Aggregates In Neurodegenerative Diseases Research Articles

Curvature Dynamics of α-Synuclein Familial Parkinson's Disease Mutants: Molecular Dynamics Simulations of the Micelle- and Membrane-Bound Forms

α-synuclein remains a protein of interest due to its propensity to form fibrillar aggregates in neurodegenerative disease and its putative function in synaptic vesicle regulation. We have performed a series of all-atom molecular dynamics simulations of wild-type α-synuclein and the three Parkinson's disease familial mutants, A30P, A53T, and E46K, bound to an SDS detergent micelle. Our analysis explains how α-synuclein adapts to, and alters, highly curved membrane surfaces through helical bending. We find that α-synuclein binding induces significant deformation in the micelle, flattening the structure and decreasing its surface area. Similar effects on biological membranes would relieve curvature stress, ameliorating the propensity to fuse, and would perhaps explain α-synuclein's role in stabilizing synaptic vesicles. Consistent with the experimentally determined behavior of A30P, the proline dramatically destabilizes the secondary structure, inducing reversible unfolding up- and down-stream of the substitution. The E46K mutation provides an additional electrostatic interaction between the protein and micelle, offering an explanation for the mutants increased affinity. In the case of the A53T mutant, recent NMR data suggested that an enthalpic interaction might be responsible for a slight rigidification of the helical structure. We show that the equilibrium structure of A53T contains a very tight hydrogen-bond between the threonine's hydroxyl and the backbone carbonyl of Val49. We speculate as to the potential effects of these dynamic and structural changes, on α-synuclein's role in neural signaling. In order to assess the applicability of these results to biological membranes, we have performed simulations of the wild-type and mutants on a DOPS bilayer. These simulations reproduce the major conclusions of the micelle-bound simulations, suggesting that the detergent micelle is an adequate model system.

Fluorescent N-Arylaminonaphthalene Sulfonate Probes for Amyloid Aggregation of α-Synuclein

The deposition of fibrillar structures (amyloids) is characteristic of pathological conditions including Alzheimer's and Parkinson's diseases. The detection of protein deposits and the evaluation of their kinetics of aggregation are generally based on fluorescent probes such as thioflavin T and Congo red. In a search for improved fluorescence tools for studying amyloid formation, we explored the ability of N-arylaminonaphthalene sulfonate (NAS) derivatives to act as noncovalent probes of α-synuclein (AS) fibrillation, a process linked to Parkinson's disease and other neurodegenerative disorders. The compounds bound to fibrillar AS with micromolar Kds, and exhibited fluorescence enhancement, hyperchromism, and high anisotropy. We conclude that the probes experience a hydrophobic environment and/or restricted motion in a polar region. Time- and spectrally resolved emission intensity and anisotropy provided further information regarding structural features of the protein and the dynamics of solvent relaxation. The steady-state and time-resolved parameters changed during the course of aggregation. Compared with thioflavin T, NAS derivatives constitute more sensitive and versatile probes for AS aggregation, and in the case of bis-NAS detect oligomeric as well as fibrillar species. They can function in convenient, continuous assays, thereby providing useful tools for studying the mechanisms of amyloid formation and for high-throughput screening of factors inhibiting and/or reversing protein aggregation in neurodegenerative diseases.

What determines the molecular composition of abnormal protein aggregates in neurodegenerative disease?

Abnormal protein aggregates, in the form of either extracellular plaques or intracellular inclusions, are an important pathological feature of the majority of neurodegenerative disorders. The major molecular constituents of these lesions, viz., beta-amyloid (Abeta), tau, and alpha-synuclein, have played a defining role in the diagnosis and classification of disease and in studies of pathogenesis. The molecular composition of a protein aggregate, however, is often complex and could be the direct or indirect consequence of a pathogenic gene mutation, be the result of cell degeneration, or reflect the acquisition of new substances by diffusion and molecular binding to existing proteins. This review examines the molecular composition of the major protein aggregates found in the neurodegenerative diseases including the Abeta and prion protein (PrP) plaques found in Alzheimer's disease (AD) and prion disease, respectively, and the cellular inclusions found in the tauopathies and synucleinopathies. The data suggest that the molecular constituents of a protein aggregate do not directly cause cell death but are largely the consequence of cell degeneration or are acquired during the disease process. These findings are discussed in relation to diagnosis and to studies of to disease pathogenesis.

