Protofibril Formation Research Articles

Determination of critical nucleation number for a single nucleation amyloid-β aggregation model

Aggregates of amyloid-β (Aβ) peptide are known to be the key pathological agents in Alzheimer disease (AD). Aβ aggregates to form large, insoluble fibrils that deposit as senile plaques in AD brains. The process of aggregation is nucleation–dependent in which the formation of a nucleus is the rate–limiting step, and controls the physiochemical fate of the aggregates formed. Therefore, understanding the properties of nucleus and pre-nucleation events will be significant in reducing the existing knowledge–gap in AD pathogenesis. In this report, we have determined the plausible range of critical nucleation number (n*), the number of monomers associated within the nucleus for a homogenous aggregation model with single unique nucleation event, by two independent methods: A reduced-order stability analysis and ordinary differential equation based numerical analysis, supported by experimental biophysics. The results establish that the most likely range of n* is between 7 and 14 and within, this range, n* = 12 closely supports the experimental data. These numbers are in agreement with those previously reported, and importantly, the report establishes a new modeling framework using two independent approaches towards a convergent solution in modeling complex aggregation reactions. Our model also suggests that the formation of large protofibrils is dependent on the nature of n*, further supporting the idea that pre-nucleation events are significant in controlling the fate of larger aggregates formed. This report has re-opened an old problem with a new perspective and holds promise towards revealing the molecular events in amyloid pathologies in the future.

The strengthening role of D:D interactions in fibrin self-assembly under oxidation.

The effect on ozone-induced oxidation on the self-assembly of fibrin in the presence of fibrin-stabilizing factor FXIIIa of soluble cross-linked fibrin oligomers was studied in a medium containing moderate urea concentrations. It is established that fibrin oligomers were formed by the protofibrils cross-linked through γ-γ dimers and the fibrils additionally cross-linked by through α-polymers. The oxidation promoted both the accumulation of greater amounts of γ-γ dimers and the formation of protofibrils, fibrils, and their dissociation products emerging with increasing urea concentrations, which have a high molecular weight. It is concluded that the oxidation enhances the axial interactions between D-regions of fibrin molecules.

Polyphosphate delays fibrin polymerisation and alters the mechanical properties of the fibrin network.

SummaryPolyphosphate (polyP) binds to fibrin(ogen) and alters fibrin structure, generating a heterogeneous network composed of ‘knots’ interspersed by large pores. Here we show platelet-derived polyP elicits similar structural changes in fibrin and examine the mechanism by which polyP alters fibrin structure. Polymerisation of fibrinogen with thrombin and CaCl2 was studied using spinning disk confocal (SDC) microscopy. PolyP delayed fibrin polymerisation generating shorter protofibrils emanating from a nucleus-type structure. Consistent with this, cascade blue-polyP accumulated in fibrin ‘knots’. Protofibril formation was visualized by atomic force microscopy (AFM) ± polyP. In the presence of polyP abundant monomers of longer length were visualised by AFM, suggesting that polyP binds to monomeric fibrin. Shorter oligomers form in the presence of polyP, consistent with the stunted protofibrils visualised by SDC microscopy. We examined whether these structural changes induced by polyP alter fibrin’s viscoelastic properties by rheometry. PolyP reduced the stiffness (G’) and ability of the fibrin network to deform plastically G’’, but to different extents. Consequently, the relative plastic component (loss tangent (G’’/G’)) was 61 % higher implying that networks containing polyP are less stiff and more plastic. Local rheological measurements, performed using magnetic tweezers, indicate that the fibrin dense knots are stiffer and more plastic, reflecting the heterogeneity of the network. Our data show that polyP impedes fibrin polymerisation, stunting protofibril growth producing ‘knotted’ regions, which are rich in fibrin and polyP. Consequently, the mechanical properties of the fibrin network are altered resulting in clots with overall reduced stiffness and increased ability to deform plastically.

