, 2003, Luk et al., 2009, Murray et al., 2003 and Serpell et al., 2000). Thus, we asked whether human α-syn pffs composed of α-syn-1-120, α-syn-1-89, α-syn-58-140, or α-syn-NAC could seed formation of LBs and LNs in neurons. We observed that α-syn-1-120 and α-syn-1-89 pffs induced robust accumulation of endogenous p-α-syn aggregates that were Tx-100-insoluble (Figure 2A; data not shown), and they are morphologically indistinguishable from those formed by α-syn-hWT pffs. α-syn-58-140 pffs also seeded formation of endogenous mouse α-syn aggregates that were hyperphosphorylated

Metformin mw (Figure 2A). Moreover, pffs composed of only the central hydrophobic α-syn-NAC domain also resulted in endogenous mouse α-syn fibrillar LB-like aggregates that were Tx-100-insoluble. Overall, our data demonstrate that α-syn pffs containing only the central, hydrophobic portion of α-syn-hWT are sufficient to seed conversion of endogenous α-syn into pathological aggregates. Mice typically do not develop LBs except in the case of transgenic lines overexpressing mutant human α-syn. We thus asked whether the formation of LB-like aggregates required human α-syn or whether they can be seeded by α-syn

pffs generated from recombinant mouse WT α-syn (α-syn-mWT) (Touchman et al., 2001). Immunoblots demonstrated that 14 days treatment of primary neurons with α-syn-mWT pffs induced appearance of p-α-syn in the Tx-100-insoluble fraction (Figure 2B). Immunofluorescence also showed that α-syn-mWT pffs induced formation of p-α-syn aggregates in neurites and somata. Thus, pathological PD-like α-syn aggregates can be induced by α-syn-mWT pffs and does not require the human protein. Examination PLX4032 of the α-syn aggregates using transmission and immuno-EM demonstrated abundant filaments in neurons treated with either α-syn-hWT or α-syn-1-120 pffs (Figure 3A) for 14 days, but not PBS-treated tuclazepam neurons (data

not shown). Remarkably, inclusions composed of 14- to 16-nm-thick filaments were seen throughout the cytoplasm, visualized by transmission EM. Two different immuno-EM detection systems, horse radish peroxidase (HRP) and immunogold amplification, were used to demonstrate that fibrils composed of p-α-syn are found throughout the neuron. P-α-syn-positive fibrils were seen in the soma (Figures 3B and 3C), adjacent to the active zone of presynaptic terminals (Figure 3D) and the postsynaptic terminal (Figure 3F) and throughout processes (Figure 3E). These data establish that the seeding and recruitment of endogenous mouse α-syn into hyperphosphorylated insoluble, filamentous aggregates recapitulate features of LBs and LNs in PD and other human synucleinopathies. To determine the temporal sequence of α-syn aggregate formation, α-syn-hWT pffs were added to the neurons at DIV5. P-α-syn immunostaining was not detectable until 4 days later when small aggregates began to appear, exclusively in the neurites, albeit at low levels (Figure 4A, upper series).