not determined which moieties in perlecan heparan sulfate GAG chains may be responsible for the observed effects and whether other GAGs were also capable of a similar enhancement of Abeta fibril formation as observed with perlecan GAGs. In the present study, thioflavin T fluorometry (over a 1-week period) was used to extend our previous studies and to test the hypothesis that the sulfate moiety is critical for the enhancing effects of heparin/heparan sulfate GAGs on Abeta 1-40 fibrillogenesis. This hypothesis was confirmed when removal of all sulfates from heparin (i.e., completely desulfated N-acetylated heparin) led to a complete loss in the enhancement of Abeta fibrillogenesis as demonstrated in both thioflavin T fluorometry and Congo red staining studies. On the other hand, removal of O-sulfate from heparin (i.e., completely desulfated N-sulfated heparin), and to a lesser extent N-sulfate (i.e., N-desulfated N-acetylated heparin), resulted in only a partial loss of the enhancement of Abeta 1-40 fibril formation. These studies indicate that the sulfate moieties of GAGs are critical for enhancement of Abeta amyloid fibril formation. In addition, other sulfated molecules such as chondroitin-4-sulfate, dermatan sulfate, dextran sulfate, and pentosan polysulfate all significantly enhanced (greater than twofold by 3 days) Abeta amyloid fibril formation. These latter findings indicate that deposition and accumulation of other GAGs at sites of Abeta amyloid deposition in Alzheimer's disease brain may also participate in the enhancement of Abeta amyloidosis.     

Inhibition of glycosaminoglycan synthesis and protein glycosylation with WAS-406 and azaserine result in reduced islet amyloid formation in vitro

Deposition of islet amyloid polypeptide (IAPP) as amyloid in the pancreatic islet occurs in approximately 90% of individuals with Type 2 diabetes and is associated with decreased islet beta-cell mass and function. Human IAPP (hIAPP), but not rodent IAPP, is amyloidogenic and toxic to islet beta-cells. In addition to IAPP, islet amyloid deposits contain other components, including heparan sulfate proteoglycans (HSPGs). The small molecule 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha-D-xylo-hexopyranose (WAS-406) inhibits HSPG synthesis in hepatocytes and blocks systemic amyloid A deposition in vivo. To determine whether WAS-406 inhibits localized amyloid formation in the islet, we incubated hIAPP transgenic mouse islets for up to 7 days in 16.7 mM glucose (conditions that result in amyloid deposition) plus increasing concentrations of the inhibitor. WAS-406 at doses of 0, 10, 100, and 1,000 microM resulted in a dose-dependent decrease in amyloid deposition (% islet area occupied by amyloid: 0.66 +/- 0.14%, 0.10 +/- 0.06%, 0.09 +/- 0.07%, and 0.004 +/- 0.003%, P < 0.001) and an increase in beta-cell area in hIAPP transgenic islets (55.0 +/- 2.6 vs. 60.6 +/- 2.2% islet area for 0 vs. 100 microM inhibitor, P = 0.05). Glycosaminoglycan, including heparan sulfate, synthesis was inhibited in both hIAPP transgenic and nontransgenic islets (the latter is a control that does not develop amyloid), while O-linked protein glycosylation was also decreased, and WAS-406 treatment tended to decrease islet viability in nontransgenic islets. Azaserine, an inhibitor of the rate-limiting step of the hexosamine biosynthesis pathway, replicated the effects of WAS-406, resulting in reduction of O-linked protein glycosylation and glycosaminoglycan synthesis and inhibition of islet amyloid formation. In summary, interventions that decrease both glycosaminoglycan synthesis and O-linked protein glycosylation are effective in reducing islet amyloid formation, but their utility as pharmacological agents may be limited due to adverse effects on the islet.     

