Monoclonal antibodies selective for α‐synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α‐synuclein transgenic mice with the disease‐causing A30P mutation

Inclusions of intraneuronal alpha‐synuclein (α‐synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α‐synuclein is a central feature of the disease pathogenesis. Among the different α‐synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α‐synuclein oligomers were generated. These antibodies, which do not bind amyloid‐beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α‐synuclein transgenic mice than linear epitope antibodies. An oligomer‐selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α‐synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α‐synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer‐selective α‐synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.     

Gelsolin co-occurs with Lewy bodies in vivo and accelerates α-synuclein aggregation in vitro

Deposition of fibrillar α-synuclein as Lewy bodies is the neuropathological hallmark of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Apart from α-synuclein, these intraneuronal inclusions contain over 250 different proteins. The actin binding protein gelsolin, has previously been suggested to be part of the Lewy body, but its potential role in α-synuclein aggregation remains unknown. Here, we studied the association between gelsolin and α-synuclein in brain tissue from PD and DLB patients as well as in a cell model for α-synuclein aggregation. Moreover, the potential effect of gelsolin on α-synuclein fibrillization was also investigated. Our data demonstrate that gelsolin co-occured with α-synuclein in Lewy bodies from affected human brain as well as with Lewy body-like inclusions in α-synuclein over expressing cells. Furthermore, in the presence of calcium chloride, gelsolin was found to enhance the aggregation rate of α-synuclein in vitro. Moreover, no apparent structural differences could be observed between fibrils formed in the presence or absence of gelsolin. Further studies on gelsolin and other Lewy body associated proteins are warranted to learn more about their potential role in the α-synuclein aggregation process.     

Ubiquitination of alpha-synuclein in Lewy bodies is a pathological event not associated with impairment of proteasome function

Lewy bodies are intracellular fibrillar inclusions composed of alpha-synuclein. They constitute the pathological hallmark of Parkinson's disease, dementia with Lewy bodies, and other neurodegenerative diseases. Although the majority of Lewy bodies are stained for ubiquitin by immunohistochemistry, the substrate for this modification is poorly understood. Insoluble, urea-soluble alpha-synuclein was separated from soluble fractions and subjected to two-dimensional gel electrophoresis to further characterize pathogenic alpha-synuclein species from disease brains. By using this approach, we found that in sporadic Lewy body diseases a highly modified, disease-associated 22-24-kDa alpha-synuclein species is ubiquitinated. Conjugation of one, two, and, to a lesser extent, three ubiquitins was detected. This 22-24-kDa alpha-synuclein species represents partly phosphorylated protein. Furthermore, no generalized impairment of the proteolytic activity of the proteasome was detected in brain regions with Lewy body pathology. Because unmodified alpha-synuclein is degraded by the proteasome in a ubiquitin-independent manner, these data suggest that accumulation of modified 22-24-kDa alpha-synuclein is a disease-specific event which may overwhelm the proteolytic system, leading to aberrant ubiquitination. Accordingly, carboxyl-terminal-truncated alpha-synuclein, presumably the result of aberrant proteolysis, is found only in association with alpha-synuclein aggregates.     

An N-terminal fragment of ProSAAS (a granin-like neuroendocrine peptide precursor) is associated with tau inclusions in Pick's disease

The deposition of aggregated tau in cytoplasmic inclusions is one of the common neuropathological features in various dementing neurodegenerative disorders. At present, it remains unclear whether tau inclusions exert neurotoxicity or they are simply the consequence of neurodegeneration. In our approach for the analysis of the composition of tau inclusions, we detected the intense binding of anti-diacylglycerol kinase-zeta (DGK-zeta) antibodies to Pick bodies (PBs), which represent tau inclusions in Pick's disease. The polyclonal antibodies were found to cross-react with a 21-kDa protein, but not with tau or ubiquitin, on Western blots of normal human brain extracts. Analysis of the 21-kDa protein by two-dimensional-gel electrophoresis and mass-spectrometry revealed that the protein is an N-terminal fragment of proSAAS (a human granin-like neuroendocrine peptide precursor). Our results suggest that sequestration of the N-terminal fragment of proSAAS in intracellular PBs may cause a functional disturbance of neurons in Pick's disease.     

Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.     

The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve

The ontogenetic appearance of the individual triplet polypeptides that comprise mammalian neurofilaments was studied in the developing rat optic nerve. Triton-insoluble cytoskeletal preparations from the optic nerves of rats of postnatal ages 1 Day (P1), 6 days (P6), 10 days (P10), 20 days (P20), and 3 months (adult) were analyzed for protein composition by one and two-dimensional gel electrophoresis. Results indicate that at P1, both the 150- and 68-kDa neurofilament subunit proteins are present. The 200-kDa subunit first becomes discernible at P20, but, at this age, it is still present in considerably less quantity than in the adult. Immunocytochemical verification of the presence of neurofilament protein was accomplished by staining tissue sections with specific antibodies against the 150- and the 68-kDa neurofilament subunits using the peroxidase-antiperoxidase technique. Results of the morphological analyses have shown that neurofilaments are not present in quantity until P10, which coincides with the time when the 68-kDa subunit increases in quantity by one dimensional gel analysis. Thus, the 150- and 68-kDa subunits can be detected prior to the appearance of neurofilaments, and the 200-kDa protein is not observed until sometime later. The potential physiological significance of the differential subunit transport is discussed with respect to neuronal differentiation in the developing mammalian CNS.     

Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson‘s disease, dementia with Lewy bodies and Lewy body variant of Alzheimer‘s disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how 13-sheets assemble into α-synuclein fibrils on a nanometer scale.     

In vitro aggregation assays for the characterization of α-synuclein prion-like properties

Aggregation of α-synuclein plays a crucial role in the pathogenesis of synucleinopathies, a group of neurodegenerative diseases including Parkinson disease (PD), dementia with Lewy bodies (DLB), diffuse Lewy body disease (DLBD) and multiple system atrophy (MSA). The common feature of these diseases is a pathological deposition of protein aggregates, known as Lewy bodies (LBs) in the central nervous system. The major component of these aggregates is α-synuclein, a natively unfolded protein, which may undergo dramatic structural changes resulting in the formation of β-sheet rich assemblies. In vitro studies have shown that recombinant α-synuclein protein may polymerize into amyloidogenic fibrils resembling those found in LBs. These aggregates may be uptaken and propagated between cells in a prion-like manner. Here we present the mechanisms and kinetics of α-synuclein aggregation in vitro, as well as crucial factors affecting this process. We also describe how PD-linked α-synuclein mutations and some exogenous factors modulate in vitro aggregation. Furthermore, we present a current knowledge on the mechanisms by which extracellular aggregates may be internalized and propagated between cells, as well as the mechanisms of their toxicity.     

Proteomic approach to studying parkinson’s disease

Parkinson’s disease is a common age-related neurodegenerative disease characterized pathologically by a loss of dopaminergic neurons in the substantia nigra with resultant depletion of striatal dopamine and presence of Lewy bodies in the remaining neurons. The Lewy body contains numerous functional and structural proteins, including α-synuclein and ubiquitin; aggregation of α-synuclein is thought to be important in Lewy body formation as well as neurodegeneration, although the detailed mechanisms remain to be defined. Increasing evidence has suggested that mitochondrial dysfunction, increased oxidative stress, and dysfunction of the ubiquitin-proteasome system may be involved in α-synuclein aggregation, Lewy body formation, and neurodegeneration. However, how these processes are related to each other is not fully understood, given that there are Parkinsonian animal models as well as human diseases with significant nigral neurodegeneration regardless of whether Lewy bodies form or not. This review summarizes the current related research fields and proposes a proteomic approach to investigate the mechanisms that may dictate α-synuclein aggregation, Lewy body formation, and neurodegeneration.     

