Amyloid β-protein oligomers upregulate the β-secretase, BACE1, through a post-translational mechanism involving its altered subcellular distribution in neurons

Quantification of fluorescence intensities in axons and dendrites. After double immunofluorescent staining of primary neurons with anti-BACE1 (green) and anti-MAP2 (red) antibodies, specimens were examined under a LSM780 microscope. BACE1 fluorescence intensities along MAP2-positive dendrites (red line) and MAP2-negative axons (blue line) were quantified as described in Methods. Scale bar = 10 μm. (PDF 66 kb

Amyloid fibrils formed by selective N-, C-terminal sequences of mouse apolipoprotein A-II

In mice, amyloidogenic type C apolipoprotein A-II (apoA-II) forms amyloid fibrils in age-associated amyloidosis. To understand the mechanism of amyloid fibril formation by apoA-II, we examined the polymerization of synthetic partial peptides of apoA-II in vitro. None of the partial apoA-II peptides polymerized into amyloid fibrils when tested as a single species mixture. We found a unique mechanism in which N- and C-terminal peptides associated into amyloid fibrils in a 1:1 ratio at pH 2.5. The 11-residue amino acid sequence (6-16), which is a common sequence of type B apoA-II and type C apoA-II proteins in amyloidosis-resistant mice and amyloidosis-susceptible mice, respectively, was critical for polymerization into amyloid fibrils. The 18-residue-long amino acid sequence (48-65) is also necessary for nucleation, but not for the extension phase. These findings suggest that there may be different mechanisms underlying the nucleation and extension phases of apoA-II amyloid fibril formation. We also found that amino acid substitutions between type B apoA-II (Pro5, Val38) and type C apoA-II (Gln5, Ala38) did not affect either phase. The strategy of using synthetic partial peptides of amyloidogenic proteins in vitro is a useful system for understanding amyloid fibril formation and for the development of novel therapies.ArticleBIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS. 1794(10):1517-1529 (2009)journal articl

The effect of truncation on prion-like properties of α-synuclein

Increasing evidence suggests that α-synuclein (αS) aggregates in brains of individuals with Parkinson\u27s disease and dementia with Lewy bodies can spread in a prion-like manner. Although the initial αS nuclei are pivotal in determining αS fibril polymorphs and resulting phenotypes, it is not clear how the initial fibril seeds are generated. Previous studies have shown that αS truncation might have an important role in αS aggregation. However, little is known about how this truncation influences αS\u27s propagation properties. In the present study, we generated αS fibrils from a series of truncated human αS constructs, characterized their structures and conformational stabilities, and investigated their ability to convert the conformation of full-length αS in vitro, in cultured cells, and in WT mice. We show that both C- and N-terminal truncations of human αS induce fibril polymorphs and exhibit different cross-seeding activities. N-terminally 10- or 30-residue–truncated human αS fibrils induced more abundant αS pathologies than WT fibrils in mice, whereas other truncated fibrils induced less abundant pathologies. Biochemical analyses of these truncated fibrils revealed that N-terminal 10- or 30-residue truncations of human αS change the fibril conformation in a manner that increases their structural compatibility with WT mouse αS fibrils and reduces their stability. C-terminally 20-residue–truncated fibrils displayed enhanced seeding activity in vitro. Our findings imply that truncation of αS can influence its prion-like pathogenicity, resulting in phenotypic diversity of α-synucleinopathies

AL amyloidosis with non-amyloid forming monoclonal immunoglobulin deposition; a case mimicking AHL amyloidosis

BackgroundImmunoglobulin heavy-and-light-chain amyloidosis (AHL amyloidosis) is a newly established disease entity where both the immunoglobulin heavy-chain and light-chain compose amyloid fibrils. The immunoglobulins responsible for the amyloid fibrils are generally identified by immunostaining and/or laser microdissection-liquid chromatography-tandem mass spectrometry (LMD-LC-MS/MS). However, both techniques do not biochemically differentiate immunoglobulins that formed amyloid fibrils from non-responsible immunoglobulins.Case presentationWe herein report a case of 67-year-old female patient with renal amyloidosis due to lymphoplasmacytic lymphoma secreting monoclonal immunoglobulin M (IgM)-kappa. Renal immunostaining monotypically positive for IgM-kappa and LMD-LC-MS/MS identification of mu heavy-chain and kappa light-chain were consistent with the diagnosis of AHL amyloidosis. In order to confirm that both the immunoglobulin heavy-chain and light-chain were forming amyloid fibrils, we performed LC-MS/MS of renal amyloid fibrils isolated by the traditional amyloid purification method. The additional LC-MS/MS identified kappa light-chain only without any heavy-chain component. These results were suggestive that amyloid fibrils were composed by kappa light-chain only and that the mu heavy-chain identified by immunostaining and LMD-LC-MS/MS was derived from the non-specific co-deposition of monoclonal IgM-kappa.ConclusionThe case was AL amyloidosis with non-amyloid forming monoclonal immunoglobulin deposition. While immunostaining and LMD-LC-MS/MS are irreplaceable techniques to classify amyloidosis, confident exclusion of the present condition should be required to diagnose AHL amyloidosis

