A novel "molecular tweezer" inhibitor of α-synuclein neurotoxicity in vitro and in vivo

Abstract

Aggregation of α-synuclein (α-syn) is implicated as being causative in the pathogenesis of Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Despite several therapies that improve symptoms in these disorders, none slow disease progression. Recently, a novel "molecular tweezer" (MT) termed CLR01 has been described as a potent inhibitor of assembly and toxicity of multiple amyloidogenic proteins. Here we investigated the ability of CLR01 to inhibit assembly and toxicity of α-syn. In vitro, CLR01 inhibited the assembly of α-syn into β-sheet-rich fibrils and caused disaggregation of pre-formed fibrils, as determined by thioflavin T fluorescence and electron microscopy. α-Syn toxicity was studied in cell cultures and was completely mitigated by CLR01 when α-syn was expressed endogenously or added exogenously. To determine if CLR01 was also protective in vivo, we used a novel zebrafish model of α-syn toxicity (α-syn-ZF), which expresses human, wild-type α-syn in neurons. α-Syn-ZF embryos developed severe deformities due to neuronal apoptosis and most of them died within 48 to 72 h. CLR01 added to the water significantly improved zebrafish phenotype and survival, suppressed α-syn aggregation in neurons, and reduced α-syn-induced apoptosis. α-Syn expression was found to inhibit the ubiquitin proteasome system in α-syn-ZF neurons, resulting in further accumulation of α-syn. Treatment with CLR01 almost completely mitigated the proteasome inhibition. The data suggest that CLR01 is a promising therapeutic agent for the treatment of Parkinson's disease and other synucleinopathies.

Fig. 1

Structure of the molecular tweezers CLR01 and CLR03.

Fig. 2

CLR01 inhibits α-synuclein (α-syn) assembly and disaggregates α-syn in vitro. (a) β-Sheet formation in α-syn in the absence or presence of different concentrations of CLR01 or CLR03 was followed by measuring thioflavin T (ThT) (Sigma-Aldrich) fluorescence. (b) Electron micrographs of α-syn in the presence or absence of 10-fold molar excess of CLR01 or CLR03 at the end of the reactions shown in panel A. Scale bars denote 100 nm. (c) Disaggregation of α-syn fibrils by CLR01 was initiated at 2 time points: on day 8 (dissociation reaction D1) or on day 24 (D2), and the reactions were monitored using ThT fluorescence. Electron micrographs were obtained periodically and show the morphology of α-syn at the indicated time points. Scale bars: 100 nm. The data are an average of 3 independent experiments.

Fig. 3

CLR01 inhibits α-synuclein (α-syn) toxicity in cell culture. (a) CLR01 inhibits endogenously expressed α-syn toxicity in HEK293 cells. α-Syn expression was induced by adding doxyclycline (Dox) in the absence or presence of CLR01. Cell numbers and cell death measured using propidium iodide were determined by flow cystometry (N = 12 per condition; *p < 0.0003; **p < 0.007). (b) Differentiated PC-12 cells were treated with 20 μM α-syn incubated in the absence or presence of increasing concentrations of CLR01 for 48 h and cell viability was measured using the MTT assay. Inset: Viability of PC-12 cells treated similarly with α-syn in the presence or absence of 10-fold molar excess of CLR01 or CLR03. The data are an average of at least 3 independent experiments with 6 wells per condition. Not significant (NS) = xxx; PI = xxxx.

Fig. 4

Overexpression of α-synuclein (α-syn) in neurons causes dysmorphic zebrafish (ZF) embryos and death. (a–f) DNA constructs were injected into ZF eggs at the 1 cell stage and embryos imaged at 48 hpf. Bright-field images are shown in (a–c) and fluorescent images in (d–f). HuC-α-syn-T2A-dsRed injected embryos were \clearly dysmorphic with reduced numbers of DsRed and α-syn expressing neurons (b, c, e, and f). HuC-dsRed control injected embryos were larger, normal appearing, with abundant DsRed-expressing neurons (a and d). (g) Survival of HuC-α-syn-T2A-dsRed injected embryos was nearly identical to HuC-α-syn injected embryos confirming that the toxicity was caused by -α-syn expression.

Fig. 5

CLR01 ameliorates α-synuclein (α-syn) neurotoxicity in zebrafish (ZF). (a) ZF embryos were treated with CLR01 at 8 hpf and were monitored for abnormal appearance and survival. Bright-field and fluorescent overlay images were taken at 72 hpf (top). Green bars represent normal-appearing embryos and red bars represent abnormal embryos (N = 132/condition). (b) CLR01 prevents α-syn-induced apoptosis. ZF embryos expressing DsRed or α-syn were incubated in acridine orange 24 hpf and apoptotic cells were counted (N = 6 per condition). Ten μM CLR01 reduced α-syn-induced apoptosis to control levels (*p < 0.007 Syn-DsRed vs Syn-DsRed/CLR01 and DsRed control). Representative images are shown on the right.

Fig. 6

CLR01 prevents α-synuclein (α-syn) aggregation and proteasome inhibition. (a–e) ZF embryos expressing α-syn-DsRed (72 hpf) were subjected to immunohistochemistry (IHC). Green represents anti-α-syn antibody binding, red is DsRed, and blue is DAPI-stained nuclei. (a, b) Representative neurons in untreated ZF. (c, d) CLR01-treated embryos. (e) Merged image of panels (c and d). (f) α-Syn expression inhibits the UPS in ZF embryos. Embryos were lysed and proteins subjected to WB analysis (10 embryos per condition, N = 4 experiments). Lane 1, DsRed control; lane 2, α-syn-DsRed untreated; lane 3, α-syn-DsRed CLR01 treated; lane 4, α-syn-DsRed CLR01 and lactocystin treated; lane 5, wild-type untreated embryos. Optical densities for α-syn (green bars) and DsRed (red bars) were normalized to actin and expressed as the percentage of untreated controls (*p < 0.0002). g) CLR01 prevents α-syn aggregate-induced inhibition of the ubiquitin-proteasome system (UPS). HuC-syn-DsRed (N = 19) or HuC-DsRed (N = 13, control) constructs were co-injected with HuC-GFP^U to determine UPS activity in neurons overexpressing α-syn. Values represent the mean number of fluorescent cells in the tail region per embryo at 48 hpf. Green bars represent the number of GFP^U-positive cells (low UPS activity), and red bars represent the number of DsRed-positive cells (high UPS activity). Representative images are shown on the right. (*p < 0.0001 vs DsRed-CLR01; **p < 0.0002 vs α-syn-CLR01).