Dysfunction of amyloid precursor protein signaling in neurons leads to DNA synthesis and apoptosis

The classic neuropathological diagnostic markers for AD are amyloid plaques and neurofibrillary tangles, but their role in the etiology and progression of the disease remains incompletely defined. Research over the last decade has revealed that cell cycle abnormalities also represent a major neuropathological feature of AD. These abnormalities appear very early in the disease process, prior to the appearance of plaques and tangles; and it has been suggested that neuronal cell cycle regulatory failure may be a significant component of the pathogenesis of AD. The amyloid precursor protein (APP) is most commonly known as the source of the beta-amyloid (Abeta) peptides that accumulate in the brains of patients with AD. However, a large body of work supports the idea that APP is also a signaling receptor. Most recently, it has been shown that familial AD (FAD) mutations in APP or simple overexpression of wild type APP cause dysfunction of APP signaling, resulting in initiation of DNA synthesis in neurons and consequent apoptosis. In this article, we review the evidence that APP has the potential to activate aberrant neuronal cell cycle re-entry in AD, and we describe a signal transduction pathway that may mediate this abnormal activation of the cell cycle.     

Intensive protein synthesis in neurons and phosphorylation of beta-amyloid precursor protein and tau-protein are triggering factors of neuronal amyloidosis and Alzheimer’s disease

Studies of Alzheimer’s disease have become particularly important and attract now much attention of scientists all over the world due to worldwide dissemination of this dangerous disorder. Causes of this pathology still remain unknown, while the final image, originally obtained on microscopic brain sections from patients with this disease more than a hundred years ago, is well familiar to clinicians. This includes deposition of amyloid-β (Aβ) in the brain tissue of senile plaques and fibrils. Many authors believe that the deposition of Aβ provokes secondary neuronal changes, responsible for death of neurons. Other authors associate the death of neurons with hyperphosphorylation of tau-proteins, which form neurofibrillar tangles inside nerve cells and cause their death. Creation of methods of preclinical diagnostics and effective treatment of Alzheimer’s disease requires novel knowledge: on the nature of triggering factors of sporadic forms of Alzheimer’s disease, on cause-effect relationships of phosphorylation of amyloid precursor protein with formation of pathogenic beta-amyloids, on the relationship between these factors underlying tau-protein hyperphosphorylation and neuron death. In this review we have analyzed reports describing increased intensity of protein synthesis in neurons under normal and various stress conditions, possibility of development of energy imbalance of neurons and activation of their protective systems. Phosphorylation and hyperphosphorylation of tau-proteins is also tightly associated with protective mechanisms of cells and with processes of evacuation of phosphates, adenosine monophosphates and pyrophosphates from the region of protein synthesis. Prolonged highly intensive protein synthesis causes overload of protective mechanisms and impairments in concerted metabolic processes. This leads to neuronal dysfunction, transport collapse, and death of neurons.     

The structural basis for amyloid formation by plasma apolipoproteins: a review

The formation of amyloid and other protein deposits in vivo is synonymous with many pathological conditions such as Alzheimer's disease, Creutzfeldt-Jakob disease and Parkinson's disease. Interestingly, many plasma apolipoproteins are also associated with amyloid deposits, including apolipoprotein (apo) A-I, apoA-II and apoE. Apolipoproteins share a number of structural and conformational properties, namely a large proportion of class A amphipathic alpha-helices and limited conformational stability in the absence of lipid. Other proteins that form amyloid such as alpha-synuclein and serum amyloid A also contain amphipathic alpha-helical domains similar to those found in apolipoproteins. In this review we develop a hypothesis to account for the widespread occurrence of apolipoproteins in amyloid deposits. We describe the conformational stability of human apoC-II and the stabilization of alpha-helical structure in the presence of phospholipid. We propose that lipid-free apoC-II forms partially folded intermediates prone to amyloid formation. Parameters that affect apolipoprotein lipid binding in vivo, such as protein and lipid oxidation or protein truncations and mutations, could promote apolipoprotein-related pathologies including those associated within amyloid deposits of atherosclerotic plaques.     