Contribution of Red Blood Cells and Clot Structure to Thrombosis

The blood clot or thrombus is composed of red blood cells, platelets and white cells that interact with the fibrin meshwork produced by the coagulation system. The red blood cells form polyhedrocytes that seal the clot, platelets provide forces for clot contraction, and white cells contribute neutrophil extracellular traps, cytokines and complement activation. The fibrin network contributes clot elasticity and provides the proteinaceous backbone structure that stabilises the clot. Previous studies from our laboratory and others have shown that dense fibrin networks demonstrating small pores and increased resistance to fibrinolysis associate with thrombosis. However, the mechanisms underpinning this have not been fully understood. Recent studies using atomic force microscopy from our laboratory have shown that fibrinogen interacts with red cells with comparable affinity as that with platelets. A patient with mutations in the β3 integrin subunit showed no binding between red cells and fibrinogen, demonstrating that a β3-related integrin receptor is involved in the interaction. Mutations in the fibrinogen α-chain integrin binding sites (D97E and D574E) reduced frequency of red cell interactions with fibrinogen. Interestingly, a naturally occuring splice variant of the fibrinogen γ-chain that reduces binding to the platelets, fibrinogen γ', increased binding interactions between fibrinogen and red blood cells. Fibrinogen γ' is a naturally occurring splice variant of fibrinogen, in which the C-terminal AGDV residues of the more common γA-chain (85%) are replaced with a negatively charched VRPEHPAETEYDSLYPEDDL sequence of the γ' chain (15%). Fibrinogen γ' induced clustering of fibrin fibres into tightly interknit nuclei of fibrin fibres, interspersed by large pores that extend over more than 50 μm within the fibrin network structure. The effects of fibrinogen γ' on fibrin clot structure was independent of thrombin and FXIII as demonstrated using snake venom enzyme. Previously we showed impaired fibrin protofibril formation with fibrinogen γ' using atomic force microscopy. Using turbidimetric analysis of fibrin intrafibrillar structure, we show that fibrinogen γ' reduces protofibil packing per fibrin fiber. Furthermore, we find that reduced protofibril packing diminishes fibrin stiffness as analysed with magnetic tweezers both in purified systems as well as in plasma at (patho)physiological fibrinogen γ' levels that range from 3-40%, and in whole blood as analysed with thromboelastography. In conclusion, our data show that red blood cells and fibrinogen γ' play major roles in the regulation of clot structure and stability, and that these effects on clot structure are major determinants of the functional properties of the blood clot. Modulating fibrin clot structure and its interactions with blood cells may represent major new targets for the treatment of thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Cu(II) promotes amyloid pore formation

The aggregation of α-synuclein is associated with dopamine neuron death in Parkinson's disease. There is controversy in the field over the question of which species of the aggregates, fibrils or protofibrils, are toxic. Moreover, compelling evidence suggested the exposure to heavy metals to be a risk of PD. Nevertheless, the mechanism of metal ions in promoting PD remains unclear. In this research, we investigated the structural basis of Cu(II) induced aggregation of α-synuclein. Using transmission electron microscopy experiments, Cu(II) was found to promote in vitro aggregation of α-synuclein by facilitating annular protofibril formation rather than fibril formation. Furthermore, neuroprotective baicalein disaggregated annular protofibrils accompanied by considerable decrease of β-sheet content. These results strongly support the hypothesis that annular protofibrils are the toxic species, rather than fibrils, thereby inspiring us to search novel therapeutic strategies for the suppression of the toxic annular protofibril formation.

Plasma clot properties in patients with a mild-to-moderate bleeding tendency of unknown cause.

In a large proportion of patients with mild bleeding disorders (MBDs) no diagnosis can be established by routine coagulation tests. We investigated whether alterations in plasma clot properties account for MBDs of unknown cause. Ninety-five patients with MBDs of unknown origin and 98 age- and sex-matched healthy controls were investigated. Furthermore, data of 25 patients with a deficiency of factor VIII were analyzed. Plasma clot characteristics in the absence and presence of recombinant tissue plasminogen activator (rtPA) represented by the lag phase, rate of protofibril formation (Vmax), fibrin structure (ΔAbs), time to peak (TTP), half lysis time (t50 and area under the curve (AUC) were measured in turbidometric clot formation and lysis assays. In the fibrinolysis assay, Vmax was lower in patients than in healthy controls. No differences in the other parameters of clot formation and lysis were detected between the groups. There was no clear association of plasma clot properties with the clinical severity of bleeding in patients with MBDs. Patients with known decreased factor VIII levels also showed a lower Vmax. Fibrinogen levels were positively associated with each of the assessed parameters in both groups, with the strongest association with ΔAbs, indicating altered fibrin structure. Factor VIII activity correlated with altered clot characteristics similar to fibrinogen, especially in patients, with the strongest positive correlation to Vmax. This cohort of patients with MBDs of unknown origin showed a lower rate of fibrin formation in the fibrinolysis assay, but otherwise similar plasma clot properties compared to healthy controls.