Biochemical classification of tauopathies by immunoblot, protein sequence and mass spectrometric analyses of sarkosyl-insoluble and trypsin-resistant tau

Intracellular filamentous tau pathology is the defining feature of tauopathies, which form a subset of neurodegenerative diseases. We have analyzed pathological tau in Alzheimer’s disease, and in frontotemporal lobar degeneration associated with tauopathy to include cases with Pick bodies, corticobasal degeneration, progressive supranuclear palsy, and ones due to intronic mutations in MAPT. We found that the C-terminal band pattern of the pathological tau species is distinct for each disease. Immunoblot analysis of trypsin-resistant tau indicated that the different band patterns of the 7–18 kDa fragments in these diseases likely reflect different conformations of tau molecular species. Protein sequence and mass spectrometric analyses revealed the carboxyl-terminal region (residues 243–406) of tau comprises the protease-resistant core units of the tau aggregates, and the sequence lengths and precise regions involved are different among the diseases. These unique assembled tau cores may be used to classify and diagnose disease strains. Based on these results, we propose a new clinicopathological classification of tauopathies based on the biochemical properties of tau

Desulfation of Heparan Sulfate by Sulf1 and Sulf2 Is Required for Corticospinal Tract Formation

Heparan sulfate (HS) has been implicated in a wide range of cell signaling. Here we report a novel mechanism in which extracellular removal of 6-O-sulfate groups from HS by the endosulfatases, Sulf1 and Sulf2, is essential for axon guidance during development. In Sulf1/2 double knockout (DKO) mice, the corticospinal tract (CST) was dorsally displaced on the midbrain surface. In utero electroporation of Sulf1/2 into radial glial cells along the third ventricle, where Sulf1/2 mRNAs are normally expressed, rescued the CST defects in the DKO mice. Proteomic analysis and functional testing identified Slit2 as the key molecule associated with the DKO phenotype. In the DKO brain, 6-O-sulfated HS was increased, leading to abnormal accumulation of Slit2 protein on the pial surface of the cerebral peduncle and hypothalamus, which caused dorsal repulsion of CST axons. Our findings indicate that postbiosynthetic desulfation of HS by Sulfs controls CST axon guidance through fine-tuning of Slit2 presentation

Structure-based classification of tauopathies

Ordered assembly of tau protein into filaments characterizes multiple neurodegenerative diseases, which are called tauopathies. We previously reported that by electron cryo-microscopy (cryo-EM), tau filament structures from Alzheimer’s disease (1,2), Pick’s disease (3), chronic traumatic encephalopathy (CTE) (4) and corticobasal degeneration (CBD) (5) are distinct. Here we show that the structures of tau filaments from progressive supranuclear palsy (PSP) define a novel three-layered fold. Moreover, the tau filament structures from globular glial tauopathy (GGT) are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs from the above and resembles the four-layered CBD fold. The AGD fold is also observed in aging-related tau astrogliopathy (ARTAG). Tau protofilament structures from inherited cases with mutations +3 or +16 in intron 10 of MAPT, the microtubule-associated protein tau gene, are also identical to those from AGD, suggesting that relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, tau filament structures from cases of familial British dementia (FBD) and familial Danish dementia (FDD) are the same as those from Alzheimer’s disease and primary age-related tauopathy (PART). These findings suggest a hierarchical classification of tauopathies based on their filament folds, which complements clinical diagnosis and neuropathology, and allows identification of new entities, as we show for a case diagnosed as PSP, but with filament structures that are intermediate between those of GGT and PSP