Environment- and mutation-dependent aggregation behavior of Alzheimer amyloid beta-protein

The deposition of amyloid beta-protein in the brain is a fundamental process in the development of Alzheimerís disease; however, the mechanism underlying aggregation of amyloid beta-protein remains to be determined. Here, we report that a membrane-mimicking environment, generated in the presence of detergents or a ganglioside, is sufficient per se for amyloid fibril formation from soluble amyloid beta-protein. Furthermore, hereditary variants of amyloid beta-protein, which are caused by amyloid precursor protein gene mutations, including the Dutch (E693Q), Flemish (A692G) and Arctic (E693G) types, show mutually different aggregation behavior in these environments. Notably, the Arctic-type amyloid beta-protein, in contrast to the wild-type and other variant forms, shows a markedly rapid and higher level of amyloid fibril formation in the presence of sodium dodecyl sulfate or GM1 ganglioside. These results suggest that there are favorable local environments for fibrillogenesis of amyloid beta-protein.     

Formation of amyloid fibers triggered by phosphatidylserine-containing membranes

Protein misfolding has been shown to be the direct cause of a number of highly devastating diseases such as Alzheimer's disease, Parkinson's disease, and Creutzfeldt-Jacob syndrome, affecting the aging population globally. The deposition in tissues of amyloid fibrils is a characteristic of all these diseases, and the mechanisms by which these protein aggregates form continue to be intensively investigated. In only a fraction of cases is an underlying mutation responsible, and accordingly, what initiates amyloid formation in vivo is the major question that is addressed. In this study, we show that membranes containing phosphatidylserine (PS), a negatively charged phospholipid, induce a rapid formation of fibers by a variety of proteins, viz., lysozyme, insulin, glyceraldehyde-3-phosphate dehydrogenase, myoglobin, transthyretin, cytochrome c, histone H1, and alpha-lactalbumin. Congo red staining of these fibers yields the characteristic light green birefringence of amyloid, and fluorescent lipid tracers further reveal them to include phospholipids. Our results suggest that PS as well as other acidic phospholipids could provide the physiological low-pH environment on cellular membranes, enhancing protein fibril formation in vivo. Interestingly, all the proteins mentioned above either are cytotoxic or induce apoptosis. PS-protein interaction could be involved in the mechanism of cytotoxicity of the aggregated protein fibrils, perturbing membrane functions. Importantly, our results suggest that this process induced by acidic phospholipids may provide an unprecedented and generic connection between three current major areas of research: (i) mechanism(s) triggering amyloid formation, (ii) cytotoxicity of amyloidal protein aggregates, and (iii) mechanism(s) of action of cytotoxic proteins.     

Functional amyloid

Evidence is growing at an increasing pace that amyloid fibers are not just the result of aberrant protein folding associated with neurodegenerative diseases, but are widespread in Nature for beneficial reasons. Amyloid is an attractive building material because its robust design and simple repetitive structure makes for very durable and metabolically cheap material. But this requires that the production of amyloid be put under firm control. This appears to involve the use of 4-5 chaperones which are expressed under the control of the same promoter as the amyloid proteins. Significant progress has been made in deciphering this process in E. coli’s csg operon, also found in Salmonella. Recently, we have discovered a new and unrelated operon (fap) responsible for amyloid production in Pseudomonas, which also confers biofilm forming properties to E. coli. Intriguingly, this operon shares a number of features with csg, namely two homologous proteins (one of which, FapC, has been shown to be directly involved in amyloid build-up) and a small number of auxiliary proteins. However, FapC seems to be less economically structured than its E. coli counterpart, with a smaller number of repeats and very large and variable linker regions. Furthermore, the putative chaperones are not homologous to their csg counterparts and have intriguing homologies to proteins with other functions. These findings suggest that controlled amyloid production has arisen on many independent occasions due to the usefulness of the product and offers the potential for intriguing insights into how Nature disarms and reconstructs a potentially very dangerous weapon.