Examination of Ataxin-3 (atx-3) Aggregation by Structural Mass Spectrometry Techniques: A Rationale for Expedited Aggregation upon Polyglutamine (polyQ) Expansion*

Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73–96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided.

Limited proteolysis of fibrinogen by fibrinogenase from Echis multisquamatis venom.

Previously we purified fibrinogenase from venom of Echis multisquamatis and showed that the enzyme predominantly cleaves BβArg42-Ala43 peptide bond of fibrinogen. A much slower hydrolysis of its Aα-chain was also shown. To evaluate the accessibility of the hydrolysis sites to fibrinogenase's hydrolytic action, the pathway of cleavage of Aα- and Bβ-chains of fibrinogen, monomeric and polymeric fibrin desA and desAB has been investigated using western blot with monoclonal antibodies to Bβ 26-42 and Aα 20-78 of fibrinogen. The data indicated that the BβArg42-Ala43 peptide bond is available for cleavage in all forms of fibrin(ogen) with the exception of polymerized fibrin desAB. This is direct evidence of BβN-domain involvement in formation of protofibrils that makes it inaccessible to protease. The Aα-chain of fibrinogen remained intact after 3 min of incubation with fibrinogenase. Further incubation resulted in cleaving of the fibrin(ogen) αC-regions with the formation of two kinds of degradation products (~30 and ~60 kDa). In the case of monomeric fibrin desA or desAB we observed simultaneous hydrolysis of Aα and Bβ-chains and the cleavage of Aα-chain was more apparent for both forms of polymeric fibrin.

Effect of incubation temperature on the self-assembly of regenerated silk fibroin: A study using AFM

Understanding effect of temperature on the molecular self-assembly process will be helpful to unravel the structure–function relationship of biomolecule and to provide important information for the bottom-up approach to nanotechnology. In this work, the effect of incubation temperature on the secondary structures and morphological structures of regenerated silk fibroin (RSF) was systematically studied using atomic force microscopy and Fourier Transform infrared spectroscopy. The effect of incubation temperature on RSF self-assembly was dependent on RSF concentration. For the RSF solution with relatively low concentrations (15μg/mL and 60μg/mL), the increase of the incubation temperature mainly accelerated the formation and aggregation of antiparallel β-sheet protofibrils and decreased the formation of random coil protofilaments/globule-like molecules. For the RSF solution with relatively high concentrations (300μg/mL and 1.5mg/mL), the increase of the incubation temperature mainly accelerated the formation and aggregation of antiparallel β-sheet RSF features (protofibrils and globule-like features) and decreased the formation of random coil bead-like features. This work implies that the morphology and conformation of biomacromolecules could be tuned by controlling the incubation temperature. Further, it will be beneficial to basic understanding of the nanoscale structure formation in different silk-based biomaterials.

Solution NMR studies of recombinant Aβ(1-42): from the presence of a micellar entity to residual β-sheet structure in the soluble species.

Amyloid-β (Aβ) peptide is the major component found in senile plaques of Alzheimer's disease patients. The 42-residue fragment Aβ(1-42) is proposed to be one of the most pathogenic species therein. Here, the soluble Aβ(1-42) species were analyzed by various liquid-state NMR methods. Transient formation of a micelle species was observed at the onset of the aggregation kinetics. This micelle is dissolved after approximately one day. Subsequent loss of this species and the formation of protofibrils are proposed to be the route of fibril formation. Consequently, the observed micelle species is suggested to be on an off-pathway mechanism. Furthermore, characterization of the NMR-observable soluble species shows that it is a random-coil-like entity with low propensities for four β-strands. These β-strands correlate with the β-strand segments observed in Aβ fibrils. This finding indicates that the 3D structure of the fibrils might already be predisposed in the soluble species.

Does topology drive fiber polymerization?