Binding of microtubule-associated protein 2 and tau to the intermediate filament reassembled from neurofilament 70-kDa subunit protein. Its regulation by calmodulin

Two major brain microtubule-associated proteins (MAPs), MAP2 and tau, were found to bind to the intermediate filaments reassembled from neurofilament 70-kDa subunit protein (= 70-kDa filaments). The binding was saturable. The apparent dissociation constant (KD) for the binding of MAP2 to the 70-kDa filaments was estimated to be 4.8 X 10(-7) M, and the maximum binding reached 1 mol of MAP2/approximately 30 mol of 70-kDa protein. The apparent KD for the tau binding was 1.6 X 10(-6) M, and the maximum binding was 1 mol of tau/approximately 3 mol of 70-kDa protein. It was also found that MAP2 and tau did not compete with each other for binding to the 70-kDa filaments. Most interestingly, calmodulin, a ubiquitous Ca2+-binding protein in eukaryotic cells, was found to inhibit the binding of MAP2 and tau to the 70-kDa filaments. The inhibition by calmodulin was regulated by changes in Ca2+ concentration around 10(-6) M, and was canceled by trifluoperazine, a calmodulin inhibitor.     

Characterization of a cyclic nucleotide- and calcium-independent neurofilament protein kinase activity in axoplasm from the squid giant axon

The phosphorylation activity associated with a neurofilament-enriched cytoskeletal preparation isolated from the squid giant axon has been studied and compared to the phosphorylation activities in intact squid axoplasm. The high molecular weight (greater than 300 kDa) and 220-kDa neurofilament proteins are the major endogenous substrates for the kinases in the axoplasm and the neurofilament preparation, whereas 95- and less than 60-kDa proteins are the major phosphoproteins in the ganglion cell preparation. The squid axon neurofilament (SANF) protein kinase activity appeared to be both cAMP and Ca2+ independent and could phosphorylate both casein (Km = 40 microM) and histone (Km = 180 microM). The SANF protein kinase could utilize either ATP or GTP in the phosphotransferase reaction, with a Km for ATP of 58 microM and 129.4 microM for GTP when casein was used as the exogenous substrate; and 25 and 98.1 microM for ATP and GTP, respectively, when the endogenous neurofilament proteins were used as substrates. The SANF protein kinase activity was only slightly inhibited by 2,3-diphosphoglycerate and various polyamines at high concentrations and was poorly inhibited by heparin (34% inhibition at 100 micrograms/ml). The failures of heparin to significantly inhibit and the polyamines to stimulate the SANF protein kinase indicate that it is not a casein type II kinase. The relative efficacy of GTP as a phosphate donor indicates that SANF protein kinase differs from known casein type I kinases. Phosphorylated (32P-labeled) neurofilament proteins were only slightly dephosphorylated in the presence of axoplasm or stellate ganglion cell supernatants, and the neurofilament-enriched preparation did not dephosphorylate 32P-labeled neurofilament proteins. The axoplasm and neurofilament preparations had no detectable protein kinase inhibitor activity, but a strong inhibitor activity, which was not dialyzable but was heat inactivatable, was found in ganglion cells. This inhibitor activity may account for the low phosphorylation activity found in the stellate ganglion cells and may indicate inhibitory regulation of SANF protein kinase activity in the ganglion cell bodies.     

Microtubule-associated protein 1B is a component of cortical Lewy bodies and binds alpha-synuclein filaments