The distribution of karyopherin β proteins in Alzheimer's disease

Electron microscopic studies of Alzheimer's disease (AD) revealed an intimate relationship between paired helical filaments and nuclear pores, structures with a key role in bidirectional nucleocytoplasmic transport. This association led us to explore potential abnormalities in transport that may contribute to tangle formation and other protein aggregates in neurodegenerative disease. Previously, we found that antibodies to several nucleoporins highlighted neuronal nuclear contour and irregularity in AD. Immunohistochemical study of nuclear transport factor 2 (NTF2) revealed increased cytoplasmic label in neurons and RanBP2/nup358 antibody labeled tangle‐bearing neurons and Hirano bodies. In the present study, we examined the distribution of karyopherinβ (Kapβ) in AD hippocampus. This group of proteins is variously involved in docking to the nuclear pore complex and importing ribosomal and other proteins into the nucleus. Using antibodies to Kapβ1, β2, and β3, we labeled sections in 3 AD and 3 control cases. Kapβ1 and Kapβ3 displayed finely granular cytoplasmic immunopositivity, the latter with extensive dendritic label. Kapβ2 antibody revealed striking large cytoplasmic granules in many hippocampal neurons, more prominent in AD than controls. Kapβ2 also was present in some tangle‐bearing cells. Further characterization of transport proteins and their potential role in AD is warranted.

Multiple proteins implicated in neurodegenerative diseases accumulate in axons after brain trauma in humans

Studies in animal models have shown that traumatic brain injury (TBI) induces the rapid accumulation of many of the same key proteins that form pathologic aggregates in neurodegenerative diseases. Here, we examined whether this rapid process also occurs in humans after TBI. Brain tissue from 18 cases who died after TBI and from 6 control cases was examined using immunohistochemistry. Following TBI, widespread axonal injury was persistently identified by the accumulation of neurofilament protein and amyloid precursor protein (APP) in axonal bulbs and varicosities. Axonal APP was found to co-accumulate with its cleavage enzymes, beta-site APP cleaving enzyme (BACE), presenilin-1 (PS1) and their product, amyloid-β (Aβ). In addition, extensive accumulation of α-synuclein (α-syn) was found in swollen axons and tau protein was found to accumulate in both axons and neuronal cell bodies. These data show rapid axonal accumulation of proteins implicated in neurodegenerative diseases including Alzheimer's disease and the synucleinopathies. The cause of axonal pathology can be attributed to disruption of axons due to trauma, or as a secondary effect of raised intracranial pressure or hypoxia. Such axonal pathology in humans may provide a unique environment whereby co-accumulation of APP, BACE, and PS1 leads to intra-axonal production of Aβ as well as accumulation of α-syn and tau. This process may have important implications for survivors of TBI who have been shown to be at greater risk of developing neurodegenerative diseases.

Serum macroglobulin induces prion protein transition

The accumulation of the protease-resistant (PrPRes) abnormal form of the cellular prion protein (PrPC) plays a central role in Transmissible Spongiform Encephalopathies. In this report we describe a blood serum protein, which is precipitated by 9% PEG-6000, migrates in a sucrose gradient with a density of 1.21–1.17 g/ml, has a molecular mass of approximately 720 kDa and amino acid sequence as α2-Macroglobulin (α2-M). This protein can potentiate the conversion of the human recombinant prion protein (23–231 AA) as well as PrPC from the brain of mouse, hamster or cow from the sensitive to the resistant form. Under physiological conditions (pH 7.5; 0.15 M NaCl; 37°C for 30 min; a nanomolar concentration range of PrP) the small, highly structured RNA (shs RNA) may activate the conversion process. We describe the design of an assay for screening for the biomolecules that prevent the PrP transition. Based on our experimental data we discuss the role of the α-helix to β-sheet conformational change in the PrPSen to PrPRes conversion as well as the role of the α2-M during the initial stage of amyloidogenesis as well as the shs RNAs involvement in the formation of amyloid aggregates in neurodegenerative diseases.