We have developed new procedures to examine the early steps in fibrin polymerization. First, we isolated fibrinogen monomers from plasma fibrinogen by gel filtration. Polymerization of fibrinogen monomers differed from that of plasma fibrinogen. The formation of protofibrils was slower and the transformation of protofibrils to fibers faster for the fibrinogen monomers. Second, we used formaldehyde to terminate the polymerization reactions. The formaldehyde-fixed products obtained at each time point were examined by dynamic light scattering and transmission electron microscopy (TEM). The data showed the formaldehyde-fixed products were stable and representative of the reaction intermediates. TEM images showed monomers, short oligomers, protofibrils, and thin fibers. The amount and length of these species varied with time. Short oligomers were less than 5% of the molecules at all times. Third, we developed models that recapitulate the TEM images. Fibrin monomer models were assembled into protofibrils, and protofibrils were assembled into two-strand fibers using Chimera software. Monomers were based on fibrinogen crystal structures, and the end-to-end interactions between monomers were based on D-dimer crystal structures. Protofibrils assembled from S-shaped monomers through asymmetric D:D interactions were ordered helical structures. Fibers were modeled by duplicating a protofibril and rotating the duplicate 120° around its long axis. No specific interactions were presumed. The two protofibrils simply twisted around one another to form a fiber. This model suggests that the conformation of the protofibril per se promotes the assembly into fibers. These findings introduce a novel mechanism for fibrin assembly that may be relevant to other biopolymers.

Inhibition of fibrin polymerization by synthetic peptides corresponding to Aα195-205 and γ69-77 sites of fibrin molecule.

Using the idea of "proline brackets" we have found four sites in fibrin amino acid sequence, and appropriate peptides were synthesized: γ69NPDESSKPN77, Bβ228QPDSSVKPY236, Bβ455RPFFPQ460 and Aα195LPSRDRQHLPL205. Turbidity and electron-microscopy analyses have demonstrated that synthetic peptide Aα195-205 specifically inhibited the stage of fibrin protofibril formation and peptide γ69-77 - the stage offibrin protofibril lateral association. The data obtained testify that there are the sites involved in these processes in the appropriate amino acid sequences of fibrin molecule.

Arctic mutant Aβ40 aggregates on α7 nicotinic acetylcholine receptors and inhibits their functions

Amyloid β protein (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Point mutations within the Aβ sequence associated with familial AD (FAD) are clustered around the central hydrophobic core of Aβ. Several types of mutations within the Aβ sequence have been identified, and the 'Arctic' mutation (E22G) has a purely cognitive phenotype typical of AD. Previous studies have shown that the primary result of the 'Arctic' mutation is increased formation of Aβ protofibrils. However, the molecular mechanism underlying this effect remains unknown. Aβ42 binds to a neuronal nicotinic acetylcholine receptor subunit, neuronal acetylcholine receptor subunit alpha-7 (CHRNA7), with high affinity and, thus, may be involved in the pathogenesis of AD. Therefore, to clarify the molecular mechanism of Arctic mutation-mediated FAD, we focused on CHRNA7 as a target molecule of Arctic Aβ. We performed an in vitro binding assay using purified CHRNA7 and synthetic Arctic Aβ40, and demonstrated that Arctic Aβ40 specifically bound to CHRNA7. The aggregation of Arctic Aβ40 was enhanced with the addition of CHRNA7. Furthermore, the function of CHRNA7 was detected by measuring Ca(2+) flux and phospho-p44/42 MAPK (ERK1/2) activation. Our results indicated that Arctic Aβ40 aggregation was enhanced by the addition of CHRNA7, which destabilized the function of CHRNA7 via inhibition of Ca(2+) responses and activation of ERK1/2. These findings indicate that Arctic Aβ mutation may be involved in the mechanism underlying FAD. This mechanism may involve binding and aggregation, leading to the inhibition of CHRNA7 functions.

Capillary electrophoresis for the analysis of the effect of sample preparation on early stages of Aβ1-40 aggregation.

