Furthermore, neurons generated from other strains of mice as well as rats developed LB- and LN-like inclusions when treated with α-syn-hWT pffs, supporting the hypothesis

that induction of α-syn pathology is a general feature selleck compound of primary rodent neurons (data not shown). P-α-syn-positive aggregates (as detected by 81A) did not form in astrocytes (Figure S1B). Moreover, the appearance of α-syn pathology required the presence of endogenous α-syn since α-syn-hWT pffs did not induce any pathology in primary neurons from α-syn −/− mice (Figure 1C). Furthermore, monomeric α-syn did not induce α-syn inclusions (data not shown), demonstrating that α-syn pffs alone seed the aggregates. Immunoblot analyses were conducted on neuron lysates sequentially

extracted with 1% Tx-100, followed by 2% SDS (Figure 1B). In contrast to PBS-treated neurons, those treated with α-syn-hWT pffs for 14 days showed > 80% reduction of α-syn in the Tx-100-soluble fraction accompanied by a concomitant appearance of α-syn in the SDS-extractable fraction. Immunoblots of the SDS-extractable fraction also showed insoluble p-α-syn. A mouse-specific anti-α-syn antibody did not detect α-syn-hWT pffs (Figure 1B, first lane on left), but detected bands in the neuron lysates similar to those labeled by the C terminus specific http://www.selleckchem.com/products/pexidartinib-plx3397.html α-syn antibody and mAB 81A. In addition, higher-molecular-weight species of α-syn were detected in the SDS fraction of all α-syn pffs-treated cultures, and likely correspond to oligomeric and/or ubiquitinated α-syn (Li et al., 2005, Luk et al., 2009 and Sampathu et al., 2003). Sequential extractions of primary hippocampal neurons from α-syn −/− mice 14 days after addition of α-syn-hWT pffs confirmed the absence of pathological α-syn or any other species of immunoreactive α -syn (Figure S1C).

Thus, these data demonstrate that α-syn pffs induced recruitment of soluble endogenous α-syn into insoluble, hyperphosphorylated α-syn aggregates. Since α-syn is ubiquitinated in LBs and LNs, we studied α-syn aggregates that formed 14 days after addition of α-syn-hWT pffs and showed they were also ubiquitin no positive (Figure 1D), and colocalized with p-α-syn. Because the exogenous α-syn-hWT pffs are not ubiquitinated or phosphorylated (Luk et al., 2009), these posttranslational modifications must occur intracellularly as endogenous mouse α-syn accumulates. Thus, these α-syn aggregates share hallmark features of PD-like LNs and LBs allowing us to conclude that misfolded α-syn pffs seed and recruit normal, endogenous α-syn to form pathologic aggregates. Previous in vitro studies have shown that recombinant α-syn protein lacking N- or C-terminal residues, or a synthetic peptide containing only the NAC domain (amino acid residues 61-95), assemble into α-syn amyloid fibrils, and nucleate full-length α-syn fibrillization (Giasson et al., 2001, Han et al.