Lewy bodies, neuropathological hallmarks of Parkinson's disease and dementia with Lewy bodies, comprise alpha-synuclein filaments and other less defined proteins. Characterization of Lewy body proteins that interact with alpha-synuclein may provide insight into the mechanism of Lewy body formation. Double immunofluorescence labeling and confocal microscopy revealed approximately 80% of cortical Lewy bodies contained microtubule-associated protein 1B (MAP-1B) that overlapped with alpha-synuclein. Lewy bodies were isolated using an immunomagnetic technique from brain tissue of patients dying with dementia with Lewy bodies. Lewy body proteins were resolved by polyacrylamide gel electrophoresis. Immunoblotting confirmed the presence of MAP-1B and alpha-synuclein in purified Lewy bodies. Direct binding studies revealed a high affinity interaction (IC(50) approximately 20 nm) between MAP-1B and alpha-synuclein. The MAP-1B-binding sites were mapped to the last 45 amino acids of the alpha-synuclein C terminus. MAP-1B also bound in vitro assembled alpha-synuclein fibrils. Thus, MAP-1B may be involved in the pathogenesis of Lewy bodies via its interaction with monomeric and fibrillar alpha-synuclein.     

alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD. alpha-Synuclein fibrils similar to the Lewy body filaments can be formed in vitro, and we have shown recently that both PD-linked mutations accelerate their formation. This study addresses the mechanism of alpha-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated alpha-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to alpha-synuclein concentration, and (iii) mutant alpha-synuclein can seed the aggregation of wild type alpha-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with alpha-synuclein mutations will contain both, the mutant and the wild type protein. Finally (iv), we show that wild type and mutant forms of alpha-synuclein do not differ in their critical concentrations. These results suggest that differences in aggregation kinetics of alpha-synucleins cannot be explained by differences in solubility but are due to different nucleation rates. Consequently, alpha-synuclein nucleation may be the rate-limiting step for the formation of Lewy body alpha-synuclein fibrils in Parkinson's disease.     

Dityrosine Cross-links are Present in Alzheimer’s Disease-derived Tau Oligomers and Paired Helical Filaments (PHF) which Promotes the Stability of the PHF-core Tau (297–391) In Vitro

A characteristic hallmark of Alzheimer’s Disease (AD) is the pathological aggregation and deposition of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs). Oxidative stress is an early event during AD pathogenesis and is associated with tau-mediated AD pathology. Oxidative environments can result in the formation of covalent dityrosine crosslinks that can increase protein stability and insolubility. Dityrosine cross-linking has been shown in Aβ plaques in AD and α-synuclein aggregates in Lewy bodies in ex vivo tissue sections, and this modification may increase the insolubility of these aggregates and their resistance to degradation. Using the PHF-core tau fragment (residues 297 – 391) as a model, we have previously demonstrated that dityrosine formation traps tau assemblies to reduce further elongation. However, it is unknown whether dityrosine crosslinks are found in tau deposits in vivo in AD and its relevance to disease mechanism is unclear. Here, using transmission electron microscope (TEM) double immunogold-labelling, we reveal that neurofibrillary NFTs in AD are heavily decorated with dityrosine crosslinks alongside tau. Single immunogold-labelling TEM and fluorescence spectroscopy revealed the presence of dityrosine on AD brain-derived tau oligomers and fibrils. Using the tau (297–391) PHF-core fragment as a model, we further showed that prefibrillar tau species are more amenable to dityrosine crosslinking than tau fibrils. Dityrosine formation results in heat and SDS stability of oxidised prefibrillar and fibrillar tau assemblies. This finding has implications for understanding the mechanism governing the insolubility and toxicity of tau assemblies in vivo.     

Roscovitine对C型尼曼-皮克病小鼠球状神经轴突形成的影响

目的探讨细胞周期依赖性蛋白激酶抑制剂roscovitine对C型尼曼-皮克病(NPC)小鼠(npc-/-)球状神经轴突形成的影响。方法采用不同浓度的roscovitine(72,144,300 and 600nmoles/day)对5.5周龄的npc-/-小鼠进行两周侧脑室灌注,以DMSO灌注的同龄npc-/-小鼠为对照。抗非磷酸化神经细丝蛋白抗体SMI32和抗有丝分裂期磷酸化表位抗体MPM-2免疫染色检测小鼠脑内球状神经轴突的数量,Western blot检测细胞骨架蛋白磷酸化程度的变化。结果Roscovitine组球状神经轴突数量较DMSO组显著减少,神经细丝和tau蛋白的过度磷酸化程度较对照组明显下降(P<0.05)。结论细胞周期依赖性蛋白激酶抑制剂能明显改善NPC的神经元细胞骨架损害。     

Topographic regulation of neuronal intermediate filaments by phosphorylation, role of peptidyl-prolyl isomerase 1: significance in neurodegeneration

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.     