Thrombin and Prothrombin Are Expressed by Neurons and Glial Cells and Accumulate in Neurofibrillary Tangles in Alzheimer Disease Brain

Thrombin is a serine protease that is generated by proteolytic cleavage of its precursor, prothrombin. We previously showed that thrombin proteolyses the microtubule-associated protein tau and that phosphorylation of tau inhibits this process. To characterize further the role of thrombin in the brain, we investigated prothrombin and thrombin expression in cultured brain cells and in brains of control, Alzheimer disease (AD) and parkinsonism-dementia complex of Guam (PDCG). We show by reverse transcriptase-polymerase chain reaction that prothrombin mRNA is expressed in brain tissues, neuroblastoma cells, and cultured human astrocytes, oligodendrocytes, and microglial cells. We also show by immunohistochemistry that the proteins prothrombin and thrombin are present in brain using specific monoclonal and polyclonal antibodies for both proteins. All antibodies stained residual serum in blood vessels, as well as normal pyramidal neurons and their processes, and some astrocytes. Additionally, in AD and PDCG cases, all antibodies stained extra- and intracellular neurofibrillary tangles (NFTs), senile plaques, and reactive microglial cells. The ubiquitous expression of prothrombin and thrombin in brain cells suggests that thrombin plays an important physiological role in normal brain. The accumulation of thrombin and prothrombin in NFTs supports the hypothesis that thrombin may be involved in tau proteolysis and that failure to metabolize tau may lead to its aggregation in neurodegenerative diseases.

α-Synuclein aggregation in neurodegenerative diseases and its inhibition as a potential therapeutic strategy

There is strong evidence for the involvement of α-synuclein in the pathologies of several neurodegenerative disorders, including PD (Parkinson's disease). Development of disease appears to be linked to processes that increase the rate at which α-synuclein forms aggregates. These processes include increased protein concentration (via either increased rate of synthesis or decreased rate of degradation), and altered forms of α-synuclein (such as truncations, missense mutations, or chemical modifications by oxidative reactions). Aggregated forms of the protein are toxic to cells and one therapeutic strategy would be to reduce the rate at which aggregation occurs. To this end we have designed several peptides that reduce α-synuclein aggregation. A cell-permeable version of one such peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with α-synuclein (A53T), a familial PD-associated mutation.

α-Synuclein aggregation in neurodegenerative diseases and its inhibition as a potential therapeutic strategy

There is strong evidence for the involvement of alpha-synuclein in the pathologies of several neurodegenerative disorders, including PD (Parkinson's disease). Development of disease appears to be linked to processes that increase the rate at which alpha-synuclein forms aggregates. These processes include increased protein concentration (via either increased rate of synthesis or decreased rate of degradation), and altered forms of alpha-synuclein (such as truncations, missense mutations, or chemical modifications by oxidative reactions). Aggregated forms of the protein are toxic to cells and one therapeutic strategy would be to reduce the rate at which aggregation occurs. To this end we have designed several peptides that reduce alpha-synuclein aggregation. A cell-permeable version of one such peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with alpha-synuclein (A53T), a familial PD-associated mutation.

