Lysine modification increases αSyn immunoreactivity by strengthening attachment to blot membranes.

<p><b>A.</b> HEL cells were crosslinked <i>in vivo</i> with the cleavable crosslinker DSP (1 mM) in comparison to DMSO-only treatment (-). Cytosols were prepared (post-20,000 <i>g</i>) and boiled in sample buffer with 5% (v/v) β-mercaptoethanol (βME), which cleaves the disulfide bond of DSP (DSP_ßME: reduced DSP). Samples (30 µg) were blotted with αSyn antibody 15G7. Equal protein loading was visualized by blotting for DJ-1 and by Ponceau (Ponc.) staining. <b>B.</b> Primary cortical rat neurons (13 days <i>in vitro</i>) were treated with 1 mM DSP <i>in vitro</i> (cytosolic lysates) in comparison to DMSO-only (-) treatment. Cytosols (post 100,000 <i>g</i>) were boiled in sample buffer with 5% βME and blotted with αSyn antibody 2F12, as well as antibodies to βSyn, γSyn and β-tubulin (β-tub.). Ponceau (Ponc.) stain of the blot membrane is shown on the left. <b>C.</b> Human (H.s.; 15 µg) and mouse (M.m., 30 µg) brain homogenates (PBS soluble fraction; post 100,000 <i>g</i>) were treated <i>in vitro</i> with DSP or DMSO-only (-), followed by boiling in sample buffer containing 5% βME. Blots were developed with αSyn antibody Syn1 as well as antibodies to β-tubulin, β-actin, GAPDH and DJ-1. <b>D.</b> Purified recombinant αSyn and purified hen-egg lysozyme (lyz) were crosslinked at a concentration of 500 ng/µL with 2 mM DSP (DSP_ßME) in comparison to DMSO only (-), followed by boiling in sample buffer with 5% βME. After separation by SDS-PAGE, samples were transferred to PVDF membranes which were dried immediately (left panel: ‘no wash’) or after washing overnight in PBST (‘wash’). Dried membranes were briefly stained with Coomassie Blue. 14, 17 and 28 kDa molecular weight markers are visible in the outer left lane.</p

<i>In vitro</i> incubation of lysates with 2 mM DSP followed by βME reduction allows optimal αSyn detection.

<p><b>A.</b> HEL intact cells (<i>in vivo</i>) or lysates (<i>in vitro</i>) were incubated with DMSO only (-) as well as gradients of 0.5, 2.0 and 8.0 mM DSP and DTBP, respectively. Cytosols (post-100,000 <i>g</i>) were boiled in sample buffer/5% βME and blotted with αSyn antibody 2F12. Identical exposures of the same blot are shown; film was cut at dotted line. <b>B.</b> HEL cell cytosols (post-100,000 <i>g</i>) at three different protein concentrations (2.4, 3.3., 4.5 µg/µL) were incubated with DMSO only (-) as well as a gradient of 0.5, 2.0 and 8.0 mM DSP and DSG. Samples were normalized to 2.4 µg after quenching, then boiled in sample buffer plus 5% βME and blotted with αSyn antibodies Syn1 and 15G7 as well as an antibody to GAPDH. <b>C.</b> HEL cell cytosols (post-100,000 <i>g</i>) were treated with DMSO-only (-) or DSP, quenched, boiled in sample buffer plus 5% βME and analyzed by blotting for αSyn (mAb Syn1 and pAb C20), Calmodulin, DJ-1, Ran, 14-3-3 and β-actin.</p

DSP/βME treatment facilitates easy detection of αSyn from cultured cells.

