Several groups have reported inhibitors of α-syn assembly and toxicity, including N
-methylated peptides [28
] dopamine and L-dopa [29
], rifampicin [30
], curcumin [31
], and various other compounds [32
]. In most cases, the compounds have been shown to inhibit α-syn aggregation in vitro
, whereas inhibition of toxicity was not studied. A few studies have reported assembly inhibitors, including peptides and small molecules [35
] that protected cells against α-syn-induced toxicity, and encouraging results were reported with the green tea-derived polyphenol (–)epigallocatechin gallate [36
]. A difficult problem has been to rationally design or select effective inhibitors with desired druggable characteristics. In most cases, the mechanism of action of the inhibitors used has been poorly understood. The importance of understanding the mechanism of inhibition has recently been highlighted [37
] following a study suggesting that many inhibitors of fibrillogenesis may act nonspecifically, likely making them unsuitable for treating aberrant protein assembly-related diseases [38
Here, we used CLR01, a general inhibitor of amyloidogenic protein assembly with a well-defined mechanism of action, which was selected based on first principles. We demonstrate that CLR01 interferes with α-syn aggregation and mitigates its toxicity. Importantly, CLR01 was found to block the toxicity of both extracellular and intracellular α-syn at similar concentrations to those needed for inhibiting α-syn aggregation in vitro, supporting the hypothesis that CLR01 protected cells against α-syn toxicity by inhibiting α-syn aggregation. One novel aspect of this report is the generation of a ZF model of α-syn neurotoxicity. We used a T2A bicistronic construct leading to α-syn expression as a fusion protein with DsRed, which rapidly is cleaved, producing native α-syn and at the same time facilitating the monitoring of the expression of α-syn. After CLR01 treatment, α-syn concentrations in α-syn-ZF embryos were reduced, whereas DsRed levels did not change. These observations suggested that CLR01 led to improved clearance of α-syn rather than affecting its expression. Indeed, in living embryos we found that the UPS was inhibited in neurons expressing α-syn, and that this inhibition was prevented in ZF treated with CLR01.
The neurotoxicity of α-syn is well established, but the mechanism of this toxicity and the pathogenic species that confer toxicity are less clear. Proposed mechanisms include impaired protein degradation (both UPS and autophagy), toxicity to synaptic terminals, inflammation, and induction of mitochondrial dysfunction [39
]. Although α-syn fibrils have been found in both humans and animal models of synucleinopathies, several lines of evidence suggest that nonfibrillar soluble oligomers of α-syn are the most toxic species [40
Inhibition of the UPS system has been reported not only for α-syn oligomers [42
], but also with toxic assemblies of other amyloidogenic proteins, including Aβ [44
] and islet amyloid polypeptide [45
]. A recent report demonstrated that overexpression of polyglutamine results in transient UPS inhibition in mice, but the activity returned when large inclusions formed [47
]. Thus, amyloidogenic protein oligomers appear to act in a vicious manner akin to the human immunodeficiency virus, shutting down the very system expected to rid them. Here, we demonstrate for the first time in an in vivo
vertebrate model that α-syn overexpression leads to UPS inhibition and that preventing α-syn aggregation preserves UPS activity.
Several lines of evidence support a role for UPS dysfunction and the development of PD. Two known genetic causes of PD involve aspects of UPS function (Parkin and UCH-L1), and α-syn is a substrate for the UPS. Reduced UPS activity has been found in brains of sporadic PD patients [48
] and some investigators have found that administration of UPS inhibitors to rodents can recreate some of the features of PD, although these models remain controversial [50
]. Finally, we have found that several commonly used pesticides inhibit the UPS and are associated with an increased risk of developing PD [7
The finding that CLR01 promotes α-syn clearance by maintaining α-syn in a soluble, nonaggregated form, and alleviating UPS inhibition is also important when potential toxicity of CLR01 is considered. Ostensibly, Lys-specific MTs, including CLR01, might bind to exposed Lys residues in proteins other than those they are expected to inhibit. MTs were selected as inhibitors based on the hypothesis that the molecular interactions leading to formation of oligomers and nuclei of amyloidogenic proteins were sufficiently weak (hence, nucleus formation is rare and oligomer structure is metastable) to be inhibited by compounds that bind with moderate affinity. As the structure of naturally folded proteins has been optimized by evolution and is substantially more stable, this allows for less strict specificity requirements, because binding with moderate (micromolar) affinity to structurally stable proteins is not expected to affect their structure or activity, whereas labile binding of MTs to naturally unstructured proteins, such as α-syn inhibits their aberrant assembly and prevents their toxicity. Because proteasomal degradation depends on attachment of ubiquitin to free Lys residues, the results presented here demonstrate that despite binding specifically to Lys, CLR01 does not prevent ubiquitination of αS, supporting our hypothesis.
It has also been reported that α-syn could impair autophagy, especially in dopamine-producing cells [56
]. Inhibition of autophagy is unlikely the cause of toxicity in the ZF model used here because treatment of the embryos with an UPS inhibitor along with CLR01 restored α-syn levels. In addition, α-syn requires the interaction with dopamine for inhibiting chaperone-mediated autophagy, and here α-syn was expressed primarily in nondopamine-producing neurons [56
In summary, we show here that CLR01 inhibits α-syn aggregation and toxicity both in vitro and in vivo. Encouragingly, CLR01 dramatically improved clearance of α-syn in a vertebrate model by restoring UPS activity, suggesting that maintaining α-syn in a benign, unaggregated form may be sufficient for alleviating its neurotoxic effects. Our findings have important therapeutic implications because α-syn aggregation is believed to be central to the pathogenesis of PD and other synucleinopathies, and CLR01 inhibits this process without any apparent adverse affects.