Aggregation of the amyloid-β protein (Aβ) contributes to the neurodegeneration characteristic of Alzheimer's disease. Of particular importance are the early stages of aggregation, which involve the formation of soluble oligomers and protofibrils. In these studies, we demonstrate the potential for CE with UV detection using a polyethylene oxide separation matrix to identify the evolution of various oligomeric species of Aβ1-40 . To demonstrate the efficacy of this technique, UV-CE was utilized to compare two methods commonly used to prepare Aβ for aggregation experiments and their effect on the formation of early aggregates. SEC-purified Aβ1-40 initially contained more small species, including monomer, than did freshly dissolved Aβ1-40 pretreated with hexafluoroisopropanol. Strikingly, the lag time to oligomer formation for SEC-isolated Aβ1-40 samples was ∼23 h shorter compared to freshly dissolved Aβ1-40 samples. Furthermore, oligomers formed from the aggregation of SEC-purified Aβ1-40 persisted within solution for a longer period of time. These results indicate that the initial sample preparation has a drastic influence on the early stages of Aβ1-40 aggregation. This is the first report of the use of UV-CE with a separation matrix to study the effect of sample preparation on early aggregation of Aβ1-40 . UV-CE was also used in parallel with dot blot analysis and inhibitory compounds to discern structural characteristics of individual oligomer peaks, demonstrating the capacity of UV-CE as a complimentary technique to further understand the aggregation process.

SAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing α-Synuclein Oligomeric Species Toxicity

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.

Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis

Serum amyloid A (SAA) represents an evolutionarily conserved family of inflammatory acute-phase proteins. It is also a major constituent of secondary amyloidosis. To understand its function and structural transition to amyloid, we determined a structure of human SAA1.1 in two crystal forms, representing a prototypic member of the family. Native SAA1.1 exists as a hexamer, with subunits displaying a unique four-helix bundle fold stabilized by its long C-terminal tail. Structure-based mutational studies revealed two positive-charge clusters, near the center and apex of the hexamer, that are involved in SAA association with heparin. The binding of high-density lipoprotein involves only the apex region of SAA and can be inhibited by heparin. Peptide amyloid formation assays identified the N-terminal helices 1 and 3 as amyloidogenic peptides of SAA1.1. Both peptides are secluded in the hexameric structure of SAA1.1, suggesting that the native SAA is nonpathogenic. Furthermore, dissociation of the SAA hexamer appears insufficient to initiate amyloidogenic transition, and proteolytic cleavage or removal of the C-terminal tail of SAA resulted in formation of various-sized structural aggregates containing ∼5-nm regular repeating protofibril-like units. The combined structural and functional studies provide mechanistic insights into the pathogenic contribution of glycosaminoglycan in SAA1.1-mediated AA amyloid formation.

Protective and toxic roles of dopamine in Parkinson's disease

The molecular mechanisms causing the loss of dopaminergic neurons containing neuromelanin in the substantia nigra and responsible for motor symptoms of Parkinson's disease are still unknown. The discovery of genes associated with Parkinson's disease (such as alpha synuclein (SNCA), E3 ubiquitin protein ligase (parkin), DJ-1 (PARK7), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1), serine/threonine-protein kinase (PINK-1), leucine-rich repeat kinase 2 (LRRK2), cation-transporting ATPase 13A1 (ATP13A), etc.) contributed enormously to basic research towards understanding the role of these proteins in the sporadic form of the disease. However, it is generally accepted by the scientific community that mitochondria dysfunction, alpha synuclein aggregation, dysfunction of protein degradation, oxidative stress and neuroinflammation are involved in neurodegeneration. Dopamine oxidation seems to be a complex pathway in which dopamine o-quinone, aminochrome and 5,6-indolequinone are formed. However, both dopamine o-quinone and 5,6-indolequinone are so unstable that is difficult to study and separate their roles in the degenerative process occurring in Parkinson's disease. Dopamine oxidation to dopamine o-quinone, aminochrome and 5,6-indolequinone seems to play an important role in the neurodegenerative processes of Parkinson's disease as aminochrome induces: (i) mitochondria dysfunction, (ii) formation and stabilization of neurotoxic protofibrils of alpha synuclein, (iii) protein degradation dysfunction of both proteasomal and lysosomal systems and (iv) oxidative stress. The neurotoxic effects of aminochrome in dopaminergic neurons can be inhibited by: (i) preventing dopamine oxidation of the transporter that takes up dopamine into monoaminergic vesicles with low pH and dopamine oxidative deamination catalyzed by monoamino oxidase (ii) dopamine o-quinone, aminochrome and 5,6-indolequinone polymerization to neuromelanin and (iii) two-electron reduction of aminochrome catalyzed by DT-diaphorase. Furthermore, dopamine conversion to NM seems to have a dual role, protective and toxic, depending mostly on the cellular context. Dopamine oxidation to dopamine o-quinone, aminochrome and 5,6-indolequinone plays an important role in neurodegeneration in Parkinson's disease since they induce mitochondria and protein degradation dysfunction; formation of neurotoxic alpha synuclein protofibrils and oxidative stress. However, the cells have a protective system against dopamine oxidation composed by dopamine uptake mediated by Vesicular monoaminergic transporter-2 (VMAT-2), neuromelanin formation, two-electron reduction and GSH-conjugation mediated by Glutathione S-transferase M2-2 (GSTM2).

