Silver staining (Campbell-Switzer) of neuronal α-synuclein assemblies induced by multiple system atrophy and Parkinson's disease brain extracts in transgenic mice.

Synucleinopathies [Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)] share filamentous α-synuclein assemblies in nerve cells and glial cells. We compared the abilities of brain extracts from MSA and PD patients to induce neuronal α-synuclein assembly and neurodegeneration following intracerebral injection in heterozygous mice transgenic for human mutant A53T α-synuclein. MSA extracts were more potent than PD extracts in inducing α-synuclein assembly and in causing neurodegeneration. MSA assemblies were Campbell-Switzer- and Gallyas-silver-positive, whereas PD assemblies were only Campbell-Switzer-positive, in confirmation of previous findings. However, induced α-synuclein inclusions were invariably Campbell-Switzer-positive and Gallyas-negative, irrespective of whether MSA or PD brain extracts were injected. The α-synuclein inclusions of non-injected homozygous mice transgenic for A53T α-synuclein were also Campbell-Switzer-positive and Gallyas-negative. These findings demonstrate that transgene expression and its intracellular environment dominated over the silver staining properties of the conformers of assembled α-synuclein

Assembly of α-synuclein and neurodegeneration in the central nervous system of heterozygous M83 mice following the peripheral administration of α-synuclein seeds.

Peripheral administration (oral, intranasal, intraperitoneal, intravenous) of assembled A53T α-synuclein induced synucleinopathy in heterozygous mice transgenic for human mutant A53T α-synuclein (line M83). The same was the case when cerebellar extracts from a case of multiple system atrophy with type II α-synuclein filaments were administered intraperitoneally, intravenously or intramuscularly. We observed abundant immunoreactivity for pS129 α-synuclein in nerve cells and severe motor impairment, resulting in hindlimb paralysis and shortened lifespan. Filaments immunoreactive for pS129 α-synuclein were in evidence. A 70% loss of motor neurons was present five months after an intraperitoneal injection of assembled A53T α-synuclein or cerebellar extract with type II α-synuclein filaments from an individual with a neuropathologically confirmed diagnosis of multiple system atrophy. Microglial cells changed from a predominantly ramified to a dystrophic appearance. Taken together, these findings establish a close relationship between the formation of α-synuclein inclusions in nerve cells and neurodegeneration, accompanied by a shift in microglial cell morphology. Propagation of α-synuclein inclusions depended on the characteristics of both seeds and transgenically expressed protein

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

Involvement of Cellular Prion Protein in a-Synuclein Transport in Neurons

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of ß-amyloid. Their interaction is mandatory for neurotoxic effects of ß-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of a-synuclein. Results demonstrate that Prnp expression is not mandatory for a-synuclein spreading. However, although the pathological spreading of a-synuclein can take place in the absence of Prnp, a-synuclein expanded faster in PrPC-overexpressing mice. In addition, a-synuclein binds strongly on PrPC-expressing cells, suggesting a role in modulating the effect of a-synuclein fibrils

TDP-43 in the hypoglossal nucleus identifies amyotrophic lateral sclerosis in behavioral variant frontotemporal dementia

The hypoglossal nucleus was recently identified as a key brain region in which the presence of TDP-43 pathology could accurately discriminate TDP-43 proteinopathy cases with clinical amyotrophic lateral sclerosis (ALS). The objective of the present study was to assess the hypoglossal nucleus in behavioral variant frontotemporal dementia (bvFTD), and determine whether TDP-43 in this region is associated with clinical ALS. Twenty-nine cases with neuropathological FTLD-TDP and clinical bvFTD that had not been previously assessed for hypoglossal TDP-43 pathology were included in this study. Of these 29 cases, 41% (n = 12) had a dual diagnosis of bvFTD-ALS at presentation, all 100% (n = 12) of which demonstrated hypoglossal TDP-43 pathology. Of the 59% (n = 17) cohort that presented with pure bvFTD, 35% (n = 6) were identified with hypoglossal TDP-43 pathology. Review of the case files of all pure bvFTD cases revealed evidence of possible or probable ALS in 5 of the 6 hypoglossal-positive cases (83%) towards the end of disease, and this was absent from all cases without such pathology. In conclusion, the present study validates grading the presence of TDP-43 in the hypoglossal nucleus for the pathological identification of bvFTD cases with clinical ALS, and extends this to include the identification of cases with possible ALS at end-stage

Ubiquitination of alpha-synuclein filaments by Nedd4 ligases.