<p><b>A.</b> Cytosols (post-100,000 <i>g</i>) of 3D5 αSyn tet-off cells in induced (3D5_I) or repressed state (3D5_R) as well as lysates of the parental neuroblastoma cell line M17D were crosslinked with 2 mM DSP, followed by boiling in sample buffer with 5% βME. Western blot analysis was performed for αSyn (C20), DJ-1 and β-actin. (Note that the DJ-1 blot shows residual C20 signal from a previous exposure.) <b>B.</b> Cytosols (post-100,000 <i>g</i>) of neuroblastoma cell lines M17D and SH-SY5Y as well as the PBS-soluble fraction of human brain homogenates were incubated in 2 mM DSP, followed by boiling in sample buffer with 5% βME and blotting for αSyn (2F12) as well as β-tubulin. A long and a short exposure of the 2F12 blot are shown. <b>C.</b> Cytosols (post-100,000 <i>g</i>) of SH-SY5Y cells were crosslinked with 2 mM DSP, followed by boiling in sample buffer/5% βME and blotting for the indicated antibodies. Membranes were cut at dotted lines after protein transfer and the left half was incubated in 0.4% PFA for 30 min, while the right half was blocked immediately. Membranes were reassembled before film development; PFA-treated and -untreated halves shown were exposed identically. <b>D.</b> M17D cells analyzed analogously to SH-SY5Y cells in Fig. 4c. In addition to αSyn and UCH-L1, histone protein H3 was detected using a total H3 as well as an H3 lysine 27-methylation-specific antibody (H3 K27met).</p

Discrepancy between immunoblotting of αSyn from crosslinker-treated and untreated lysates.

<p><b>A.</b> Intact primary cortical rat neurons (13 days <i>in vitro</i>) were incubated with DMSO only (-) or a gradient of 0.1, 0.5 and 1.0 mM DSP or DSG, sonicated in PBS supplemented with protease inhibitors and spun at 100,000 <i>g</i> for 60 min. The resultant cytosols (30 µg) were blotted with αSyn antibody Syn1 (left panel: short exposure; middle panel: long exposure) and with anti-DJ-1 as a control (right panel). Arrows indicate the crosslinker-trapped αSyn species that have been described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081314#pone.0081314-Dettmer1" target="_blank">[7]</a> as well as the DJ-1 dimer (2-mer) and monomer (1-mer). <b>B.</b> Densitometric analysis of the blots shown in Fig. 1a using ImageJ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081314#s2" target="_blank">Materials and Methods</a>). Bars indicate the total immunoreactivity of each lane as a percentage of the signal from DMSO-only treated sample (lane 1), after subtraction of the background from the empty lane 5.</p

5‑Nitro-1,2-benzothiazol-3-amine and <i>N</i>‑Ethyl-1-[(ethylcarbamoyl)(5-nitro-1,2-benzothiazol-3-yl)amino]formamide Modulate α‑Synuclein and Tau Aggregation

Protein misfolding results in a plethora of known diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, transthyretin-related amyloidosis, type 2 diabetes, Lewy body dementia, and spongiform encephalopathy. To provide a diverse portfolio of therapeutic small molecules with the ability to reduce protein misfolding, we evaluated a set of 13 compounds: 4-(benzo[d]thiazol-2-yl)aniline (BTA) and its derivatives containing urea (1), thiourea (2), sulfonamide (3), triazole (4), and triazine (5) linker. In addition, we explored small modifications on a very potent antioligomer 5-nitro-1,2-benzothiazol-3-amine (5-NBA) (compounds 6–13). This study aims to define the activity of BTA and its derivatives on a variety of prone-to-aggregate proteins such as transthyretin (TTR81–127, TTR101–125), α-synuclein (α-syn), and tau isoform 2N4R (tau 2N4R) through various biophysical methods. Thioflavin T (ThT) fluorescence assay was used to monitor fibril formation of the previously mentioned proteins after treatment with BTA and its derivatives. Antifibrillary activity was confirmed using transmission electron microscopy (TEM). Photoreactive cross-linking assay (PICUP) was utilized to detect antioligomer activity and lead to the identification of 5-NBA (at low micromolar concentration) and compound 13 (at high concentration) as the most promising in reducing oligomerization. 5-NBA and not BTA inhibited the inclusion formation based on the cell-based assay using M17D neuroblastoma cells that express inclusion-prone αS-3K::YFP. 5-NBA abrogated the fibril, oligomer, and inclusion formation in a dose-dependent manner. 5-NBA derivatives could be the key to mitigate protein aggregation. In the future, the results made from this study will provide an initial platform to generate more potent inhibitors of α-syn and tau 2N4R oligomer and fibril formation