Molecular origin of polyglutamine aggregation in neurodegenerative diseases

Expansion of polyglutamine (polyQ) tracts in proteins results in protein aggregation and is associated with cell death in at least nine neurodegenerative diseases. Disease age of onset is correlated with the polyQ insert length above a critical value of 35–40 glutamines. The aggregation kinetics of isolated polyQ peptides in vitro also shows a similar critical-length dependence. While recent experimental work has provided considerable insights into polyQ aggregation, the molecular mechanism of aggregation is not well understood. Here, using computer simulations of isolated polyQ peptides, we show that a mechanism of aggregation is the conformational transition in a single polyQ peptide chain from random coil to a parallel β-helix. This transition occurs selectively in peptides longer than 37 glutamines. In the β-helices observed in simulations, all residues adopt β-strand backbone dihedral angles, and the polypeptide chain coils around a central helical axis with 18.5 ± 2 residues per turn. We also find that mutant polyQ peptides with proline-glycine inserts show formation of antiparallel β-hairpins in their ground state, in agreement with experiments. The lower stability of mutant β-helices explains their lower aggregation rates compared to wild type. Our results provide a molecular mechanism for polyQ-mediated aggregation.

3' untranslated region in a light neurofilament (NF-L) mRNA triggers aggregation of NF-L and mutant superoxide dismutase 1 proteins in neuronal cells.

The pathogenesis of neurodegenerative diseases is believed to involve abnormal aggregation of proteins, but the mechanisms initiating protein aggregation are unclear. Here we report a novel phenomenon that could be instrumental in triggering protein aggregation in neurodegenerative diseases. We show that the 3' untranslated region (3'UTR) of a light neurofilament (NF-L) transcript enhances the reactivity of its own translated product and leads to loss of solubility and aggregation of NF-L protein and to coaggregation of mutant superoxide dismutase 1 (SOD1) protein. Full-length mouse NF-L cDNAs, with and without NF-L 3'UTR, were fused to the C terminus of a green fluorescent protein (GFP) reporter gene, and the GFP-tagged NF-L proteins were examined in transfected Neuro2a cells. The GFP-tagged NF-L protein expressed from the transgene containing NF-L 3'UTR, but not from the transgene lacking NF-L 3'UTR, colocalizes with endogenous heavy neurofilament protein and, at high-level expression, leads to loss of solubility and aggregation of GFP-tagged NF-L protein. Aggregation of GFP-tagged NF-L protein triggers coaggregation and loss of solubility of coexpressed DsRed-tagged mutant (G93A) SOD1 protein but not wild-type SOD1 protein. Deletional mutagenesis maps the RNA sequence causing aggregation of GFP-tagged NF-L protein to the proximal 45 nucleotides of NF-L 3'UTR. This is the site of a major destabilizing element in NF-L RNA and binding site for RNA-binding proteins. Our findings support a working model whereby NF-L RNA, or cognate RNA-binding factors, enhances the reactivity of NF-L protein and provides a triggering mechanism leading to aggregation of NF-L and other proteins in neurodegenerative diseases.

Aggregates in neurodegenerative disease: crowds and power?

Aggregates in neurodegenerative disease: crowds and power?

Oxidative stress induces amyloid-like aggregate formation of NACP/alpha-synuclein in vitro.

The precursor of non-amyloid beta protein component of Alzheimer's disease amyloid (NACP/alpha-synuclein), found in Lewy bodies of Parkinson's disease (PD), is a presynaptic protein genetically linked to some familial types PD. Mechanisms of abnormal NACP/alpha-synuclein aggregation in neurodegenerative diseases are unclear. Since oxidative stress might play a role in PD pathogenesis, we investigated the role of iron and peroxide in NACP/alpha-synuclein aggregation. Immunoblot analysis showed that human NACP/alpha-synuclein (but not beta-synuclein) aggregated in the presence of ferric ion and was inhibited by the iron chelator deferoxamine. Ferrous ion was not effective by itself, but it potentially aggregated NACP/alpha-synuclein in the presence of hydrogen peroxide. NACP/ alpha-synuclein aggregates displayed strong thioflavine-S and congo-red reactivity, reminiscent of amyloid. This study suggests that NACP/alpha-synuclein aggregation might be closely related to oxidative reactions which may play a critical role in neurodegeneration in disorders with Lewy bodies.