Immunohistochemical evidence for reorganization of tau in the plaques and tangles in Alzheimer''s dissease

Summary Cytochemical and biochemical techniques have been used to assess the relationship of epitopes on the microtubuleassociated protein, tau, to the cytoskeletal pathology of Alzheimer's disease. The main probes were Tau-1 and Alz-50, two monoclonal antibodies which recognize tau and a potentially related 68kDa protein. Sequential treatment of tissue slices with combinations of the antibodies showed that each blocked the binding of the other to neurofibrillary tangles and neuritic plaques but not to normal axons. Western blot analysis of tau proteins isolated from Alzheimer's disease brains did not reveal such blocking patterns. The issue of steric hindrance affecting antibody binding in tissue sections was addressed by using Alz-50 in combination with Tau-2, another monoclonal antibody recognizing tau on blots and in Alzheimer's disease pathology. Neither antibody blocked the binding of the other to neurofibrillary tangles and neuritic plaques. These data suggest that the Alz-50 and Tau-1 epitopes are selectively organized in the tangles and plaques to be in close proximity which supports the hypothesis that in Alzheimer's disease pathology, tau is modified.     

Immunohistochemical localization of microtubule-associated proteins in the nervous system of the small intestine of guinea pig

Summary Layers containing Auerbach's and Meissner's plexuses were dissected from the small intestine of guinea pig and immunostained with affinity-purified antibodies against brain-specific microtubule-associated proteins (MAPs): MAP1, MAP2 and tau and a MAP with a molecular weight of 190000 dalton purified from bovine adrenal cortex (190-kDa MAP). MAP1 antibody stained the network of nerve fibers and the cell bodies of enteric neurons in both Auerbach's and Meissner's plexuses. Staining with anti-tau antibody gave the same results. Antibody against MAP2 stained neuronal cell bodies and short thin processes extending from them. Interganglionic strands composed mainly of long processes were unstained. Anti-190-kDa MAP antibody stained both the neuronal cell bodies and bundles of nerve fibers. However, the staining was less intense than that with anti-MAP1 and tau antibodies. Differentiation in the structure of the cytoskeleton probably exists in the neuronal processes of the enteric neurons as is shown in the dendrites and axons in some neurons of the central nervous system. Thus, enteric neurons possess axon-like processes containing MAP1, tau and probably lower amounts of 190-kDa MAP. Cell bodies and dendrite-like structures of these neurons contain MAP2 in addition to MAP1, tau and 190-kDa MAP.     

Mechanisms in dominant parkinsonism: The toxic triangle of LRRK2, α‐synuclein,and tau

Parkinson's disease (PD) is generally sporadic but a number of genetic diseases have parkinsonism as a clinical feature. Two dominant genes, α‐synuclein (SNCA) and leucine‐rich repeat kinase 2 (LRRK2), are important for understanding inherited and sporadic PD. SNCA is a major component of pathologic inclusions termed Lewy bodies found in PD. LRRK2 is found in a significant proportion of PD cases. These two proteins may be linked as most LRRK2 PD cases have SNCA‐positive Lewy bodies. Mutations in both proteins are associated with toxic effects in model systems although mechanisms are unclear. LRRK2 is an intracellular signaling protein possessing both GTPase and kinase activities that may contribute to pathogenicity. A third protein, tau, is implicated as a risk factor for PD. We discuss the potential relationship between these genes and suggest a model for PD pathogenesis where LRRK2 is upstream of pathogenic effects through SNCA, tau, or both proteins.