How to Determine the Size of Folding Nuclei of Protofibrils from the Concentration Dependence of the Rate and Lag-Time of Aggregation. I. Modeling the Amyloid Protofibril Formation

The question about the size of nuclei of formation of protofibrils (which constitute mature amyloid fibrils) formed by different proteins and peptides is yet open and debatable because of the absence of solid knowledge of underlying mechanisms of amyloid formation. In this work, a kinetic model of the process of formation of amyloid protofibrils is suggested, which allows calculation of the size of the nuclei using only kinetic data. In addition to the stage of primary nucleation, which is believed to be present in many protein aggregation processes, the given model includes both linear growth of protofibrils (proceeding only at the cost of attaching of monomers to the ends) and exponential growth of protofibrils at the cost of growth from the surface, branching, and fragmentation with the secondary nuclei. Theoretically, only the exponential growth is compatible with the existence of a pronounced lag-period (which can take much more time then the growth of aggregates themselves). The obtained analytical solution allows us to determine the size of the primary and secondary nuclei from the experimentally obtained concentration dependences of the time of growth and the new parameter-the ratio Lrel of the lag-time duration to the time of growth of amyloid protofibrils.

Perspectives on future Alzheimer therapies: amyloid-β protofibrils - a new target for immunotherapy with BAN2401 in Alzheimer's disease.

The symptomatic drugs currently on the market for Alzheimer’s disease (AD) have no effect on disease progression, and this creates a large unmet medical need. The type of drug that has developed most rapidly in the last decade is immunotherapy: vaccines and, especially, passive vaccination with monoclonal antibodies. Antibodies are attractive drugs as they can be made highly specific for their target and often with few side effects. Data from recent clinical AD trials indicate that a treatment effect by immunotherapy is possible, providing hope for a new generation of drugs. The first anti-amyloid-beta (anti-Aβ) vaccine developed by Elan, AN1792, was halted in phase 2 because of aseptic meningoencephalitis. However, in a follow-up study, patients with antibody response to the vaccine demonstrated reduced cognitive decline, supporting the hypothesis that Aβ immunotherapy may have clinically relevant effects. Bapineuzumab (Elan/Pfizer Inc./Johnson & Johnson), a monoclonal antibody targeting fibrillar Aβ, was stopped because the desired clinical effect was not seen. Solanezumab (Eli Lilly and Company) was developed to target soluble, monomeric Aβ. In two phase 3 studies, Solanezumab did not meet primary endpoints. When data from the two studies were pooled, a positive pattern emerged, revealing a significant slowing of cognitive decline in the subgroup of mild AD. The Arctic mutation has been shown to specifically increase the formation of soluble Aβ protofibrils, an Aβ species shown to be toxic to neurons and likely to be present in all cases of AD. A monoclonal antibody, mAb158, was developed to target Aβ protofibrils with high selectivity. It has at least a 1,000-fold higher selectivity for protofibrils as compared with monomers of Aβ, thus targeting the toxic species of the peptide. A humanized version of mAb158, BAN2401, has now entered a clinical phase 2b trial in a collaboration between BioArctic Neuroscience and Eisai without the safety concerns seen in previous phase 1 and 2a trials. Experiences from the field indicate the importance of initiating treatment early in the course of the disease and of enriching the trial population by improving the diagnostic accuracy. BAN2401 is a promising candidate for Aβ immunotherapy in early AD. Other encouraging efforts in immunotherapy as well as in the small-molecule field offer hope for new innovative therapies for AD in the future.