1,4-Diurea- and 1,4-Dithiourea-Substituted Aromatic Derivatives Selectively Inhibit α‑Synuclein Oligomer Formation <i>In Vitro</i>

Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting the elderly population worldwide. In PD, the misfolding of α-synuclein (α-syn) results in the formation of inclusions referred to as Lewy bodies (LB) in midbrain neurons of the substantia nigra and other specific brain localizations, which is associated with neurodegeneration. There are no approved strategies to reduce the formation of LB in the neurons of patients with PD. Our drug discovery program focuses on the synthesis of urea and thiourea compounds coupled with aminoindole moieties to abrogate α-syn aggregation and to slow down the progression of PD. We synthesized several urea and thiourea analogues with a central 1,4-phenyl diurea/thiourea linkage and evaluated their effectiveness in reducing α-syn aggregation with a special focus on the selective inhibition of oligomer formation among other proteins. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), as well as M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. Our results identified compound 1 as the best compound in reducing α-syn fibril formation via ThT assays. The antioligomer formation of compound 1 was subsequently superseded by compound 2. Both compounds selectively curtailed the oligomer formation of α-syn but not tau 4R isoforms (0N4R, 2N4R) or p-tau (isoform 1N4R). Compounds 1 and 2 failed to abrogate tau 0N3R fibril formation by ThT and atomic force microscopy. Compound 2 was best at reducing the formation of recombinant α-syn fibrils by TEM. In contrast to compound 2, compound 1 reduced the formation of α-syn inclusions in M17D neuroblastoma cells in a dose-dependent manner. Compound 1 may provide molecular scaffolds for the optimization of symmetric molecules for its α-syn antiaggregation activity with potential therapeutic applications and development of small molecules in PD

Evaluation of N- and O‑Linked Indole Triazines for a Dual Effect on α‑Synuclein and Tau Aggregation

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder underlying dementia in the geriatric population. AD manifests by two pathological hallmarks: extracellular amyloid-β (Aβ) peptide-containing senile plaques and intraneuronal neurofibrillary tangles comprised of aggregated hyperphosphorylated tau protein (p-tau). However, more than half of AD cases also display the presence of aggregated α-synuclein (α-syn)-containing Lewy bodies. Conversely, Lewy bodies disorders have been reported to have concomitant Aβ plaques and neurofibrillary tangles. Our drug discovery program focuses on the synthesis of multitarget-directed ligands to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to slow the progression of AD and related dementias. To this end, we synthesized 11 compounds with a triazine-linker and evaluated their effectiveness in reducing α-syn, tau isoform 2N4R, and p-tau isoform 1N4R aggregation. We utilized biophysical methods such as thioflavin T (ThT) fluorescence assays, transmission electron microscopy (TEM), photoinduced cross-linking of unmodified proteins (PICUP), and M17D intracellular inclusion cell-based assays to evaluate the antiaggregation properties and cellular protection of our best compounds. We also performed disaggregation assays with isolated Aβ-plaques from human AD brains. Our results demonstrated that compound 10 was effective in reducing both oligomerization and fibril formation of α-syn and tau isoform 2N4R in a dose-dependent manner via ThT and PICUP assays. Compound 10 was also effective at reducing the formation of recombinant α-syn, tau 2N4R, and p-tau 1N4R fibrils by TEM. Compound 10 reduced the development of α-syn inclusions in M17D neuroblastoma cells and stopped the seeding of tau P301S using biosensor cells. Disaggregation experiments showed smaller Aβ-plaques and less paired helical filaments with compound 10. Compound 10 may provide molecular scaffolds for further optimization and preclinical studies for neurodegenerative proteinopathies