Alpha-synuclein can form beta-sheet filaments, the accumulation of which plays a key role in the development of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. It has previously been shown that alpha-synuclein is a substrate for the HECT domain-containing ubiquitin ligase Nedd4, and is subject to ubiquitin-mediated endosomal degradation. We show here that alpha-synuclein filaments are much better substrates for ubiquitination in vitro than monomeric alpha-synuclein, and that this increased susceptibility cannot be mimicked by the mere clustering of monomers. Recognition by Nedd4 family enzymes is not through the conventional binding of PPxY-containing sequences to WW domains of the ligase, but it also involves C2 and HECT domains. The disease-causing alpha-synuclein mutant A53T is a much less efficient substrate for Nedd4 ligases than the wild-type protein. We suggest that preferential recognition, ubiquitination and degradation of beta-sheet-containing filaments may help to limit toxicity, and that A53T alpha-synuclein may be more toxic, at least in part because it avoids this fate

Ligase-specific recognition of alpha-synuclein filaments.

<p>(A) Cartoon of the modular architecture of Nedd4-type E3 ligases and overview of the constructs used in this study. (B) Western blots of <i>in vitro</i> ubiquitination assays comparing different Itch versions (full-length, ΔC2, WW mutants [175nM, left panel]) and full-length or HECT-only versions of Nedd4, Nedd4L and Smurf2 [100nM, right panel] in their ability to ubiquitinate alpha-synuclein [600nM, upper panels]. The lower panels (anti-ubiquitin blot) show overall ubiquitination, demonstrating the activity of the E3 ligase constructs by their ability to auto-ubiquitinate. Note that full-length Smurf2 is auto-inhibited in the presence of monomeric alpha-synuclein. (C) Activation of autoinhibited Smurf2 by alpha-synuclein filaments. (D) Western blots of <i>in vitro</i> ubiquitination assays comparing full-length, ΔC2 or HECT-only versions of Nedd4, Itch and Smurf2 [100nM] in their ability to ubiquitinate alpha-synuclein [400nM], indicating ligase-specific recognition of filaments (antibody Syn-1). Three sections of the same exposure of the same gel are shown.</p

Efficient <i>in vitro</i> ubiquitination of alpha-synuclein filaments by Nedd4.

<p>Ubiquitination assays of Nedd4 with alpha-synuclein (antibody Syn-1). (A) Coomassie-stained gels of purified wild-type (WT) and A53T mutant alpha-synuclein. (B) Time course of polymerization of WT and mutant alpha-synuclein assayed by thioflavin dye fluorescence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200763#sec007" target="_blank">Methods</a>). (C) Coomassie-stained gel of purified Nedd4 ligase. (D) <i>In vitro</i> ubiquitination assays using Nedd4 [200nM] with alpha-synuclein [200nM] showing efficient ubiquitination of filamentous (fil.) wild-type alpha-synuclein with strongly reduced modification of the filamentous A53T mutant and hardly any detectable ubiquitination of the monomeric forms (mo.). (E) Ubiquitination assays of Nedd4 [150nM] with alpha-synuclein filaments [600nM] in the presence of ubiquitin chain-type specific deubiquitinases as indicated [UbiCREST, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200763#pone.0200763.ref014" target="_blank">14</a>]] demonstrating mainly K63, but also some K29 and K33, linkages.</p